Handbook of Stem Cell Therapy [1 ed.] 9789811926549, 9789811926556, 9811926549

Table of contents :
Foreword
Contents
About the Editor
Section Editors
Contributors
Part I: Cell-Based Therapy Approach: Mesenchymal Stem Cell-Based Therapy
1 Human Mesenchymal Stem Cells: The Art to Use Them in the Treatment of Previously Untreatable
Introduction
Mesenchymal Stem Cells Current Status: The ``Problems´´ That in Reality Do Not Exist
Practical Approaches: The Golden Standards, i.e., ``Good Biological Praxis´´ (GBP) Using the Historical Laboratory Experience
Data Extrapolation from Small Animal Studies to Humans Is Unfounded and Without Relevance
Current Research Paradigm: MSCs in Advanced Phases of Clinical Trials Although Understandable Is Far from Personalized Medicine
A Case of Autologous Versus Allogeneic MSCs, an Unanswered Question
A Case for Autologous Cells
Autologous Cells from Patients Are Also ``sick´´
Autologous Cells in Elderly Patients Are Also Aged
A Case for Allogeneic Cells
Logistic Advantage of Allogenic MSCs
Allogenic MSCs Overcome the Limitations of Aging and Sickness of Autologous Cells
Quality Control of MSCs Preparation: A Prerequisite for Optimal Prognosis
Conclusion
References
2 Sources and Therapeutic Strategies of Mesenchymal Stem Cells in Regenerative Medicine
Introduction
Mesenchymal Stem Cells
Sources of Mesenchymal Stem Cells
Bone Marrow-Derived MSCs
Advantages of BM-Derived MSCs
Limitations of BM-Derived MSCs
Adipose Tissue-Derived MSCs
Advantages of Adipose Tissue-Derived MSCs
Limitations of Adipose Tissue-Derived MSCs
Isolation of Adipose Tissue-Derived MSCs
Clinical Applications of Adipose Tissue-Derived MSCs
Umbilical Cord-Derived MSCs
Human Umbilical Cord Structure and Function
Advantages of WJ-Derived MSCs
Limitations of WJ-Derived MSCs
Isolation Methods of WJ-Derived MSCs
Clinical Applications of WJ-Derived MSCs
Therapeutic Strategies of Using MSCs
Tissue Regeneration
Immunomodulation of MSCs
Extracellular Vesicles/Exosomes Derived from MSCs
MSCs Act as an Efficient Tool for Gene Therapy
References
3 Considerations for Clinical Use of Mesenchymal Stromal Cells
Introduction
Biological Characteristics
Phenotypic Profile
MSCs´ Proliferation
Differentiation Capacity
Cellular Transformation
Mechanism of Action
Cell Migration Toward Damaged Tissues
Tissue Repair
Immunomodulatory Potential
Preclinical Applications
MSC-Based Therapy for Autoimmune Diseases
MSCs for Cancer Treatment
The Anti-tumor Activity of MSCs
The Pro-tumor Activity of MSCs
The Imprint of Disease on MSCs
Considerations for Clinical Applications
Safety Concerns
Cell Manufacturing for Clinical Use
Considerations for Cellular Medicament
General Considerations
Attempts to Improve the Therapeutic Outcomes of Cellular Medicament
Concluding Remarks and Future Perspective
Cross-References
References
4 Stem Cells in Wound Healing and Scarring
Introduction
Wound-Healing Process
Normal Wound Healing
Impaired Wound Healing
Excessive Wound Healing
Stem Cell Subtypes in Skin Regeneration
Embryonic Stem Sells
Induced Pluripotent Stem Cells
Mesenchymal Stem Cells
Epithelial (Epidermal) Stem Cells
Applications in Defective Wound Healing
Stem Cells in Overscarring
Stem Cell Therapy in Pathological Scarring
Conclusion
Cross-References
References
5 Mesenchymal Stem Cells
Introduction
Mesenchymal Stem Cells: Fist Description in Bone Marrow
Bone Marrow-Derived MSCs
Isolation of MSCs from Different Sources
Adipose Tissue
Umbilical Cord
Umbilical Cord Blood
Wharton´s Jelly
Placenta
Characterization MSCs Before Culture
STRO-1
CD271
SSEA-4
CD146
MSCs in the Clinical Perspective
MSCs-Based Therapy for Cardiovascular Pathologies
MSCs-Based Therapy for Acute Graft-Versus-Host Disease
MSCs-Based Therapy for Crohn´s Disease
MSCs-Based Therapy for Multiple Sclerosis
MSCs-Based Therapy for Rheumatoid Arthritis and Osteoarthritis
Exosomes-Based Therapeutic Intervention
MSCs-Derived Exosome for Therapy ``Without Cells´´
MSCs-Derived Exosomes and Acute Graft-Versus-Host Disease
MSCs-Derived Exosomes for Inflammatory Bowel Disease
MSCs-Derived Exosomes for Rheumatoid Arthritis and Knee Osteoarthritis
MSCs-Derived Exosomes for the Heart
Multiple Sclerosis
Conclusion
References
6 Mesenchymal Stromal Cells for COVID-19 Critical Care Patients
Introduction
Mechanism of Infection and Immune Response
The Rationale for the Clinical Use of MSCs for COVID-19 Patients
Progress in MSC-Based Therapy for COVID-19
Other Cell-Based Innovation for COVID-19
Side Effects of Mesenchymal Stem Cell-Based Therapy
Unmet Challenges of Adoptive MSCs Therapy
Study Design
Source of Cells
Route of Cell Administration
Dosage Strategies
Risks Associated with Stem Cell Therapy
Concluding Remarks
References
7 Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease
Introduction
IBD Therapy with MSCs
MSCs Properties
Mechanisms of MSCs´ Action
Preclinical Studies
MSCs-Derived Exosomes for Experimental IBD Therapy
Clinical Studies
Completed Clinical Studies
Local MSCs Injections
Intravenous MSCs Administration
Proceeding Clinical Studies
Conclusion
References
8 Extracellular Vesicles-Based Cell-Free Therapy for Liver Regeneration
Introduction
Liver and Heart Connected and Non-connected Defects
Status on Cell-Based Therapy for Liver Diseases
Experimental Animal Studies
Clinical Studies
Cell-Free Therapy Approaches for the Liver
Diagnostic and Therapeutic Role of EVs in Liver Diseases
Conclusion and Future Perspectives
References
9 Current State of Stem Cell Therapy for Heart Diseases
Introduction
Aging of the Heart
Heart Disease
Conventional Therapies for Heart Disease
Mechanisms of Action of Stem Cells in Restoring the Heart Function
Clinical Evidence of Cell-Based Intervention in Ameliorating Heart Disease
Acute Myocardial Infraction
Heart Failure
Refractory Angina
Limitations of Stem Cell Therapy
Future Perspectives
Combination with Gene Therapy
Exosomes
Biomaterials
Conclusion
Cross-References
References
10 Mesenchymal Stem Cells for Cardiac Repair
Introduction
BM-Derived MSCs
Isolation and Characterization of MSCs
Important Characteristics of MSCs
Differentiation Potential of MSCs
Trophic Functions of MSCs
Immunomodulatory Characters of MSCs
Preclinical Studies with MSCs
Small Experimental Animal Studies
Large Animal Experimental Models: Translational Studies
MSCs in the Clinical Perspective
Randomized Clinical Trials with Autologous or Allogenic BM-Derived MSCs
The POSEIDON Trial
The PROMETHEUS Clinical Trial
The TAC-HTF Clinical Trial
Mesenchymal Stromal Cell for IHD
Intravenous Allogenic MSC Nonischemic Cardiomyopathy
Conclusions and Future Perspective
References
11 Preclinical Research of Mesenchymal Stem Cell-Based Therapy for Ocular Diseases
Introduction
Models of Corneal Diseases
Models of Lens Diseases
Retinal Models
Model Damage to the N-methyl-d-aspartate Receptor (NMDA)
Models of Transient Ischemia
Transgenic RGC Loss Models
Model of Experimental Autoimmune Encephalopathy (EAE)
Models of Hereditary Diseases Associated with Optic Nerve Damage
Glaucoma Models
Animal Models of Primary Angle-Closure Glaucoma (PACG)
References
12 Stem Cell for Cartilage Repair
Introduction
Cartilage
Chondrocyte
Collagen
Fluid
Chondrocyte Degeneration and Loss of Articular Cartilage
The Articular Cartilage Defects Treatments
Traditional Surgical Regeneration Techniques
Microfracture
Osteochondral Transplantation (OCT)
Osteochondral Autologous Transplantation (OAT)
Osteochondral Allograft Transplantation (OCA)
Particulated Articular Cartilage Implantation (PACI)
Tissue-Engineered Articular Cartilage
``Cell-Scaffold Construct´´ Strategies
Cell-Free Strategies
Scaffold-Free Strategies
Common Cell Types for Articular Cartilage Regeneration
Mature Cells
Stem Cells
Mesenchymal Stem Cells
BM-MSCs
WJ-MSCs
UCB-MSCs
SMSCs/SF-MSCs
Adipose Tissue-Derived MSCs
PB-MSCs
Scaffolds for Cartilage Treatment
Bioreactors
Spinner Flasks
Rotating Wall Vessels
Perfusion Bioreactors
Magnetic Field Bioreactors
Ultrasonic Bioreactors
Conclusion
Cross-References
References
13 Direct Reprogramming Strategies for the Treatment of Nervous System Injuries and Neurodegenerative Disorders
Introduction
Direct Neural Reprogramming Strategies
Reprogramming Factors
Transcription Factors
miRNA
Small Molecules
Approaches for Determining the Optimal Combination of Reprogramming Factors
Comparative Gene Expression Analyses
Trial and Error Screening of Libraries
CRISPR Activation-Based Screening
Employing Single-Cell RNAseq and Computational Analysis
Implications of the Cell Source
Fibroblasts
Astrocytes
Hepatocytes
Pericytes
Urine Epithelial Cells
Generation of Distinct Neural Cell Subtypes
Different Subtypes of Astrocytes
Different Subtypes of Oligodendrocytes
Potential Pitfalls in Autologous Cell Transplantation
Treating CNS Disorders Using Transdifferentiated Neural Cells
Spinal Cord Injury
Traumatic Brain Injury
Ischemic Stroke
Multiple Sclerosis
Parkinson´s Disease
Alzheimer´s Disease
Conclusions
Cross-References
References
14 Therapeutic Effects of Mesenchymal Stem Cells on Cognitive Deficits
Introduction
MSCs and Their Exosomes as New Therapeutic Agents in Regenerative Neurology and Neuroimmunology
Therapeutic Effects of MSCs and Their Exosomes in the Treatment of Alzheimer´s Disease
Improvement of Cognitive Impairment Following Brain Damage and Ischemia in MSC-Treated Mice
Therapeutic Effects of MSCs and MSC-Exos in the Treatment of Parkinson´s Disease, Schizophrenia, and Autism
Conclusion
References
15 Augmenting Mesenchymal Stem Cell-Based Therapy of the Infarcted Myocardium with Statins
Introduction
Stem Cell-Based Therapy for Myocardial Repair
Mesenchymal Stem Cells in Myocardial Repair and Regeneration
Poor Survival of Stem Cells Postengraftment
Strategies to Enhance Donor Cell Survival
Stem Cell Preconditioning
Augmenting the Myocardial Microenvironment Favorable for Donor Cells
Statins as Preconditioning Mimetic and Modifier of Myocardial Microenvironment
Introduction to Statins
Functional Mechanism of Statins
The Impact of Statins on Mesenchymal Stem Cells
Statins and Stem Cell Preconditioning
Statins and In Vitro Experimental Studies
Statins and in vivo Experimental Animal Models
Combined Statin and MSCs-Based Cell Therapy
Using Statin-Preconditioned MSCs to Treat AMI
Statin-Based Preconditioning of MSCs and Exosomes
Combined Statin and Cell-Based Therapy for AMI Patients
Conclusion and Future Perspective
Cross-References
References
16 Unraveling the Mystery of Regenerative Medicine in the Treatment of Heart Failure
Introduction
Heart Failure
Regenerative Potential of Stem Cells
Endogenous Cardiac Regeneration
Outcomes of Clinical Studies
Pluripotent Stem Cells
Skeletal Myoblasts
Bone Marrow-Derived Stem Cells
Mesenchymal Stem Cells
Cardiac Stem Cells
Understanding the Factors Affecting Cell-Based Therapy
Engraftment, Survival, and Rejection
Dosage
Cell Type
Route of Administration
Hippo-YAP Pathway
Ethical Issues in Regenerative Medicine
What Does the Future Hold for Cardiac Regenerative Medicine?
Cell-Free Strategies
Bioengineering
Conclusion and Future Perspectives
References
17 Therapeutic Uses of Stem Cells for Heart Failure: Hype or Hope
Introduction
Pathophysiology of Heart Failure
Significance to the Research
Introduction to Stem Cells
Embryonic Stem Cells
Induced Pluripotent Stem Cells
Adult Stem Cells
Endogenous Regeneration of Cardiomyocytes
Landmark Clinical Trials
Adult Stem Cells
Cardiac Stem Cells
Bone Marrow-Derived Cells
Mesenchymal Stem Cells
Skeletal Myoblasts
Pluripotent Stem Cells
Embryonic Stem Cells
Induced Pluripotent Stem Cells
Insights into Ccell-Based Therapy Approaches
What Is the Proposed Mechanism of Action?
Paracrine Signaling
Direct Regeneration
Optimizing Cardiac Stem Cell Therapy
What Is the Optimal Cell Type?
What Is the Optimal Method of Delivery?
What Is the Appropriate Timing of Administration?
Patient Characteristics and Sourcing
What Are Some Challenges that Remain?
What Are Some Ethical Considerations in Using Stem Cells?
False Claims
Conclusion
References
18 Mesenchymal Stem Cell-Probiotic Communication: Beneficial Bacteria in Preconditioning
Introduction
Identification, Isolation, and Biology of OMT-Derived Stem Cells
Preconditioning Strategies
Probiotics and their Health Benefits Associated with Gut Microbiota
Mesenchymal Stem Cells-Probiotic Interaction
Future Perspective and Conclusion
References
19 Effects of 3D Cell Culture on the Cell Fate Decisions of Mesenchymal Stromal/Stem Cells
Introduction
3D Cell Culture of MSCs
Impact of 3D Cell Culture on Cell Fate Decisions of MSCs
Viability and Proliferation
Cellular Senescence
Osteogenic Differentiation
Adipogenic Differentiation
Chondrogenic Differentiation
MSC-Secretome, Immunomodulation, and Anti-Inflammatory Potential
Conclusions
Cross-References
References
Part II: Cell-Based Therapy Approach: Non-Mesenchymal Stem Cell-Based Therapy
20 The Function of Stem Cells in Ocular Homeostasis
Introduction
Cornea
Corneal Epithelium
Corneal Stroma
Corneal Endothelium
Conjunctiva
Iris
Ciliary Body
Trabecular Meshwork
Lens
Retina
Retinal Pigment Epithelium
Retinal Muller Cells
Summary
Cross-References
References
21 The Potential of Stem Cells in Ocular Treatments
Introduction
Cornea
Corneal Epithelium
Corneal Stroma
Stem Cell-Based Treatment for Corneal Edema
Trabecular Meshwork
Ocular Lens
The Retina
Conclusion
Cross-References
References
22 Myoblast Therapies Constitute a Safe and Efficacious Platform Technology of Regenerative Medicine for the Human Health Indu...
Introduction
Gene Defect: The Original Sin
Genetic Diseases Account for >80% of Human Death
Approaches Towards Treatment
Autografts Cannot Treat Genetic Diseases
Treatment Design and Mechanisms
Why Cell Therapy
Myoblasts: Nature´s Chest of Gene Medicine
MTT Platform Technology
Replenishing Dead Cells
Repairing Hereditary Degenerative Cells
Engineering Genetic Mosaicism with MTT: Supply of Normal Genome via Injection of Myoblasts
Original Testing of Idea
Pertinent Muscle Developmental Biology
Myogenesis
Neuromuscular Transmission
Excitation Contraction Coupling
Number of Myofibers and Strength
Parameters Governing Cell Fusion
Controlled Cell Fusion
Mitotic Cardiomyocytes via In Vitro Controlled Cell Fusion
Injection Methods Regulate Cell Distribution and Fusion
Cyclosporine Immunosuppression
Muscular Dystrophy
Etiology
Pathogenesis: Cell Membrane Defect
Duchenne Muscular Dystrophy
Natural History
Strategizing DMD Treatment
Induction of Genetic Mosaicism
Normal and Dystrophic Minced Muscle Mixes
Newborn Muscle Transplant
Mesenchymal Cell Transplant
MTT with Primary Myoblast Culture
MTT in DMD Subjects
Single Muscle Treatment (SMT): Phase I Trial
Lower Body Treatment (LBT): Phase II Trial
Whole Body Treatment (WBT): Phase II and III Trials
Rationale
Dose Escalation
Objective
Study Design
UBT/LBT Randomization
Myoblast/Placebo Randomization
Donors
Myoblast Preparation
The 25 Billion Myoblast WBT Protocol
The 50 Billion Myoblast WBT Protocol
WBT Significance
Heart Muscle Degeneration
Severe Myocardial Infarction
Proof-of-Concept for AMT
AMT Is Safe and Efficacious with MI Patients
AMT Is Safe and Efficacious with End-Stage HF Subjects
Regulatory
Case Selection
Patient Selection
Manufacture of Allogeneic Human Myoblasts
Cyclosporine Immunosuppression
Clinical Research Procedures
AMT
Data Analysis and Interpretation
Statistical Analyses
Safety Assessment
Efficacy Assessment
Understanding and Debating the Study Outcomes
Perspectives
Conclusion
Angiomyogenesis
Autonomous Robotic Cell Injection Catheter System
Cellular and Molecular Phases of MTT Development
MTT Corrects Gene Defects in Type II Diabetes
Type II Diabetes
MTT in Type II Diabetes Human Study
MTT Use in Drug Discovery
Multiple Usage of MTT in Genetic Diseases
Preliminary Safety and Efficacy Data of MTT in Cancer Treatment
Identification of the Polygenic Defects of Various Cancer
Myoblasts Inhibit Metastasis, Tumor Growth, and Induce Cancer Apoptosis
Myoblasts Fuse with Cancer Stem Cell
Plausible Sequence of Events
MTT in Breast and Uterine Cancers
Conclusion
Antiaging Aesthetica
In Law´s Enchanting World of MTT
Personalized Medicine for Each Family
Biologic Skin
Body Sculpture
Be More Macho
Be More Feminine
Tendon Repair for Injured Athletes
Loose Teeth and Bone Fracture Repair
Stimulation Therapies and Fractal Dynamics to Complement MTT Treatment of Diseases
MTT Replenishes Mitochondria and Regenerates Energy Network of Life
Intellectual Property Portfolio of Professor Peter K. Law
FDA, EMA Approved MTT IND´s
Conclusion
References
23 Adipose Tissue-Derived Regenerative Cell-Based Therapies: Current Optimization Strategies for Effective Treatment in Aesthe...
Introduction
AT-Derived Regenerative Cells
Adipose-Derived Stem Cells
Current Regulatory Considerations for the Production and Clinical Application of SVF/ASCs
Assessing the Therapeutic Potential of SVF/ASCs in Aesthetic Surgery
Safety Concerns with the Use of Culture-Expanded ASCs for Cell Therapy
Clinical Application of SVF/ASCs in Aesthetic Surgery
Soft Tissue Reconstruction
Use of ASCs in Re-contouring Depressed Facial Lesions
ASCs in Wound Healing
Bone Reconstruction
Cross-References
References
24 Molecular Signature of Stem Cells Undergoing Cardiomyogenic Differentiation
Introduction
Basic Structure of Cardiomyocytes
Atrial and Ventricular Cardiomyocytes
Stem Cells and Cardiac Regenerative Therapy
Embryonic Stem Cells and Their Derivative CMs
iPSCs and Their Derivative Cardiomyocytes
Adult Tissue-Derived Stem/Progenitor Cells
Molecular Profiling of Cardiomyocytes
Cardiomyocyte Lineage Commitment and Differentiation
Bone Morphogenetic Proteins (BMPs)
Wnt Pathway
Fibroblast Growth Factor Signaling Pathway
Cardiac-Specific Transcription Factors
GATA Family of Transcription Factors
Myocyte Enhancer Factor-2 (MEF-2) Family
NKX-2.5
Serum Response Factor and Myocardin
HAND Transcription Factors
Two-Dimensional (2D) Differentiations of Stem Cells into Cardiomyocytes
Status of iPSC-Derived Cardiomyocytes
Current Technical Limitations
State of the Art
Regulation of Lineage Differentiation in Stem Cells
Differentiation Cues
Small Molecule Inhibitors
Role of Small Molecule Inhibitors in Stem Cell Biology
Microenvironment
Exosomes for In Vitro and In Vivo Cardiac Applications
Detailed Methodology
Functional Maturation Analysis
Morphological Analysis
Functional Analysis
Metabolic Analysis
Cardiomyocyte Maturation-Specific Gene Expression Analysis
Limitations of 2D Culture
3D Cardiac Organoids
Working on the 3D Models
Scaffolds Employed
Analytical Methods
Conclusions and Outlook
References
25 Stem Cell Applications in Cardiac Tissue Regeneration
Introduction
Engineering of Infarcted Myocardium
MSCs in Cardiac Regenerative Therapy
Natural and Synthetic Polymer Used for Regenerative Therapy
Natural Scaffolds
Extra-Cellular Matrix (ECM)
Collagen Scaffolds
Synthetic Scaffolds
Hydrogel Based Therapy
Patch-Based Cell Therapy
3D-Bioprinting
The Complexity of the Cardiac Tissue
3D-Printing Salient Points
3D Printing Techniques of Heart Tissue
Bioinks for Heart Tissue
Conclusion and Future Directions
Cross-References
References
26 Vascular Functional Recovery and Reparation by Human Endothelial Progenitor Cells
Introduction
Endothelial Progenitor Cells: Definition and Biological Function
Endothelial Progenitor Cell Dysfunction in Cardiovascular Diseases
Early Outgrowth and Endothelial Colony-Forming Progenitor Cells in Revascularization and Vascular Reparation
Animal Studies for Vascular Repair with EPCs
Clinical Studies for EPCs Applications
Conclusion and Future Prospects
References
27 Neural Stem Cells
Introduction
Historic Prospective of Neural Stem Cells
Neural Stem Cell Culture - Its Applications and Limitations
Influence of the Microenvironment and Niche on Embryonic NSCs - Intrinsic Regulation
Signaling Molecules That Affect NSC Differentiation
Extracellular Matrix (ECM) Role in Regulating Neural Stem Cell Characteristics
Anomalies in Neurogenesis and Brain Pathologies
Advances in Neural Stem Cell Transplantation and Therapy (Fig. 1 and Table 3)
Conclusions
References
28 Glial Cells in Neuroinflammation in Various Disease States
Introduction
Microglial Cells and Their Physiological Functions
Physiological Roles of Astrocytes
Reactive Gliosis in Astrocytes
Stroke, CNS Trauma, and Their Impact on Glial Cells
Roles of Glial Cells in CNS Autoimmune Conditions
Roles of Glial Cells in Neurodegenerative Disorders
Current Therapies for CNS Disorders
Treatments for Stroke and CNS Trauma
Treatments for CNS Autoimmune Conditions
Treatments for Neurodegenerative Disorders
Cell-Based Therapies for CNS Disorders
Conclusion
References
29 Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform for Modeling Arrhythmias
Introduction
Establishing Patient-Derived hiPSC Lines
hiPSC Reprogramming
hiPSC Quality Control and Genotyping
Genome Editing Applications
hiPSC-CM Differentiation, Purification, and Maturation
Differentiation and Definition of Homogenous Cardiomyocyte Subpopulations
Maturation
Phenotypic Assays
Transcriptomic and Proteomic Analysis
Metabolic Considerations
hiPSC-CM Morphology
Contractility and Force Generation
Electrophysiological Measurements and Arrhythmia Assessment
hiPSC-CMs for Proarrhythmic Drug Assessment
Conclusion
Cross-References
References
30 Induced Pluripotent Stem Cells
Introduction
From Classical Transcription Factor Quartet Transduction to Small Molecule Manipulation Protocols
iPSCs´ Limitations - Overcoming Barriers to Clinical Translation
Low-Efficiency Reprogramming of Somatic Cells
Tumorigenicity
Immunogenicity
Economic Issues and Biobanking iPSCs
Heterogeneity
Harnessing the Potential of iPSCs
iPSC-Based Disease Modeling, Drug Discovery, and Toxicity Studies
iPSC-Based Cell Therapy - Ongoing Clinical Trials
Conclusion
Cross-References
References
31 Human Stem Cell Differentiation In Vivo in Large Animals
Introduction
Long-Term Transplantation Tolerance Following Intrauterine Transplantation (IUT)
What Is a Stem Cell?
Why a Large Animal?
IUT as an Experimental Tool to Study SC Biology or What Is a Stem Cell Revisited
The Hematopoietic SC (HSCs)
The Mesenchymal Stem Cell (MSC)
Stem Cell Plasticity
What Is Stem Cell Plasticity, and How Can It Be Explained Physiologically
IUT as a Clinical Option
Conclusion
Cross-References
References
Part III: Cell-Free Therapy Approach
32 Mesenchymal Stem Cell-Extracellular Vesicle Therapy in Patients with Stroke
Introduction
Advantages of EVs over MSCs in Stroke
Evidence of the Role of Stem Cell-Derived EVs in Stroke
Preclinical Evidence of EV Therapeutics in Stroke Models
Clinical Evidence of EV Efficacy in the Clinical Trial of MSCs in Stroke Patients
Status and Limitations of EV Therapeutics
Preclinical Studies
Clinical Studies
Considerations for the Application of EV Therapeutics in Stroke
Current Guidelines/Recommendations
Quality Control and Potency Markers
RNAs
Proteins
Other Cargos
Isolation, Dosage, Mode of Application, and Biodistribution
Production of EV Preparation
Isolation of Prepared EVs
Recent Advances in EV Therapeutics
Conclusions and Perspective
References
33 Mesenchymal Stem Cell Secretome: A Potential Biopharmaceutical Component to Regenerative Medicine
Introduction
MSCs´ Roots
MSCs´ Biological Properties
The Pitfalls of MSCs
Secretome Derived from MSCs as Cell-Free-Based Therapeutic Strategy
Composition and Characterization of MSCs´ Secretome
The Soluble Fraction: Cytokines and Growth Factors
The Vesicular Fraction: Extracellular Vesicles
The Vesicular Fraction: Coding and Non-coding RNAs
Mechanisms of Action and Principal Effects
Pre-clinical Approaches of Mesenchymal Stem Cell Secretome
Wound Healing and Cartilage Repair and Regeneration
Cardiovascular Diseases
Kidney and Lung Injuries
Central Nervous System Pathologies
Traumatic Brain Injury (TBI)
Spinal Cord Injury (SCI) and Ischemic Stroke (IS)
Neurodegenerative Disorders
Clinical Approaches Based on Mesenchymal Stem Cell Secretome
The Benefits and Barriers of MSCs´ Secretome
Improvements Needed for Secretome Application as a Cell Transplantation-Free Tool
Conclusion
Cross-References
References
34 Exosome-Based Cell-Free Therapy in Regenerative Medicine for Myocardial Repair
Introduction
Cell-Based Therapy for the Heart
Cell-Free Therapy Approach for the Heart
Theranostic Role of Exosomes in the Heart
Diagnostic Applications of Exosomes
Exosomes as a Treatment Option for the Injured Heart
Exosome in Experimental Animal Heart Models
Exosome in Translational Experimental Studies
Exosome in Clinical Studies
Challenges and Future Perspective
Cross-References
References
35 Current Trends and Future Outlooks of Dental Stem-Cell-Derived Secretome/Conditioned Medium in Regenerative Medicine
Introduction
Dental Stem/Progenitor Cells (DMSCs)
Stem/Progenitor Cells´ Secretome/Conditioned Medium
Extracellular Vesicles (EVs)
Exosomes (EXs) and Microvesicles (MVs)
Comparison Between Secretome/CM Derived from DMSCs and MSCs from Other Tissue Sources
Secretome/Conditioned Medium (SHED-CM) Derived from Stem Cells from Exfoliated Human Deciduous Teeth
SHED-CM in Treating Cardiopulmonary Injuries
SHED-CM in Treating Hepatic Disorders
SHED-CM in Treating Diabetes Mellitus
SHED-CM in Treating Immunological Disorders
SHED-CM in Treating Dental-Pulpal Disorders
SHED-CM in Treating Neural Injuries
Secretome/Conditioned Medium Derived from Dental Pulp Stem Cells
The Osteogenic Potential of DPSCs-CM
DPSCs-CM in the Treatment of Liver Disorders
Potential of DPSCs-CM to Regenerate Dental Tissues
The Role of DPSCs-CM in the Treatment of Neurological Diseases
DPSCs-CM in the Treatment of Autoimmune Diseases
Secretome/Conditioned Medium Derived from Gingival Mesenchymal Stem/Progenitor Cells
GMSCs-CM in Treating Neural Disorders
GMSCs-CM in Treating Skin Injuries
GMSCs-CM Osteogenic Potential
Periodontal Ligaments Stem-Cell-Derived Secretome/Conditioned Medium
PDLSCs-CM in the Therapy of Neural Disorders
PDLSCs-CM Osteogenic Potential
PDLSCs-CM in Dental Tissue Regeneration
Secretome/Conditioned Medium Derived from Stem/Progenitor Cells of Apical Papilla (SCAP), Dental Follicle Stem/Progenitor Cell...
Conclusions
References
36 Extracellular Vesicles Derived from Mesenchymal Stem Cells
Introduction
Extracellular Vesicles
Mesenchymal Stem Cell-Derived Extracellular Vesicles
Role of Mesenchymal Stem Cell-Derived EVs in Regeneration
Immunosuppressive Activity of EVs
EV-Mediated Mitochondria Donation by MSCs
Approaches of Large-Scale Production of Vesicles
Cytochalasin B-Induced Membrane Vesicles
Vesicle Production by Mechanical Extrusion
Vesicle Production Using a Hyperosmotic Solution
Conclusion and Future Directions
Cross-References
References
37 Skeletal Muscle-Extricated Extracellular Vesicles: Facilitators of Repair and Regeneration
Introduction
Skeletal Muscle Repair and Regeneration
General Introduction to Skeletal Muscle Regeneration Events
The Role of (Other) Residential Cells
The Immune Response
Macrophages and Lymphocytes
The Case for Muscular Dystrophy
Myofiber Intracellular Repair
Extracellular Vesicles
Proteins and Lipids
RNA Cargo
Uptake of Vesicles by Target Cells
EVs Released by Different Cells in the Injured Muscle Environment
The Inflammatory Response via EVs and Subsequent Activation of Nearby Cells
Conclusion
References
38 Regenerative Medicine Applied to the Treatment of Musculoskeletal Pathologies
Introduction
Mesenchymal Stem Cells Based Therapies: A Useful Tool?
MSCs Secretome as an Alternative to Cell-Based Therapy
Soluble Factors Secreted by MSCs
Immunomodulation
Angiogenesis and Revascularization
Anti-apoptotic Activity
Extracellular Vesicles
Composition of EVs Cargo
Applications of the Secretome from Mesenchymal Stem Cells in the Treatment of Musculoskeletal Diseases
MSCs Secretome for Bone Regeneration
MSCs Secretome in the Treatment of Cartilage Defects and Cartilage Degeneration
MSCs Secretome in the Treatment of Tendon Injuries
Modulating Secretome Therapeutic Properties
Hypoxia Induction
Inflammatory Priming
Addition of Bioactive Factors
Modulation of Cell-Cell Interactions
Tuning Cell-Substrate Interaction (ECM and Scaffolds)
Genetic Editing
Disease-Like Priming
Future Perspectives
References
Part IV: Miscellaneous Aspects of Stem Cell Applications
39 Common Ethical Considerations of Human-Induced Pluripotent Stem Cell Research
Introduction
Safety
Reproduction
Patentability
Informed Consent and Donors´ Right
Conclusion
Cross-References
References
40 Response of the Bone Marrow Stem Cells and the Microenvironment to Stress
Introduction
Bone Marrow Microenvironment
Components of the Bone Marrow Microenvironment
Different Stem Cell Niches in the BM Microenvironment and Their Spatial Position
Dynamic Interactions in the BM Microenvironment
Bone Marrow and Stress Response
Cell and Microenvironment Response to Stressors
Hematopoietic Stress Response
Emergency Hematopoiesis
Environmental Hematopoietic Stressors
Environmental Pollutants
Benzene
Nanoparticles and particulate matter
Dioxins as Persistent Organic Pollutants (POPs)
Polycyclic Aromatic Hydrocarbons (PAH)
Heavy Metal Exposure
Detoxification Response and Signaling Pathways
Drug metabolizing enzymes Cytochrome P450 (CYP) and Glutathione S-Transferases (GSTs)
Aryl Hydrocarbon Receptor (AhR)
Toll-like receptor (TLR) Signaling
BM Microenvironment Alterations, Dysregulation, and Myeloid Leukemia
Development of Secondary Myeloid Malignancy
Therapy-Related Myeloid Malignancy
Leukemia-Predisposing Inherited BM Failure Syndromes
Fanconi anemia
Shwachman-Diamond syndrome
BM Microenvironment as a Contributor/Driver of Leukemia
Metabolic Dysregulation and the Role of Adipocytes in the Malignant Niche
Marrow Adipose Tissue and Leukemia
Environmental Stressors and Adipose Tissue
Conclusions
References
41 Advances, Opportunities, and Challenges in Stem Cell-Based Therapy
Introduction
Mesenchymal Stem Cells
Mesenchymal Stem Cells Sources
The Therapeutic Potential of Mesenchymal Stem Cells
Application of Mesenchymal Stem Cells in Medicine
MSC-Based Therapies in Graft Versus Host Disease
Mesenchymal Stem Cell-Based Therapies in Crohn´s Disease
Mesenchymal Stem Cell-Based Therapies in Cardiology
Mesenchymal Stem Cells-Based Therapies in Orthopedics
MSC-Based Therapies in Neurology
Potential Risks of MSC-Based Therapies
Conclusions
References
42 The Stem Cell Continuum Model and Implications in Cancer
Introduction
The Stem Cell Continuum
Extracellular Vesicle Basics
Extracellular Vesicle Functions
Extracellular Vesicle Pleiotropy and Regenerative Potential
Extracellular Vesicles Affect Normal Stem Cells
Extracellular Vesicles Affect Cancer Stem Cells
An Introduction to Extracellular Vesicles and Hematological Malignancies
Extracellular Vesicles in Leukemia
The Leukemic Bone Marrow Microenvironment
Predictors of Response and Promoters of Resistance in Leukemia
Extracellular Vesicles in Lymphoma
The Lymphoma Tumor Microenvironment
An Introduction to Extracellular Vesicles and Breast Cancer
Role of TEVs in Breast Cancer
Use of TEVs in Breast Cancer Treatment
Use of TEVs for Diagnostic and Predictive Markers in Breast Cancer
An Introduction to EVs and Lymphoma
Extracellular Vesicles in Primary CNS Lymphomas
The Role of EVs in Diagnosis and as a Biomarker in Primary CNS Lymphoma
Extracellular Vesicles´ Function on Lymphomagenesis and Lymphoma Treatment Response
EVs´ Effects on Lymphoma Patient Outcomes and Prognosis
Role or Target in Therapy for Lymphoma
Lymphoma-Specific EV Roles
Diffuse Large B-Cell Lymphoma (DLBCL)
Virally Mediated Lymphomas
Hodgkin´s Lymphoma (HL)
References
43 Toll-Like Receptor 3
Introduction
Mesenchymal Stem Cells
Toll-Like Receptor 3 and Its Activation Pathways
TLR3 Priming and MSC Immunomodulation
TLR3 Priming and MSC Differentiation
TLR3 Priming and MSCs´ Migration and Proliferation
TLR3-Mediated Apoptosis and Survival of MSCs
Micro-RNAs as Modulators of TLR3-Mediated Response in MSCs
Conclusion
References
44 Targeting Cancer Stem Cells: New Perspectives for a Cure to Cancer
Introduction
Role of Cancer Stem Cells (CSCs) in the Tumor
Markers in CSCS: A New Approach Against Tumor
Targeting CSCs
The Hedgehog Signaling Pathway
The Notch Signaling Pathway
The Wnt Signaling Pathway
Targeting ALDH as the Drug-Detoxify Enzymes Against CSCs
Targeting Drug Efflux Pumps
Targeting the CSC Niche and the Quiescent State
An Update on the Most Used Target Therapies Against CSCs
Oncolytic Adenovirus
Oncolytic Herpes Simplex Viruses
Oncolytic Virus and Its Effects in Recent Clinical Trials
Oncolytic Adenoviruses and Pancreatic Clinical Trials
Oncolytic Herpes Simplex Viruses and Pancreatic Clinical Trials
Stem Cell-Derived Exosomes as Therapeutic Carriers
Conclusions and Future Directions
References
45 Bioengineering Technique Progress of Direct Cardiac Reprogramming
Introduction
Pluripotent Stem Cells in Myocardial Regeneration and Repair
The Strategy of Direct Reprogramming to Generate Cardiomyocytes
Observations and Data Analysis
Synthesis and Characterization of CMBs and Lentivirus-Binding Capacity
Reprogramming of Rat CFs into iCMs by the GMT Lentivirus Vectors In Vitro
UTMD-Mediated Localized Delivery of Direct-Reprogramming GMT Lentivirus-CMB Hybrids In Vivo
Histological Assessment of Post-Infarct Ventricles After UTMD Therapy
Cardiac Function Improvement After UTMD-Mediated Direct Reprogramming Therapy
Significance of the Combinatorial Approach of UTMD with CMB-Lentiviral Hybrid
CMB-Lentivirus Hybrids in Gene Delivery
Advantages of Lentiviral Vectors for Direct Reprogramming
Improvement of Reprogramming Factors in Direct Cardiac Reprogramming
Ultrasound-Mediated Localized Direct Reprogramming via Lentivirus Delivery for Cardiac Repair
Conclusion and Future Perspective
A Summary of the Experimental Methods and Design
Vectors and Cells
Creation of CMBs and Microbubble-Virus Hybrids
Direct Reprogramming of CFs In Vitro
A Western Blot Analysis
Development of Experimental Rat Model of MI
Delivery of Microbubble-Lentivirus Hybrids Using UTMD In Vivo
Histological Examination
Cardiac Magnetic Resonance Imaging (cMRI) and Echocardiography
References
46 Future Perspectives of Exosomal Payload of miRNAs in Lung Cancer
Introduction
Exosome Composition
Biological Functions of Exosomes
Exosomes and Cancer
The Pathways of Exosomal miRNA Secretion
Exosomal miRNAs in Cancer Progression
Tumor-Derived Exosomes and Lung Cancer
Future Perspectives of Exosomal miRNAs in the Clinics
References
47 Endothelial Progenitor Cells from Bench to Antitumor Therapy and Diagnostic Imaging
Introduction
Stem Cells
An Overview of Endothelial Progenitor Cells
Endothelial Progenitor Cells in Neovascularization
Endothelial Progenitor Cells Applications
Endothelial Progenitor Cells Sources, Ex Vivo Culturing, and Implantation
Endothelial Progenitor Cells in Preclinical Cancer Studies
Side Effects and Potential Risks of EPCs Cell Therapy
Conclusion and Future Perspective
References
48 Non-cryopreserved Peripheral Stem Cell Autograft for Multiple Myeloma and Lymphoma in Countries with Low Resources
Introduction
Bibliographic Literature Search for the Chapter
Data Analysis from the Selected Studies
Cell Mobilization
Apheresis
Cell Storage
Cell Viability
Conditioning Regimen
Cell Engraftment
Posttransplant Data
Comparative Studies
The Study by Sarmiento et al. (2018)
The Study by Bittencourt et al. (2019)
The Study by Bekadja et al. (2021)
The Study by Garifullin et al. (2021)
An In-Depth Analysis of the Published Data
Cell Mobilization
Conservation and Cell Viability
Therapeutic Intensification
Donor Cell Engraftment
Conclusion and Future Perspective
References
49 A Multilevel Approach to the Causes of Genetic Instability in Stem Cells
Introduction
DNA Damage Response in Stem Cells
Factors That Influence DNA Stability in Stem Cells
Oxidative Stress
Senescence as a Factor Inducing Stem Cells Genomic Instability
Aging and Genomic Instability in Stem Cells
Telomere Damage and Genetic Instability in Stem Cells
Aneuploidy as an Expression of Genome Instability
Influence of Genomic Arrangement in the Nucleus on Aneuploidy
Lamins Alterations and Aneuploidy
Replicative Stress/Replication Timing as Causes of Genome Instability
Cell Origin as Source of Genome Instability
Adipose Stem Cells (ASC)
Embryonic Stem Cells
Induced Pluripotent Stem Cells
Effect of Reprogramming Methods on Genetic Instability
Mutations Induced During Reprogramming
Culture Conditions, the Passage Number
The Bystander Effect
Instability Factors Related to Donors
Obesity
Donor Age
Toxic Habits and Diseases in the Donors
Conclusion
Cross-References
References
Index

Citation preview

Khawaja Husnain Haider Editor

Handbook of Stem Cell Therapy

Handbook of Stem Cell Therapy

Khawaja Husnain Haider Editor

Handbook of Stem Cell Therapy With 119 Figures and 71 Tables

Editor Khawaja Husnain Haider Department of Basic Sciences Sulaiman AlRajhi University Al Bukairiyah, Saudi Arabia

ISBN 978-981-19-2654-9 ISBN 978-981-19-2655-6 (eBook) https://doi.org/10.1007/978-981-19-2655-6 © Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

To My Parents, Brothers, Sisters, Brothers-inLaw, and my wife DIYA who mothered our two sons: Mowahid who is the love of my life and Anas “the angel of paradise” whose departure from my life is a constant source of inspiration for me to do science.

Foreword

Debilitating and fatal, hereditary degenerative diseases deprive their sufferers of a normal quality of life and life span. An effective treatment must not only repair degenerating cells but replenish degenerated cells with live ones. From the first success of mesenchyme transplantation in dystrophic mice reported in Muscle & Nerve 1982; 5: 619–627 unto the current >20 chapters documenting biomedical and clinical advances of mesenchymal stem cell transplantation, it becomes apparent that cell therapy has come of age! Better still, Food and Drug Agency (FDA), European Medicines Agency (EMA), and National Medicine Products Administration (NMPA) have granted several biologic license approvals since July 14, 1990, when the world’s first human gene therapy and somatic cell therapy was published in Lancet 336:114–115. Cell therapy is a mainstream regenerative medicine characterized with transplantation of biologics including live cells, genes, factors, and/or a combination of them. It replenishes live cells to degenerative organs, and in allografts, offers the normal genome for genetic complementation treatment. It regenerates tissues and organs. Exosomes secreted from cultured cells are often formulated to become molecular medicine. Since a foreign gene and its derivatives always exert its effect on the cell, cell therapy is the common pathway to good health. Debilitating and fatal diseases with no known cure are often results of polygenic aberration, and only cell therapy rather than individual molecular medicine can be effective. Cell therapists harvesting the innovations and discoveries of developmental cell biologists since the mid-1950s should not forget their teachings. Somatic cell therapies and stem cell therapies utilize different compositions and methods of treatment. It is through continual research in cell identification, quantity, purity, viability, and potency, especially in cell differentiation and transcription, that stem cell therapies will one day overcome the inadequacy of uncontrolled differentiation and carcinogenicity. This is the seventh book Professor Haider has edited on key issues of stem cells and stem cell therapies. Professor Haider has devoted more than two decades in these arenas, publishing cutting-edge research with complete dedication and passion. In this book, he has compiled the latest advances of encompassing cell therapies from world experts toward treating cancer, heart failure, Type-II diabetes, aging, muscular dystrophies, wounds, ocular diseases, COVID-19, inflammatory bowel disease, cartilage damage, spinal cord injury, cognitive deficits, and ischemic stroke. This vii

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Foreword

book is classified as a Major Reference Work consisting of 50 chapters from nearly 45 labs around the globe, from the USA to Canada, Europe, China, Japan, Malaysia, and many other countries. With these elite Editor, Publisher, and Authors working in harmony, the world of science and medicine will look forward to new editions of this landmark undertaking in the years to come. All these drops into the bucket, some representing the life-long effort of the pioneers, others representing novel discoveries and innovations, will all be collected monumentally; for biologics are evolutionary medicine shared by all animals in the last 500 million years, only to be discovered, isolated, manipulated with human innovation, and formulated as Genetic Cell Therapies to provide mankind with longterm efficacy and lesser side effects than herbs, chemicals, surgery, or radiation. Cell Therapy Institute Wuhan, CHINA August 2022

Peter K. Law, Ph.D., Professor, Founder & Chairman

Contents

Volume 1 Part I Cell-Based Therapy Approach: Mesenchymal Stem Cell-Based Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2

1

Human Mesenchymal Stem Cells: The Art to Use Them in the Treatment of Previously Untreatable . . . . . . . . . . . . . . . . . . . . . . . Jan Lakota, Maria Dubrovcakova, and Khawaja Husnain Haider

3

Sources and Therapeutic Strategies of Mesenchymal Stem Cells in Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mohamed Kamal, Dina Kassem, and Khawaja Husnain Haider

23

3

Considerations for Clinical Use of Mesenchymal Stromal Cells . . . Abdelkrim Hmadcha, Bernat Soria, Juan R. Tejedo, Francico J. Bedoya, Jose Miguel Sempere-Ortells, and Tarik Smani

51

4

Stem Cells in Wound Healing and Scarring . . . . . . . . . . . . . . . . . . Roohi Vinaik and Marc G. Jeschke

103

5

Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maria Alvarez-Viejo and Khawaja Husnain Haider

127

6

Mesenchymal Stromal Cells for COVID-19 Critical Care Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abdelkrim Hmadcha, Tarik Smani, Jose Miguel Sempere-Ortells, Robert Chunhua Zhao, and Bernat Soria

7

8

Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mikhail Konoplyannikov, Oleg Knyazev, Peter Timashev, and Vladimir Baklaushev Extracellular Vesicles-Based Cell-Free Therapy for Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mustapha Najimi and Khawaja Husnain Haider

163

193

221

ix

x

Contents

9

Current State of Stem Cell Therapy for Heart Diseases . . . . . . . . Yong Sheng Tan, Qi Hao Looi, Nadiah Sulaiman, Min Hwei Ng, and Daniel Law Jia Xian

239

10

Mesenchymal Stem Cells for Cardiac Repair . . . . . . . . . . . . . . . . . Abdullah Murhaf Al-Khani, Mohamed Abdelghafour Khalifa, and Khawaja Husnain Haider

269

11

Preclinical Research of Mesenchymal Stem Cell-Based Therapy for Ocular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Zakirova, A. M. Aimaletdinov, A. G. Malanyeva, C. S. Rutland, and A. A. Rizvanov

12

Stem Cell for Cartilage Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anneh Mohammad Gharravi, Mohammad Reza Gholami, Saeed Azandeh, and Khawaja Husnain Haider

13

Direct Reprogramming Strategies for the Treatment of Nervous System Injuries and Neurodegenerative Disorders . . . . . . . . . . . . Katarzyna Pieczonka, William Brett McIntyre, Mohamad Khazaei, and Michael G. Fehlings

14

15

16

17

18

19

Therapeutic Effects of Mesenchymal Stem Cells on Cognitive Deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carl Randall Harrell, Ana Volarevic, and Vladislav Volarevic Augmenting Mesenchymal Stem Cell-Based Therapy of the Infarcted Myocardium with Statins . . . . . . . . . . . . . . . . . . . . . . . . Sulaiman Alnasser, Mabrouk AL-Rasheedi, Mateq A. Alreshidi, Saleh F. Alqifari, and Khawaja Husnain Haider

323

349

383

413

437

Unraveling the Mystery of Regenerative Medicine in the Treatment of Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathieu Rheault-Henry, Ian White, and Rony Atoui

471

Therapeutic Uses of Stem Cells for Heart Failure: Hype or Hope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathieu Rheault-Henry, Ian White, and Rony Atoui

511

Mesenchymal Stem Cell-Probiotic Communication: Beneficial Bacteria in Preconditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ayşegül Mendi, Büşra Aktaş, and Belma Aslım

545

Effects of 3D Cell Culture on the Cell Fate Decisions of Mesenchymal Stromal/Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . Darius Widera

565

Contents

xi

Part II Cell-Based Therapy Approach: Non-Mesenchymal Stem Cell-Based Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

585

20

The Function of Stem Cells in Ocular Homeostasis . . . . . . . . . . . . S. Amer Riazuddin, Shahid Y. Khan, and Muhammad Ali

587

21

The Potential of Stem Cells in Ocular Treatments . . . . . . . . . . . . . S. Amer Riazuddin, Shahid Y. Khan, and Muhammad Ali

607

22

Myoblast Therapies Constitute a Safe and Efficacious Platform Technology of Regenerative Medicine for the Human Health Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peter K. Law, Wenbin Li, Qibin Song, Shi Jun Song, Jun Ren, Manye Yao, Qiaoyun Li, Qizhong Shi, Keqiang Wang, Jing Wang, Lei Ye, Jian-Hua Ma, Khawaja Husnain Haider, Li-ping Su, Ping Lu, Weyland Cheng, Ming Zhang Ao, and Danlin M. Law

23

24

Adipose Tissue-Derived Regenerative Cell-Based Therapies: Current Optimization Strategies for Effective Treatment in Aesthetic Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yusuke Shimizu, Edward Hosea Ntege, and Hiroshi Sunami Molecular Signature of Stem Cells Undergoing Cardiomyogenic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kavitha Govarthanan, Piyush Kumar Gupta, Binita E. Zipporah, Vineeta Sharma, M. Rajasundari, and Khawaja Husnain Haider

625

691

725

Volume 2 25

Stem Cell Applications in Cardiac Tissue Regeneration . . . . . . . . Vineeta Sharma, Sanat Kumar Dash, Piyush Kumar Gupta, Binita E. Zipporah, Khawaja Husnain Haider, and Kavitha Govarthanan

26

Vascular Functional Recovery and Reparation by Human Endothelial Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexander E. Berezin and Alexander A. Berezin

769

799

27

Neural Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yash Parekh, Ekta Dagar, Khawaja Husnain Haider, and Kiran Kumar Bokara

821

28

Glial Cells in Neuroinflammation in Various Disease States Derek Barthels and Hiranmoy Das

.....

849

29

Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform for Modeling Arrhythmias . . . . . . . . . Lisa Lin, Tiffany Barszczewski, Patrick G. Burgon, and Glen F. Tibbits

875

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Contents

30

Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . Adegbenro Omotuyi John Fakoya, Adekunle Ebenezer Omole, Nihal Satyadev, and Khawaja Husnain Haider

895

31

Human Stem Cell Differentiation In Vivo in Large Animals . . . . . John S. Pixley

921

Part III 32

33

Cell-Free Therapy Approach

........................

Mesenchymal Stem Cell-Extracellular Vesicle Therapy in Patients with Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oh Young Bang, Eun Hee Kim, Gyeong Joon Moon, and Jae Min Cha Mesenchymal Stem Cell Secretome: A Potential Biopharmaceutical Component to Regenerative Medicine . . . . . . . Bruna Araújo, Rita Caridade Silva, Sofia Domingues, António J. Salgado, and Fábio G. Teixeira

945

947

973

34

Exosome-Based Cell-Free Therapy in Regenerative Medicine for Myocardial Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007 Khawaja Husnain Haider and Mustapha Najimi

35

Current Trends and Future Outlooks of Dental Stem-Cell-Derived Secretome/Conditioned Medium in Regenerative Medicine . . . . . . 1035 Israa Ahmed Radwan, Dina Rady, Sara El Moshy, Marwa M. S. Abbass, Khadiga Mostafa Sadek, Aiah A. El-Rashidy, Azza Ezz El-Arab, and Karim M. Fawzy El-Sayed

36

Extracellular Vesicles Derived from Mesenchymal Stem Cells M. O. Gomzikova, V. James, and A. A. Rizvanov

37

Skeletal Muscle–Extricated Extracellular Vesicles: Facilitators of Repair and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1097 Laura Yedigaryan and Maurilio Sampaolesi

38

Regenerative Medicine Applied to the Treatment of Musculoskeletal Pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1123 Alberto González-González, Daniel García-Sánchez, Ana Alfonso-Fernández, Khawaja Husnain Haider, José C. Rodríguez-Rey, and Flor M. Pérez-Campo

Part IV 39

. . . 1071

Miscellaneous Aspects of Stem Cell Applications . . . . . . .

1159

Common Ethical Considerations of Human-Induced Pluripotent Stem Cell Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161 Adekunle Ebenezer Omole, Adegbenro Omotuyi John Fakoya, Kinglsey Chinonyerem Nnawuba, and Khawaja Husnain Haider

Contents

xiii

40

Response of the Bone Marrow Stem Cells and the Microenvironment to Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179 Duygu Uçkan-Çetinkaya and Bihter Muratoğlu

41

Advances, Opportunities, and Challenges in Stem Cell-Based Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229 Renata Szydlak

42

The Stem Cell Continuum Model and Implications in Cancer . . . 1255 Theo Borgovan, Ari Pelcovitz, Rani Chudasama, Tom Ollila, and Peter Queseneberry

43

Toll-Like Receptor 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279 Mohamed Mekhemar, Johannes Tölle, Christof Dörfer, and Karim M. Fawzy El-Sayed

44

Targeting Cancer Stem Cells: New Perspectives for a Cure to Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303 Beatrice Aramini, Valentina Masciale, Giulia Grisendi, Federico Banchelli, Roberto D’Amico, Massimo Dominici, and Khawaja Husnain Haider

45

Bioengineering Technique Progress of Direct Cardiac Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1333 Dingqian Liu, Khawaja Husnain Haider, and Changfa Guo

46

Future Perspectives of Exosomal Payload of miRNAs in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1367 Beatrice Aramini, Valentina Masciale, Giulia Grisendi, Federico Banchelli, Roberto D’Amico, Massimo Dominici, and Khawaja Husnain Haider

47

Endothelial Progenitor Cells from Bench to Antitumor Therapy and Diagnostic Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1389 Tiziana Annese, Roberto Tamma, and Domenico Ribatti

48

Non-cryopreserved Peripheral Stem Cell Autograft for Multiple Myeloma and Lymphoma in Countries with Low Resources . . . . . 1421 Mohamed Amine Bekadja

49

A Multilevel Approach to the Causes of Genetic Instability in Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445 Elio A. Prieto Gonzalez

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

About the Editor

Dr. Khawaja Husnain Haider is currently a Professor of Cellular and Molecular Pharmacology (Stem cells and Gene Therapy) and Chairman of the Basic Sciences Department (Medical Program) at Sulaiman AlRajhi University. He has also served as Principal Investigator (PI) and Co-PI on various NIH-funded stem cells research projects. He has been on the editorial boards of various research journals and has served as an invited reviewer for several respected international journals. His research focuses on the use of DNA, miRNAs, and stem cells as “drugs,” a topic that has gained popularity in regenerative medicine. He has published more than 300 book chapters, abstracts, and research papers published in various books and leading research journals, including Circulation, Circulation Research, Cardiovascular Research, Journal Cellular and Molecular Medicine (JCMM), the Journal of Biological Chemistry, Cell Cycle, Basic Research in Cardiology, and Antioxidant Redox Signaling. He has also given numerous presentations and edited seven books covering various facets of stem cells and their applications from drug to drug development.

xv

Section Editors

Maria Alvarez-Viejo Unidad de Terapia Celular y Medicina Regenerativa, Servicio de Hematología y Hemoterapia Hospital Universitario Central de Asturias HUCA Instituto de Investigación Biosanitaria del Principado de Asturias and Universidad de Oviedo Asturias, Spain

Beatrice Aramini Division of Thoracic Surgery, Department of Experimental, Diagnostic and Specialty Medicine–DIMES of the Alma Mater Studiorum University of Bologna, G.B. Morgagni–L. Pierantoni Hospital Forlì, Italy

Rony Atoui Division of Cardiac Surgery, Health Sciences North Northern Ontario School of Medicine Sudbury, ON, Canada

xvii

xviii

Section Editors

Oh Young Bang Department of Neurology School of Medicine, Sungkyunkwan University Samsung Medical Center Seoul, Korea

Khawaja Husnain Haider Department of Basic Sciences Sulaiman AlRajhi University Al Bukairiyah, Saudi Arabia

Mustapha Najimi Laboratory of Pediatric Hepatology and Cell Therapy Institut de Recherche Expérimentale et Clinique (IREC) Université catholique de Louvain (UCLouvain) Brussels, Belgium

Section Editors

xix

Elio Antonio Prieto González Centre for Advanced Studies in Humanities and Health Sciences Interamerican Open University Buenos Aires, Argentina ISALUD University, Nutrition Career Buenos Aires, Argentina

S. Amer Riazuddin The Wilmer Eye Institute Johns Hopkins University School of Medicine Baltimore, MD, USA

Contributors

Marwa M. S. Abbass Oral Biology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt A. M. Aimaletdinov Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Büşra Aktaş Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Burdur Mehmet Akif Ersoy University, Burdur, Turkey Ana Alfonso-Fernández Servicio de Cirugía Ortopédica y Traumatología, Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain Muhammad Ali The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Abdullah Murhaf Al-Khani Sulaiman AlRajhi Medical School, Al Bukairiyah, Kingdom of Saudi Arabia Sulaiman Alnasser Pharmacology and Toxicology Department, Unizah College of Pharmacy, Qassim University, Unaizah, Kingdom of Saudi Arabia Saleh F. Alqifari College of Pharmacy - Department of Pharmacy Practice, University of Tabuk, Tabuk, Saudi Arabia Mabrouk AL-Rasheedi Al Bukayriah General Hospital, AlQassim, Kingdom of Saudi Arabia Mateq A. Alreshidi Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukayriyah, Kingdom of Saudi Arabia Maria Alvarez-Viejo Unidad de Terapia Celular y Medicina Regenerativa, Servicio de Hematología y Hemoterapia, Hospital Universitario Central de Asturias HUCA, Instituto de Investigación Biosanitaria del Principado de Asturias, Universidad de Oviedo, Asturias, Spain xxi

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Contributors

Tiziana Annese Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy Ming Zhang Ao Hua Zhong University of Science and Technology, Wuhan, China Bruna Araújo Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal Beatrice Aramini Division of Thoracic Surgery, Department of Experimental, Diagnostic and Specialty Medicine–DIMES of the Alma Mater Studiorum, University of Bologna, G.B. Morgagni–L. Pierantoni Hospital, Forlì, Italy Belma Aslım Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Rony Atoui Division of Cardiac Surgery, Health Sciences North, Northern Ontario School of Medicine, Sudbury, ON, Canada Saeed Azandeh Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran Vladimir Baklaushev Federal Research Clinical Center of Specialized Medical Care and Medical Technologies of the FMBA of Russia, Moscow, Russia Federico Banchelli Center of Statistic, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy Oh Young Bang Department of Neurology, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, South Korea S&E Bio, Inc, Seoul, South Korea Tiffany Barszczewski Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada Cellular and Regenerative Medicine Centre, BC Children’s Hospital Research Institute, Vancouver, BC, Canada Derek Barthels Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA Francico J. Bedoya Department of Molecular Biology and Biochemical Engineering, University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Mohamed Amine Bekadja Department of Hematology and Stem Cell Therapy, Etablissement Hospitalier Universitaire 1st November, Ahmed Benbella 1 University, Oran, Algeria Alexander E. Berezin Internal Medicine Department, State Medical University, Zaporozhye, Ukraine

Contributors

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Alexander A. Berezin Internal Medicine Department, Academy of Post-graduating Education, State Medical University, Zaporozhye, Ukraine Kiran Kumar Bokara CSIR–Centre for Cellular and Molecular Biology, Hyderabad, India Theo Borgovan Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Patrick G. Burgon Department of Chemistry and Earth Sciences, College of Arts and Sciences, Qatar University, Doha, Qatar Jae Min Cha Department of Mechatronics Engineering, College of Engineering, Incheon National University, Incheon, South Korea 3D Stem Cell Bioengineering Laboratory, Research Institute for Engineering and Technology, Incheon National University, Incheon, South Korea Weyland Cheng Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, China Rani Chudasama Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Roberto D’Amico Center of Statistic, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy Ekta Dagar CSIR–Centre for Cellular and Molecular Biology, Hyderabad, India Hiranmoy Das Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA Sanat Kumar Dash Department of Mechanical Engineering, Indian Institute of Technology-Madras, Chennai, Tamil Nadu, India Sofia Domingues Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal Massimo Dominici Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy Christof Dörfer Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian-Albrecht’s University, Kiel, Germany Maria Dubrovcakova Biomedical Center, Slovak Academy of Sciences, Bratislava, Slovakia Sara El Moshy Oral Biology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt

xxiv

Contributors

Aiah A. El-Rashidy Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Biomaterials Department, Faculty Dentistry, Cairo University, Cairo, Egypt Azza Ezz El-Arab Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Adegbenro Omotuyi John Fakoya Department of Anatomical Sciences, University of Medicine and Health Sciences, Basseterre, Saint Kitts and Nevis Karim M. Fawzy El-Sayed Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian Albrechts University, Kiel, Germany Michael G. Fehlings Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada Daniel García-Sánchez Grupo de Ingeniería de Tejidos de Cantabria, Dpto. Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IDIVAL, Santander, Cantabria, Spain Anneh Mohammad Gharravi Shahroud University of Medical Sciences, Shahroud, Iran Mohammad Reza Gholami Kermanshah University of Medical Sciences, Kermanshah, Iran M. O. Gomzikova Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Alberto González-González Grupo de Ingeniería de Tejidos de Cantabria, Dpto. Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IDIVAL, Santander, Cantabria, Spain Kavitha Govarthanan MK BHAN Young Research Fellow, Centre for Cardiovascular Biology and Disease, Institute for Stem Cell Sciences and Regenerative Medicine (inStem), Bengaluru, Karnataka, India Giulia Grisendi Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy

Contributors

xxv

Changfa Guo Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China Piyush Kumar Gupta Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India Department of Biotechnology, Graphic Era Deemed to be University, Dehradun, Uttarakhand, India Khawaja Husnain Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia Carl Randall Harrell Regenerative Processing Plant, LLC, Palm Harbor, FL, USA Abdelkrim Hmadcha Department of Biotechnology, University of Alicante, Alicante, Spain University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain V. James School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK Marc G. Jeschke Sunnybrook Research Institute, Toronto, ON, Canada Division of Plastic Surgery, Department of Surgery, University of Toronto, Toronto, ON, Canada Department of Immunology, University of Toronto, Toronto, ON, Canada Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada Mohamed Kamal Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Faculty of Pharmacy, Center for Drug Research and Development, The British University in Egypt (BUE), Cairo, Egypt Dina Kassem Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Mohamed Abdelghafour Khalifa Sulaiman AlRajhi Medical School, Al Bukairiyah, Kingdom of Saudi Arabia Shahid Y. Khan The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Mohamad Khazaei Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada Eun Hee Kim S&E Bio, Inc, Seoul, South Korea

xxvi

Contributors

Oleg Knyazev Department of Inflammatory Bowel Diseases, Moscow Clinical Scientific Center Named After A. S. Loginov, Moscow, Russia Department of Inflammatory Bowel Diseases, State Scientific Centre of Coloproctology Named After A. N. Ryzhih, Moscow, Russia Mikhail Konoplyannikov Federal Research Clinical Center of Specialized Medical Care and Medical Technologies of the FMBA of Russia, Moscow, Russia Department of Advanced Biomaterials, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia Jan Lakota Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Management, Comenius University, Bratislava, Slovakia Danlin M. Law Cell Therapy Institute, Wuhan, China Daniel Law Jia Xian Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Jalan Yaacob Latif, Kuala Lumpur, Malaysia Peter K. Law Cell and Gene Therapies, Cell Therapy Institute, Wuhan, China Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, China Qiaoyun Li Zhengzhou University, Henan, China Wenbin Li Capital Medical University, Beijing, China Lisa Lin Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada Dingqian Liu Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China Qi Hao Looi Future Cytohealth Sdn Bhd, Kuala Lumpur, Malaysia Ping Lu Cell Therapy Institute, Wuhan, China Jian-Hua Ma Nanjing Medical University, Nanjing, China A. G. Malanyeva Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Valentina Masciale Division of Thoracic Surgery, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy William Brett McIntyre Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada

Contributors

xxvii

Mohamed Mekhemar Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian-Albrecht’s University, Kiel, Germany Ayşegül Mendi Faculty of Dentistry Department of Basic Sciences, Gazi University, Ankara, Turkey Gyeong Joon Moon AMC Stem Cell Center, Asan Medical Center, Seoul, South Korea Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul, South Korea Bihter Muratoğlu Center for Stem Cell Research and Development (PEDISTEM), Hacettepe University, Ankara, Turkey Institute of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey Mustapha Najimi Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCLouvain), Brussels, Belgium Min Hwei Ng Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Jalan Yaacob Latif, Kuala Lumpur, Malaysia Kinglsey Chinonyerem Nnawuba Department of Clinical Medicine, School of Medicine, Caribbean Medical University, Willemstad, Curaçao Edward Hosea Ntege Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Tom Ollila Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Adekunle Ebenezer Omole Department of Anatomical Sciences, American University of Antigua College of Medicine, St. John’s, Antigua and Barbuda Yash Parekh CSIR–Centre for Cellular and Molecular Biology, Hyderabad, India Ari Pelcovitz Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Flor M. Pérez-Campo Grupo de Ingeniería de Tejidos de Cantabria, Dpto. Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IDIVAL, Santander, Cantabria, Spain Katarzyna Pieczonka Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada John S. Pixley Texas Tech University Health Science Center, Lubbock, TX, USA

xxviii

Contributors

Elio A. Prieto Gonzalez Centre for Advanced Studies in Humanities and Health Sciences, Interamerican Open University, Buenos Aires, Argentina ISALUD University, Nutrition Career, Buenos Aires, Argentina Peter Queseneberry Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Israa Ahmed Radwan Oral Biology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Dina Rady Oral Biology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt M. Rajasundari Centre for Advanced Research (Stem Cells), Government Stanley Hospital, Chennai, Tamil Nadu, India Jun Ren Tong Ren Hospital of Wuhan University, Wuhan, China Mathieu Rheault-Henry Division of Cardiac Surgery, Health Sciences North, Northern Ontario School of Medicine, Sudbury, ON, Canada S. Amer Riazuddin The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Domenico Ribatti Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy A. A. Rizvanov Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia José C. Rodríguez-Rey Grupo de Ingeniería de Tejidos de Cantabria, Dpto. Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IDIVAL, Santander, Cantabria, Spain С. S. Rutland Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK Khadiga Mostafa Sadek Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Biomaterials Department, Faculty Dentistry, Cairo University, Cairo, Egypt António J. Salgado Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

Contributors

xxix

Maurilio Sampaolesi Translational Cardiomyology Laboratory, Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium Histology and Medical Embryology Unit, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Sapienza University of Rome, Rome, Italy Nihal Satyadev Department of Anatomical Sciences, University of Medicine and Health Sciences, Basseterre, Saint Kitts and Nevis Jose Miguel Sempere-Ortells Department of Biotechnology, Immunology Division, University of Alicante, Alicante, Spain Vineeta Sharma Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India Qizhong Shi Xinxiang Medical University, Henan, China Yusuke Shimizu Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Rita Caridade Silva Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal Tarik Smani Cardiovascular Pathophysiology, Institute of Biomedicine of Seville (IBiS), University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain Department of Medical Physiology and Biophysics, University of Seville, Seville, Spain Biomedical Research Networking Centers of Cardiovascular Diseases (CIBERCV), Madrid, Spain Tarik Smani Cardiovascular Pathophysiology, Institute of Biomedicine of Seville, University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain Qibin Song Renmin Hospital of Wuhan University, Wuhan, China Shi Jun Song Xinxiang Medical University, Henan, China Bernat Soria University of Pablo de Olavide, Sevilla, Spain Department of Physiology, Institute of Bioengineering-Alicante Institute for Health and Biomedical Research (ISABIAL), University Miguel Hernández School of Medicine, Alicante, Spain Li-ping Su Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, USA Nadiah Sulaiman Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Jalan Yaacob Latif, Kuala Lumpur, Malaysia

xxx

Contributors

Hiroshi Sunami Center for Advanced Medical Research, School of Medicine, University of the Ryukyus, Okinawa, Japan Renata Szydlak Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland Roberto Tamma Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy Yong Sheng Tan Cardiothoracic Department, Serdang Hospital, Kajang, Selangor, Malaysia Fábio G. Teixeira Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal Juan R. Tejedo Department of Molecular Biology and Biochemical Engineering, University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Glen F. Tibbits Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada Cellular and Regenerative Medicine Centre, BC Children’s Hospital Research Institute, Vancouver, BC, Canada Peter Timashev Department of Advanced Biomaterials, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia Laboratory of Clinical Smart Nanotechnologies, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia Department of Polymers and Composites, N. N. Semenov Institute of Chemical Physics, Moscow, Russia Chemistry Department, Lomonosov Moscow State University, Moscow, Russia Johannes Tölle Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian-Albrecht’s University, Kiel, Germany Duygu Uçkan-Çetinkaya Faculty of Medicine, Department of Pediatrics, Division of Hematology, Hacettepe University, Ankara, Turkey Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Ankara, Turkey Institute of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey Roohi Vinaik Sunnybrook Research Institute, Toronto, ON, Canada

Contributors

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Ana Volarevic Department of Cognitive Psychology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia Vladislav Volarevic Department of Genetics and Department of Microbiology and immunology, Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia Jing Wang Renmin Hospital of Wuhan University, Wuhan, China Keqiang Wang Xinxiang Medical University, Henan, China Ian White Division of Cardiac Surgery, Health Sciences North, Northern Ontario School of Medicine, Sudbury, ON, Canada Darius Widera Stem Cell Biology and Regenerative Medicine Group, School of Pharmacy, University of Reading, Reading, UK Manye Yao Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou, China Lei Ye Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, USA Laura Yedigaryan Translational Cardiomyology Laboratory, Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium E. Zakirova Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Robert Chunhua Zhao Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Cell Biology, School of Life Sciences, Shanghai University, Shanghai, China Binita E. Zipporah Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India

Part I Cell-Based Therapy Approach: Mesenchymal Stem Cell-Based Therapy

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Human Mesenchymal Stem Cells: The Art to Use Them in the Treatment of Previously Untreatable Jan Lakota, Maria Dubrovcakova, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells Current Status: The “Problems” That in Reality Do Not Exist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practical Approaches: The Golden Standards, i.e., “Good Biological Praxis” (GBP) Using the Historical Laboratory Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data Extrapolation from Small Animal Studies to Humans Is Unfounded and Without Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Research Paradigm: MSCs in Advanced Phases of Clinical Trials Although Understandable Is Far from Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Case of Autologous Versus Allogeneic MSCs, an Unanswered Question . . . . . . . . . . . . . . . . Quality Control of MSCs Preparation: A Prerequisite for Optimal Prognosis . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 5 5 6 8 11 16 17 17

Abstract

Mesenchymal stem cells (MSCs) can be isolated from almost all organs and tissues in the human body. For practical purposes, there are two main sources for their isolation and ex vivo expansion – the bone marrow and fat tissue. Based on their inherent plastic adherence properties, the ex vivo expansion of J. Lakota (*) Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Management, Comenius University, Bratislava, Slovakia e-mail: [email protected] M. Dubrovcakova Biomedical Center, Slovak Academy of Sciences, Bratislava, Slovakia e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_1

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MSCs is a rather simple process. Nevertheless, the biological features (gained from decades of tissue culture experience) are contrary to bureaucratic rules, which govern the good laboratory practice. MSCs cannot be successfully used in the treatment of human diseases if they are not handled optimally akin to the conditions in their natural habitat. Moreover, extrapolation of the data obtained from animal studies (mainly rodents) to humans is unfounded and with little relevance. The therapeutic use of genetically manipulated MSCs in human can be even harmful to patients. The current research paradigm, i.e., the use of MSCs in advanced phases of clinical trials although understandable, is far from personalized medical approach. The use of allogeneic versus autologous MSC in the clinical perspective is still debatable. There is growing evidence that the autologous MSCs derived from sick patients are “ill” in contrast to MSCs derived from healthy allogeneic donors. One can observe various changes at the DNA, RNA, and protein level in these “ill” cells. However, the huge number of cells from ex vivo expanded autologous MSCs can, possibly, overcome these aberrations. The off-the-shelf availability of allogenic MSCs also contributes to their logistic superiority over autologous cells. Moreover, due to almost non-existing immunological barriers, allogenic MSCs are emerging as gold standard and near-optimal cell types for the treatment of various diseases in humans. This chapter reviews the authors experience(s) in the treatment of various diseases with autologous/allogenic MSCs handled optimally ex vivo. Keywords

Adipose tissue · Allogeneic · Autologous · Bone marrow · Ex vivo · Good biological praxis (GBP) · Human disease · MSCs · Treatment List of Abbreviations

DMEM-LG GMP GVDH LG LVAD MSCs NYHA

Dulbecco’s modified Eagle medium, low glucose Good Manufacturing Practices Graft-versus-host disease Low glucose Left ventricular assist device Mesenchymal stem cells New York Heart Association

Introduction The paper of Koç (Koç et al. 2000) symbolically “opened the door” for the mesenchymal stem cells (MSCs) in the third millennium. Here, the authors reported about the autologous blood stem cells and in tissue culture (in vitro or ex vivo) expanded bone marrow-derived MSCs in advanced breast cancer patients receiving high-dose chemotherapy. Since then, an enormous amount of material has been published (Musiał-Wysocka et al. 2019). Coming down to the molecular level, our

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knowledge each day is growing exponentially. Nevertheless, the primary question remains: What is the “therapeutic mechanism” of the applied MSCs? We call this effect as “posthypnagogic command.” After the treatment with MSCs, the effect of healing is present for months, without the proven presence of MSCs. We are not coming in detail here; the reader could educate himself in the enormous amount of literature.

Mesenchymal Stem Cells Current Status: The “Problems” That in Reality Do Not Exist Practical Approaches: The Golden Standards, i.e., “Good Biological Praxis” (GBP) Using the Historical Laboratory Experience In our opinion, it is useful to repeat the whole procedure of ex vivo expansion in detail as it has been described in part “Ex Vivo MSCs Culture” (Koç et al. 2000): “Mononuclear cells (from bone marrow) were re-suspended at 106 cells/mL in Dulbecco’s modified Eagle medium, low glucose (DMEM-LG) with 10% fetal bovine serum and 30 mL of cell suspension was plated in a 175 cm2 flask. MSCs were cultured in humidified incubators with 5% CO2 and initially allowed to adhere for 72 h, followed by media change every 3–4 days. When cultures reached more than 90% confluence, adherent cells were detached with 0.05% trypsin-EDTA.” Later, additional characterizations and refinement added some regulatory rules. These ex vivo expanded MSCs fulfilled the criteria provided (later) by the International Society for Cellular Therapy (Horwitz et al. 2005). Briefly, MSCs are defined by their plastic-adherent properties under standard culture conditions, by their ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro under a specific stimulus and by positive (CD105, CD73, and CD90) or negative (CD45, CD34, CD14, and HLA-DR) expression of specific surface markers. There are two main sources for their isolation and ex vivo expansion for practical purposes – the bone marrow and the fat tissue. This ex vivo expansion of MSCs is a rather simple process based on their inherent plastic adherence properties. The pilot paper by Le Blanc et al. (2004) described the use of “third party” (here – haploidentical) MSCs for transplantation in a patient with severe treatment-resistant grade IVacute graft versus host disease (GVHD) of the gut and liver after allogeneic stem cell transplantation. For decades, two organs (or tissues), i.e., bone marrow and fatty tissue, were the primary sources for the isolation and ex vivo expansion of MSCs. The task of using allogeneic MSCs (obtained from healthy donors) or autologous MSCs in clinical settings will be discussed later. MSCs cannot be successfully used to treat human diseases if they are not handled optimally akin to the conditions in their natural habitat. Let us discuss this in depth. As an example, we will consider the research paper published by Yau and colleagues (Yau et al. 2019). The authors claimed that “among patients with advanced heart failure, intramyocardial injection of mesenchymal precursor cells, as compared with the injections of a cryoprotective medium as sham treatment, did not improve successful temporary weaning from left ventricular assist device (LVAD) support

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at 6 months. These findings do not support the use of intramyocardial MSCs to promote cardiac recovery as measured by temporary weaning from device support.” According to the authors, the patients were randomly assigned to cell therapy group who received intramyocardial injection of 150 million MSCs and a cryoprotective medium treatment group without cells for comparison. The allogeneic MSCs were obtained from healthy donors and expanded in a Good Manufacturing Practices (GMP) certified laboratory. It is evident that the cells were thawed directly before use (“injections of mesenchymal precursor cells, compared to injections of a cryoprotective medium as sham treatment”). The cells were neither washed nor cultivated further for expansion before use. In the opinion of the authors that it is mandatory to use the MSCs that have been freshly prepared and not frozen or thawed immediately before use. After decades of expanding the MSCs (and other cells) ex vivo (in vitro), we firmly stand behind this point of view. After thawing, the cells need to be cultured at least for 48 h in humidified incubators supplemented with 5% CO2. Only after this wait period, one should start to consider further experimental (or therapeutic) work using these cells. On the other hand, one can consider growing the cells ex vivo, detaching them when 80% confluent and applying them to the patient in a short time (up to 3 h at room temperature). The practice to use freshly thawed cells (MSCs) makes the abovementioned study (and others in this fashion designed trials) from the biological point of view rather dubious and medically useless. It should be noted that the number of skeptical articles and comments about the relevance of the MSCs for cell-based therapy is growing (Gomez-Salazar et al. 2020; Curfman 2019). We strongly disagree with the emerging notion. In our opinion, it is necessary to return to the laboratory and to give the MSCs a “second chance” by consequently following the GBP developed during the decades of cell tissue culturing in vitro. We recommend returning to the praxis of small tissue culture centers associated with (or localized within) the hospitals. In coordination with the hospital departments, they could prepare fresh MSCs, which would be “on demand” prepared for use and treat the patients. Logistically, to prepare a total of 20–50
106 cells is not a difficult task. One skilled technician could obtain this amount under sterile conditions in 1–2 h. What about the tests for the differentiation and of sterility? Well, yes, one can ask a heretical, unorthodox question: Did anybody ever observe that the MSCs in vitro did not differentiate to osteoclasts, adipocytes, and chondrocytes during appropriate treatment? This has been further discussed elsewhere in the chapter.

Data Extrapolation from Small Animal Studies to Humans Is Unfounded and Without Relevance The therapeutic use of genetically manipulated MSCs in humans is even harmful to patients. The engineered (“therapeutic”) MSCs are genetically modified MSCs that contain a stable gene encoding for protein or enzyme product/s able to kill the tumor cells. A typical example of such a construct is the yeast enzyme cytosine deaminase,

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which converts the rather nontoxic 5-fluorocytosine to the cytostatic agent 5-fluorouracil (Kucerova et al. 2007). The results obtained from rodents, i.e., preclinical experimental models, are promising. In a recently published review article, Pawitan and colleagues have stated: “So far, most studies using pre-clinical cancer models have shown consistent results, i.e., the engineered MSCs could inhibit tumor growth and enhance the survival rate of the tumor-bearing animals” (Pawitan et al. 2020). And only one published clinical paper by Lakota et al. (2015) claims the opposite: “Treatment with therapeutic MSCs (i.e., genetically engineered MSCs) of this patient highlighted the following points” (Table 1): 1. There was no evidence of any therapeutic benefit after intravenous administration (not local, i.e., intra-tumoral injection) of the therapeutic MSCs. Six days after the cell administration, the metastatic process did not show any signs of regression. Moreover, 40 days after the treatment, there was a progression of the metastases. 2. After the intravenous administration, the therapeutic MSCs were probably “homing” into the bone marrow despite their adipose tissue of origin. Even a relatively low cell count (60x106) was able to cause grade 2 (resp. grade 3) thromobocytopenia (resp. neutropenia) It should be noted that this patient did not receive any systemic chemotherapy in the past. The observed bicytopenia with a nadir neutropenia occurred 48 h after administering therapeutic MSCs (with concomitant prodrug administration). Moreover, in a more recently published paper by Lakota (2018), the author claims that: (i) There was no sign of any therapeutic effect after intravenous administration (not local, i.e., intra-tumoral) of the “therapeutic” MSCs. (ii) After the intravenous administration, the “therapeutic” MSCs were “homing” into the bone marrow. (iii) There has not been any entrapment of the “therapeutic” MSCs in the lungs (after the intravenous administration). Table 1 Blood counts of the patient with head and neck tumor during and after the therapy with therapeutic MSCs. (The table is taken from Lakota et al. 2015) Day 2 0 +1 +2 +3 +4 +5 +6 +18

Leukocytes (
1012/l) 6.88 5.82 4.06 1.99 2.89 3.50 4.50 4.29 6.57

Neutrophils (
1012/l) 4.86 3.98 3.41 1.00 1.58 2.16 2.98 2.63 4.66

Erythrocytes (
1015/l) 4.41 3.71 3.80 3.41 3.72 3.72 3.79 3.89 4.06

Hb (g/l) 134 119 119 112 117 115 118 121 125

Plt (
1014/l) 179 150 119 100 115 122 129 132 191

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(iv) After local (i.e., in situ) administration, the “therapeutic” MSCs did not migrate to any “neighboring” tissue/organ (the liver, retroperitoneum, abdominal wall) or other distant organs, including the bone marrow. Thus, the data obtained from small experimental animal models (mice and rats) must not be extrapolated to the humans. Moreover, the unrestrained transfer (although understandable) of the requisitioned data obtained with the MSCs from rodents may negatively influence the research trends in the novel treatment(s) of human diseases. This kind of treatment of human patients is ineffective and can be harmful and dangerous. The systemically administered therapeutic MSCs are rather homing into the patients’ bone marrow and not into the tumor tissues. After local application (in the tumor area), their ability to destroy the whole tumor or its metastasis is limited. Nor they are moving to other metastatic tumor localizations.

Current Research Paradigm: MSCs in Advanced Phases of Clinical Trials Although Understandable Is Far from Personalized Medicine Current praxis is treating human diseases based on evidence-based medicine (Masic et al. 2008, and the Internet). Clinical trials are the cornerstone to assess novel disease treatment(s), and among the clinical trials, randomized clinical trials are considered as the most preferred design as it provides a causal relationship between the medical intervention and the desired effects (Sawchik et al. 2018). A pertinent question here is: Is the same approach valid for MSCs which is fast emerging as a biopharmaceutical? Here we present some of our (published and unpublished) data none of which has been acquired in any registered trial. All of these data were obtained according to the rules of personalized medicine. (i) Autologous bone marrow-derived MSCs were used for transplantation in ten patients with ischemic cardiomyopathy (Lakota 2014a). All patients had a welldocumented history of anterior wall acute myocardial infarction. They were in NYHA stage III or IV. The freshly prepared MSCs were injected into the left anterior descending coronary artery. All of the patients tolerated the MSCs injection well and were discharged from the hospital 3–4 days after the procedure. Six patients died 7, 9, 37, 71, 101, and 119 months after the procedure. Four patients were alive 112, 113, 120, and 121 months after the MSCs-based treatment. (ii) The allogeneic adipose tissue-derived MSCs were used for the treatment of the patient with 11 years history of ulcus cruris on his left leg, which remained stationary despite trials of conservative treatment (Lakota 2014b). The adipose tissue-derived MSCs (30
106) were applied locally (circumferentially). After a single round of MSCs treatment, the ulcus healing progressed “normally” despite repeated courses of standard and high-dose chemotherapy for patient’s oncological diagnosis (high-grade non-Hodgkin’s lymphoma). The final effect, i.e., restitutio ad integrum, has been observed during 6 months after

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Fig. 1 Healing of the ulcus cruris after local application of MSCs. (a) 11/2012, day after application; (b) 02/2013, d þ 25 after autologous stem cell transplantation; and (c) 05/2013. (The figure is taken from Lakota (2014b))

the MSCs-based treatment (Fig. 1). This course of healing did not differ from the patients with ulcus cruris who received allogeneic or autologous MSCs and who did not receive any chemotherapy (Lakota, unpublished). (iii) Ten patients after allogeneic stem cell transplantation (high-dose therapy for hematological malignant disease) and with severe steroid-resistant grade IV acute GvHD were treated with allogeneic bone marrow-derived MSCs (range 0.3–0.5
106 MSCs/kg body weight) (Lakota 2017a). GvHD was successfully resolved in four patients who received MSCs-based therapy (Table 2). In the end, the author claimed: “Moreover, it seems plausible to have the MSCs ‘in stock’ for fast ex vivo expansion to use the freshly prepared cells (rather than frozen, thawed, and immediately used).” (iv) The autologous bone marrow-derived MSCs and allogeneic adipose tissuederived MSCs were used to treat aseptic necrosis of the jaw. The treatment was performed in 30 patients over a period of 15 years. Freshly prepared MSCs (20
106) from the respective tissues were applied locally. In five patients, MSCs were used twice. The result was a restitutio ad integrum (Fig. 2) (Lakota, unpublished data and Lakota 2017b, in Slovak). (v) The allogeneic adipose tissue-derived MSCs were used for the treatment of ten patients with sclerosis multiplex. The target dose has been 0.5
106 cells/kg bodyweight. The patients were advised not to stop current “official” treatment. Freshly prepared MSCs were delivered intravenously. No adverse effects were observed. The CNS lesions in all patients but one did not progress during 15 years of follow-up after MSCs-based cell therapy (Lakota, unpublished data). We did not mention all the data obtained by the authors during the last two decades involving MSCs-based cell therapy. The authors never performed any double-blind randomized clinical trials due to logistic and economic reasons. In our hands, personalized medicine and the medical treatment followed the golden rule: “primum non nocere.” We followed the aim and principle of helping the sick

Patient No 1 2 3 4 5 6 7 8

Gender M M M F F M F M

Born 1971 1976 1971 1970 1978 1969 1973 1987

Diagnosis AML HD ALL Ph-CML HD ALL HD HD

Transplantation 16.03.04 30.01.05 13.12.05 26.04.07 13.10.09 23.02.10 21.10.10 18.10.11

MSC infusion d þ 206 dþ1 dþ9 d þ 33 d þ 60, d þ 80 d þ 168 d þ 92, d þ 178 d þ 43

Death 09.11.04 14.02.05 05.04.06 – 23.02.10 16.08.10 22.09.11 09.04.13

Reason of death cGvHD aGvHD Disease progression Alive (31.12.16) aGvHD cGvHD Infection Disease progression

HaploTx RI Tx

RI Tx

Haplo Tx

Comment

Table 2 Patients’ characteristics, date of transplantation, date of MSCs infusion, date of the patients’ death, and the reason of death. (Haplo Tx: HLA haploidentical transplantation; RI Tx: reduced intensity conditioning transplantation). (The table is taken from Lakota 2017a)

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Fig. 2 Healing of the osteonecrosis of the jaw after local application of MSCs. Left: during the surgery; right: 12 weeks after the surgery. (The figure is taken from Lakota 2017b)

and suffering patients in concert to see how serpentine can be the road to establish a new treatment in medicine. It is worth mentioning the following observation. In 2004, Vulliet and colleagues published an interesting research paper of using MSCs in a healthy dog (Vulliet et al. 2004). The authors delivered 0.5 million MSCs/kg bodyweight into the canine left circumference coronary artery. The authors observed ECG changes (ST-segment elevation) in all dogs receiving cell administration which was a characteristic of acute myocardial ischemia. Besides microscopic and macroscopic evidence of myocardial ischemia, the authors also observed increased troponin I in two dogs in which measurements were made. These data suggested that myocardial ischemia occurred after the injection of MSCs at the dose used in this study. Microinfarction was also confirmed with histological and immunocytochemical data. These results showed a potential complication of injecting MSCs, or probably any similarly sized cell, into the coronary circulation. Although differences between canine and human coronary circulation exist, and different cell types and sizes have been used for selected cytotherapeutic applications, this potential complication should be thoroughly investigated before MSCs are routinely injected into the arterial circulation of the patients. Luckily for us (Lakota 2014a) and for the patients at the time of publishing this paper (Vulliet et al. 2004) as we became aware of that we performed three intracoronary MSCs transplants and three other patients were “on the horizon.” Thus, this is another confirmation of how dubious the translation of “(pseudo)clinical” data obtained on animals to human clinics is. Two recently published reviews in this regard have elegantly discussed the donor-related factors and quality of the cell preparation as the possible determinants of the outcome of cell-based therapy in the clinics (Haider 2018; Rady et al. 2020).

A Case of Autologous Versus Allogeneic MSCs, an Unanswered Question One of the ongoing and inconclusive debates in cell-based therapy is the preference for autologous over allogeneic MSCs. However, the use of allogeneic MSCs for

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cell-based therapy is fast emerging as a new paradigm in therapeutics (Karantalis et al. 2016). Starting with the pioneering work of Orlic et al. (2001), preclinical studies have characterized syngeneic, xenogenic, allogeneic, and autologous MSCs; however, most of these studies, especially in the small animals, i.e., mice, rats, etc., have focused on the use of allogenic MSCs due to ease of availability (Orlic et al. 2001, Fukuda and Fujita 2005; Jiang et al. 2006; Haider et al. 2008; Beitnes et al. 2012). Similarly, in large animal translational studies, autologous and allogeneic MSCs have been used without any safety issues with either cell type (Poh et al. 2007; Quevedoa et al. 2009; Chen et al. 2014). But then the question remains why these data have been ignored while designing the clinical trials wherein mostly autologous MSCs have been focused on cell-based studies. As evidenced by a systematic review and meta-analysis of 82 animal studies involving 1482 animals that both autologous and allogeneic cells are equally effective (Jansen of Lorkeers et al. 2015), but they have their respective advantages and limitations in the clinical perspective. Hence, the clinical researchers should give these parameters due consideration during clinical study design for optimal therapeutic outcomes. The following section discusses in depth the pros and cons of each cell source in the light of the published data and implores that the relevant information should be given due consideration, especially during the design of clinical studies.

A Case for Autologous Cells On its face, the fear of incompatibility is alleviated with the use of autologous cells. Hence, autologous cells’ usage is advantageous as it does not require immunosuppression to support the acceptance of the transplanted cells and their derivative tissue after differentiation posttransplantation. Moreover, treatment with autologous cells is considered a step closer to the fast-emerging personalized medicine. However, on the downside of autologous cell-based therapy, the use of autologous cells is timeconsuming and labor-intensive exercise, and clinically less viable option, as it may necessitate biobanking of autologous cells for future use of the cells for every individual. On the same note, it is also not sustainable for diseases where the patient may require early intervention, i.e., myocardial infarction, stroke, etc., and the patient does not have the choice of long waiting time until the harvested cells can be purified and expanded to achieve the required cell number for transplantation. Some essential considerations in case autologous cells are as follows: Autologous Cells from Patients Are Also “sick” There is growing evidence that the autologous cells derived from the patients eligible for the cell-based treatment are “sick” compared to the ones derived from healthy allogeneic donors due to their exposure to different risk factors and comorbidities in the “sick” donors (Dimmeler and Leri 2008; Cesselli et al. 2011). Moreover, it is now well-documented that many diseases compromise the stem cell niche homeostasis and seriously affect stem cell properties such that they are rendered unsuitable for cell-based therapy (Perez et al. 2018). For example, Liu et al. have shown that MSCs from diabetic patients have impaired cardioprotective function as compared to the

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ones derived from the healthy donors, which was ascribed to the long-term exposure to hyperglycemia (Liu et al. 2013). We have performed RNA microarray analysis comparing MSCs from two patients with ischemic cardiomyopathy and two healthy donors (Lakota 2014a). Data analyses showed a significantly enhanced gene expression ratio for STAT1α/β (signal transducer and activator of transcription 1-alpha/beta) and ISG15 (ISG15 or g1p2 or ucrp; ubiquitin cross-reactive protein). The decreased ratio of gene expression has been shown for GTP-binding protein RAD. The first analyzed patient died 101 months after the procedure. The second one was alive 120+ months after the procedure. It should be noted that the MSCs in all patients treated were isolated from their bone marrow. This data clearly shows that ischemic cardiomyopathy is a “systemic” disease. One can speculate about the following fact: How is it possible that the patients with this “damaged” RNA profile in MSCs survived such a long time after the procedure, i.e., such “sick” MSCs were able to repair the damaged myocardium? Autologous Cells in Elderly Patients Are Also Aged Another critical aspect generally overlooked during the use of autologous cells is the donor age besides the age of the recipient. A considerable majority of the patient population who are candidates for cell-based therapy are elderly. Using their own (autologous) cells for cell-based therapy and transplanting them back into an aging tissue environment (which has lost the vigor of reparability due to chronological aging) accounts to double negative that significantly hampers the therapeutic outcome (Zhuo et al. 2010). It is pertinent to mention that similar to any other body cell, stem cells also undergo chronological aging, which is multifactorial, i.e., metabolic alteration, accumulation of reactive oxygen species, accumulation of DNA damage and mutations, telomere shortening, etc. (Oh et al. 2014; Schultz and Sinclair 2016). These metabolic and molecular-level changes lead to loss of their stemness characteristics with age, which is reflected in the impairment of their functionality (Kissel et al. 2007; Bustos et al. 2014). Moreover, chronological aging also leads to a significant reduction in stem cells (Maijenburg et al. 2012). Stenderup et al. carried out a direct in vitro comparison of bone marrow-derived MSCs from young donors (18–29-yearold, n ¼ 6) versus elderly donors (68–81-year-old, n ¼ 5) (Stenderup et al. 2003). The authors compared the cells for the expression of senescence markers, cell growth, and differentiation potential. It was observed that the cells from elderly donors showed significantly reduced maximal life span in terms of population doublings (PD) and PD rate, and accelerated senescence as evidenced by betagalactosidase expression as a marker. Similarly, a comparison of bone marrowderived MSCs from human donors (17–90 years age) showed a significant increase in doubling time beside increased expression of senescence markers with the advancing age of the donor (Zhou et al. 2008). Chronological aging and obesity also cause a decline in stem cell yield and their ability to hematopoiesis and bone regeneration (Pachon-Pena et al. 2016, Ambrosi et al. 2017). Therefore, it is essential that clinical researchers consider the

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consequence of donor age while opting for autologous cell sources (Stolzinga et al. 2008). A direct comparison of the reparability of bone marrow-derived MSCs showed that the rate of old donor bone marrow-derived MSCs had poor cardiomyogenic differentiation potential as compared to the MSCs derived from young donor bone marrow (Jiang et al. 2008). For comparison, the cells were transplanted in the same heart in an experimental animal model of acute myocardial infarction. Khan et al. showed that the reparability of the senescent myocardium is determined by the age of the donor (Khan et al. 2011). All these data signify that MSCs from aging patients show a drastic loss of their biological activity and bring us to an important question if the use of autologous is the real culprit for the modest outcome of the clinical trials reported to date (Shahid et al. 2016).

A Case for Allogeneic Cells Data emanating from the clinical studies have shown the safety and efficacy of allogeneic MSCs in adult and pediatric patients (Koc et al. 2002; Horwitz et al. 2002). Recent clinical application of allogeneic MSCs in ischemic cardiomyopathy patients vindicated these data and reported that allogeneic MSCs were as good as autologous MSCs in their functionality and efficacy, favorably affecting LV end-diastolic volumes, LVEF, and ventricular remodeling leading to improved quality of life (Hare et al. 2012, 2017). More importantly, these studies did not report any severe adverse reactions associated with the cell-based therapy with allogeneic cells, including immunologic responses. The safety profile of allogenic MSCs has also been substantiated during a systematic review and meta-analysis of 36 clinical studies, including 1012 participants (Lalu et al. 2012). Experimental studies assessing immunological profiling of MSCs have shown that although they are not immunopriviledged, allogeneic MSCs are weakly immunogenic, because they lack MHC class II and co-stimulatory molecules, i.e., CD40, CD80, and CD86, while they have weak MHC class I expression (Machado et al. 2013; Lohan et al. 2014). Moreover, they do show immunomodulation by suppressing the activation and proliferation of immune cells (Asari et al. 2009; Corcione et al. 2006). Their interesting immune profile tips them as a good candidate for cell-based therapy without the need for immunosuppression therapy and takes care of them not being “self” for the recipient (Kariminekoo et al. 2016). These data about the allogeneic MSCs are a step forward towards the ongoing quest for “Universal donor cells,” which should be available off-the-shelf as a ready-to-use cell preparation (Kinkaid et al. 2010). Logistic Advantage of Allogenic MSCs One of the primary advantages of allogenic MSCs is their logistic superiority over autologous cells (Zhang et al. 2015). Unlike autologous MSCs, which need to be isolated, purified, and expanded in culture before use for each patient, allogenic cells are logistically feasible as they may be readily available off-the-shelf. This ready availability makes possible their use in urgent clinical situations, which is not possible with the autologous cells as it may take 3–4 weeks of isolation, purification,

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and expansion before they could be used for delivery. For any cell-based therapy to be of routine clinical significance as a therapeutic modality, it is imperative that the cells must be available off-the-shelf akin to any other conventional pharmacological agent. Despite the fast-emerging innovative field of personalized medicine, drugs are not to be synthesized for each patient; instead, their use is tailored according to the need of the patients who are stratified to enhance therapeutic efficacy (Marshall et al. 2016). Similarly, in cell-based therapy, which is one form of personalized medicine, cells cannot be prepared for each patient before use; there have to be readily available cell preparations, which can be tailored to the need of the patient. Manufacturing large clinical grade batches of allogenic cell products using GMP, quality controlled for viability, self-renewal, and stemness characteristics will be cost-effective, timesaving, less labor-intensive, commercially favorable, and clinically more relevant for reproducible outcome. Moreover, this off-the-shelf approach fits well with the current pharmaceutical practices, scale-up manufacturing, and may involve automation to make it efficient in manufacturing, thus having a better commercial potential than autologous cells (Malik and Durdy 2015). Additionally, allogeneic MSCs may allow repeated doses of the cells which may be more beneficial than one-time treatment (Poh et al. 2007). Allogenic MSCs Overcome the Limitations of Aging and Sickness of Autologous Cells As discussed earlier, autologous cells derived from elderly patients, especially those with multiple comorbidities, may not fetch the desired results. Availability of allogeneic cells from a young healthy may be a better option for cell-based therapy. In a recently published study, which was aimed to identify a set of donors and their donated cells’ characteristics with predictive value for optimal osteoblastic differentiation, bone marrow-derived MSCs from 58 patients undergoing surgery for bone fracture were characterized for high osteogenic potential and low adipogenic potential (Kowal et al. 2021). The authors have reported a well-defined criterion that donor cells obtained from male donors, without a diagnosis of osteoporosis and containing a higher fraction of CD146+ fraction of cells, together were predictors of high osteogenic potential. Such predefined criterion is also warranted for selecting MSCs for use in the patients who are candidates for cell-based therapy for other diseases. We will not go into detail here (Lakota 2016); nevertheless, one possible explanation could be that the myocardial repair occurred because of the presence of a considerable amount of ex vivo expanded MSCs. The RNA microarray analysis reflects only the statistically up- or downregulation of specific genes. In another study, Koh and coworkers suggested that pluripotency and the secretion of trophic factors of the bone marrow-derived MSCs in amyotrophic lateral sclerosis patients were reduced in proportion to a poorer prognosis. This may suggest that allogeneic (bone marrow- or adipose tissue-derived) MSCs from healthy donors may be a better option for MSCs therapy in amyotrophic lateral sclerosis patients (Koh et al. 2012).

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Quality Control of MSCs Preparation: A Prerequisite for Optimal Prognosis Autologous or allogeneic MSCs are part of the primary prerequisite of quality of the MSCs preparation for use in the patients (Haider 2018) and remain a fundamental determinant of the outcome of any cell-based therapy procedure and its success (Haider 2017). It encompasses everything from percentage cell count and cell viability, their identification to proliferation and differentiation potential to paracrine action of the cells in the MSCs preparation to ensure that the cell preparation is the best compromise of all these properties. For example, the cells should have the proliferation capacity, but at the same time, unlimited proliferation will add the risk of tumorigenicity. Hence, validating MSCs preparation, both biologically and functionally, will ensure that the cells will do the needful, which is meant for postengraftment in the clinical settings. Unfortunately, these critical aspects of quality control of the cell preparation in general, and functional assessment in particular, have been generally overlooked during the design of the clinical studies. This has seriously impacted the efficacy of the cells, thus significantly contributing to the modest outcome of the clinical trials in most cases. For example, bone marrow-derived MSCs, both autologous and allogenic, require in vitro culturing for at least 3–4 weeks. Out from their natural habitat, they are exposed for so long to the unnatural biological environment, which is only partially emulating their natural habitat at best, is expected to alter their biological as well as functional characteristics significantly. All this leads to their senescence or aging in culture due to less than optimal culture conditions (Bonab et al. 2006; Jiang et al. 2017; Shen et al. 2018). Moreover, the long-term culture may render the cells devoid of their specific surface marker expression, i.e., CD29, CD44, CD90, and induce chromosomal instability (Furlani et al. 2009). Transplantation of these in vitro expansion showed no functional effect post-engraftment in experimentally infarcted myocardium. Although various strategies have been developed to recover the culture-induced senescence of cells in terms of their proliferation capacity (Koichi et al. 2011; Tan et al. 2021), the cells may become transformed to be tumorigenic depending upon the culture condition, thus becoming unsafe for cellbased therapy (Rosland et al. 2009; Wolf et al. 2009). Similarly, the long-term culture of the cells becomes immunogenic due to the settlement of extraneous proteins from the culture medium. These ill effects of prolonged in vitro culture are most pronounced in 2D culture conditions. It is anticipated that the advanced 3D culture conditions in vitro will go a long way in alleviating the effects of cell culture-induced cell senescence due to its biomimetic properties (Hoch and Leach 2014; Jeger-Madiot et al. 2021). Another typical example is the effect of cryopreservation of the cells in clinical settings. Although the current cryopreservation protocols are well-optimized and successfully preserve the biological and functional characteristics of the cryopreserved cells, some aspects of the technique require further refinement (Mamidi et al. 2012). Cryopreserved MSCs show altered immunomodulatory and therapeutic efficacy with a significant reduction in the number of viable cells. The rate of cell viability, as well as cell clumping, is also an important complication of

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cryopreservation that may contribute to micro-occlusions after intra-arterial delivery of the cells as compared to the freshly isolated cells with cryopreservation (Cui et al. 2016). A recent study has reported that the cryopreserved successfully maintained their multi-lineage differentiation, immunomodulatory and anti-inflammatory properties, but they lose some of their stemness characteristics in a reversible fashion, which the cells could recover within 24 h in the culture after thawing (Antebi et al. 2019). Kaplan et al. have elegantly reviewed the effect of cryopreservation of the MSCs biological and functional characteristics (Kaplan et al. 2017).

Conclusion In our opinion, the question of the use of autologous versus allogeneic MSCs remains unresolved. Autologous MSCs, although sick, are returning home after ex vivo expansion when they are used for cell-based therapy. Allogeneic healthy cells are here on demand. The immunological barriers do not (in practice) exist. According to the authors’ experience, one should not be afraid to use them in all cases when there are doubts about the current availability of autologous MSCs. No single negative effect has been ever observed. Therefore, the summary of theoretical pros and cons cannot solve up to date this problem. In conclusion, we would like to remind the reader of the old rule which governs the laboratory praxis. This is what we call “good biological praxis” (GBP). One should not create problems where they do not exist. Or, better, with humbleness, we should approach the divine principles in nature which we receive as gifts. With these gifts earlier hidden, we will be able to treat previously untreatable. MSCs are the current example. Human medicine is hotly waiting.

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Poh KK, Sperry E, Young RG, Freyman T, Barringhaus KG, Thompson CA (2007) Repeated direct endomyocardial transplantation of allogeneic mesenchymal stem cells: safety of a high dose, “off-the-shelf”, cellular cardiomyoplasty strategy. Int J Cardiol 117(3):360–364 Quevedoa HC, Hatzistergosa KE, Oskoueia BN et al (2009) Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. PNAS 106(33):14022–14027 Rady D, Abbass MMS, El-Rashidy AA, El Moshy S, Radwan IA, Dörfer CE, Fawzy El-Sayed KM (2020) Mesenchymal stem/progenitor cells: the prospect of human clinical translation. Stem Cells Int 2020:8837654. https://doi.org/10.1155/2020/8837654 Røsland GV, Svendsen A, Torsvik A et al (2009) Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation. Cancer Res 69(13):5331–5339 Sawchik J, Hamdani J, Vanhaeverbeek M (2018) Randomized clinical trials and observational studies in the assessment of drug safety. Rev Epidemiol Sante Publique 66(3):217–225. https:// doi.org/10.1016/j.respe.2018.03.133 Schultz MB, Sinclair DA (2016) When stem cells grow old: phenotypes and mechanisms of stem cell aging. Development 143(1):3–14 Shahid MS, Lasheen W, Haider KH (2016) Modest outcome of clinical trials with bone marrow cells for myocardial repair: is the autologous source of cells the prime culprit? J Thorac Dis 8(10):E1371–E1374 Shen C, Jiang T, Zhu B, Le Y, Liu J, Qin Z, Chen H et al (2018) In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response. J Biol Eng 12(1):1–2 Stenderup K, Justesen J, Clausen C, Kassem M (2003) Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone 33(6):919–926. https:// doi.org/10.1016/j.bone.2003.07.005 Stolzinga A, Jonesb T, McGonagleb D, Scutta A (2008) Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 129(3):163–173 Tan YZ, Xu XY, Dai JM, Yin Y, He X-T, Zhang Y-L, Zhu T-X et al (2021) Melatonin induces the rejuvenation of long-term ex vivo expanded periodontal ligament stem cells by modulating the autophagic process. Stem Cell Res Ther 12:254. https://doi.org/10.1186/s13287-021-02322-9 Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD (2004) Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet 363(9411): 783–784. https://doi.org/10.1016/S0140-6736(04)15695-X Wolf D, Reinhard A, Wolf D, Reinhard A, Seckinger A, Gross L (2009) Regenerative capacity of intravenous autologous, allogeneic and human mesenchymal stem cells in the infarcted pig myocardium – complicated by myocardial tumor formation. Scand Cardiovasc J 43(1):39–45 Yau TM, Pagani FD, Mancini DM, Chang HL, Lala A, Woo YJ, Acker MA, Cardiothoracic Surgical Trials Network et al (2019) Intramyocardial injection of mesenchymal precursor cells and successful temporary weaning from left ventricular assist device support in patients with advanced heart failure: a randomized clinical trial. JAMA 321(12):1176–1186. https://doi.org/ 10.1001/jama.2019.2341 Zhang J, Huang X, Wang H, Liu X, Zhang T, Yunchuan W, Hu D (2015) The challenges and promises of allogeneic mesenchymal stem cells for use as a cell-based therapy. Stem Cell Res Ther 6. https://doi.org/10.1186/s13287-015-0240-9 Zhou S, Greenberger JS, Epperly MW et al (2008) Age-related intrinsic changes in human bonemarrow-derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell 7(3):335–343 Zhuo Y, Li SH, Chen MS, Wu J, Kinkaid HY, Fazel S, Weisel RD, Li RK (2010) Aging impairs the angiogenic response to ischemic injury and the activity of implanted cells: combined consequences for cell therapy in older recipients. J Thorac Cardiovasc Surg 139(5):1286–1294

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Sources and Therapeutic Strategies of Mesenchymal Stem Cells in Regenerative Medicine Mohamed Kamal, Dina Kassem, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone Marrow-Derived MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adipose Tissue-Derived MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Umbilical Cord-Derived MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Strategies of Using MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulation of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles/Exosomes Derived from MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs Act as an Efficient Tool for Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

During the past decade, mesenchymal stem cells (MSCs) have made their mark as a potential weapon in regenerative medicine. Since their first isolation by Friedenstein in the late 1970s of the last century, MSCs have opened new avenues M. Kamal (*) Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Faculty of Pharmacy, Center for Drug Research and Development, The British University in Egypt (BUE), Cairo, Egypt e-mail: [email protected] D. Kassem Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_2

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in the field of regenerative medicine. The main fascination about MSCs lies in their ease of isolation and large ex vivo expansion capacity, as well as demonstrated multipotency and immunomodulatory activities. Basically, several reports have proved that MSCs isolated from different sources possess different characteristics and potentials. In addition, the mechanisms by which these cells can help regenerate tissues and treat several diseases have been proved to be far more complicated than ever thought of. Moreover, a growing body of research has revealed that the therapeutic effects of MSCs occur largely via paracrine signaling and secreted extracellular vesicles, which act as “signalosomes” controlling fundamental cellular functions in recipient cells. In this chapter, we will discuss how MSCs isolated from different sources such as bone marrow; the prototype MSCs, adipose tissue, and umbilical cord differ in their characteristics as potential sources of allogenic versus autologous cell therapy options. Besides, we will clarify the main documented mechanisms of action which MSCs play in regenerative medicine including their differentiation to tissues of mesenchymal versus non-mesenchymal lineages. Additionally, the immune-modulatory effects of MSCs will be discussed as an important arm in their therapeutic potential. Finally, we will discuss the potential of extracellular vesicles produced by MSCs as an emerging cell-free alternative to stem cells therapy. Keywords

Adipose tissue · Bone marrow · Cell free · Exosomes · Mesenchymal stem cells · Stem cells · Umbilical cord blood · Wharton’s jelly Abbreviations

BM CM EVs FDA FGF GMP GvHD HGF IDO IFN-γ ISCT MSCs NK cells PDL-1 PGE2 PRP SVF TGF-β1

Bone marrow Conditioned medium Extracellular vesicles Food and Drug Administration Fibroblast growth factor Good Manufacturing Practice Graft vs host rejection disease Hepatocyte growth factor Indoleamine-pyrrole 2,3-dioxygenase Interferon-γ International Society for Cellular Therapy Mesenchymal stem cells Natural killer cells Programmed death ligand-1 Prostaglandin E2 Platelet-rich plasma Stromal vascular fraction Transforming growth factor-β1

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TNF-α UC UCB WJ

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Tumor necrosis factor-α Umbilical cord Umbilical cord blood Wharton’s jelly

Introduction Mesenchymal Stem Cells During the past decade, the field of mesenchymal stem cells (MSCs)-based research has witnessed huge progress due to unraveling many of their exceptional biological features and stemness characteristics, as well as the encouraging results of several preclinical and clinical studies. Thus, it is now widely accepted that MSCs could provide a revolutionary therapeutic intervention for various diseases (Batsali et al. 2013; Wang et al. 2012). MSCs were first reported in the early 1970s when a population of plastic-adherent, fibroblast-like, non-hematopoietic cells were isolated from bone marrow (BM) (Friedenstein et al. 1974). Later, in the early 1990s, based on their properties, the term “mesenchymal stem cell” was proposed (Caplan 1991). The term “mesenchymal” is derived from the word mesenchyme, which refers to loosely organized tissue during embryonic development broadly associated with connective tissues (Viswanathan et al. 2019). Its noteworthy here that the acronym “MSCs” has also been designated for “multipotent mesenchymal stromal cells” (Horwitz et al. 2005). Nevertheless, although both terms have been commonly used interchangeably in the literature, it is pertinent to mention that the International Society for Cellular Therapy (ISCT), now called the International Society of Cell and Gene Therapy, has clarified through a couple of position statements that the term “mesenchymal stem cell” is not equivalent to “mesenchymal stromal cells.” Moreover, the latter refers to a bulk population with notable immunomodulatory as well as secretory and homing properties (Horwitz et al. 2005; Viswanathan et al. 2019). They have suggested that the term “mesenchymal stromal cells” can be used to describe the heterogeneous bulk population of cells, which includes fibroblasts, myofibroblasts, and even a relatively small proportion of stem/progenitor cells, but excludes endothelial and hematopoietic cells (Viswanathan et al. 2019). Notably, the ISCT has defined specific minimal criteria for the proper characterization of MSCs so that the research data from various research groups may be compared (Dominici et al. 2006). This criterion includes: 1. MSCs are plastic-adherent when maintained in well-defined and standardized culture conditions. 2. MSCs must express some mesenchymal cluster of differentiation (CD) surface markers like CD105, CD73, and CD90 and lack the expression of hematopoietic stem cell-specific markers like CD45, CD34, and CD14.

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Fig. 1 Isolation and characterization of MSCs from different tissue sources, including umbilical cord, adipose tissue, and bone marrow. After isolation, MSCs from different tissue sources exhibit ISCT criteria based on plastic adherence, immunophenotyping, and trilineage differentiation potential of osteogenic, adipogenic, and chondrogenic phenotype. (Created with Biorender.com)

3. MSCs should exhibit multilineage differentiation potential to adopt adipogenic, osteogenic, and chondrogenic phenotypes in vitro in the presence of specific chemical cues. These essential characterization criteria are summarized in Fig. 1.

Sources of Mesenchymal Stem Cells So far, MSCs have been successfully isolated from various tissues, including adult tissues such as adipose tissue, BM, peripheral blood, dental pulp, skin, muscle, endometrium, ovary, as well as perinatal/fetal sources, like the placenta, umbilical cord blood (UCB), and umbilical cord (UC) matrix/tissue (Da Silva Meirelles et al. 2006; Jackson et al. 2010; Ma et al. 2014; Vasandan et al. 2014; Niezgoda et al. 2017; Berebichez-Fridman and Montero-Olvera 2018; Ghamari et al. 2021; Zolbin et al. 2021). Despite all of these being MSCs from different tissue sources, they significantly differ from each other in terms of their proliferation, self-renewal, paracrine action, and even differentiation potential. For example, MSCs obtained from UCB are rich in a more primitive cell population than those obtained from the BM (Hass et al. 2011; Wang et al. 2016b). Similarly, adipose tissue-derived MSCs (Ad-MSCs) have specific features, such as their transcriptome, proteome, and immunoregulatory properties as compared to the BM-derived counterparts (Strioga et al. 2012; Li et al. 2015a). Similarly, the contributing factors causing divergence in MSCs’ characteristics include age, sex, the health status of the donor, etc.

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For example, MSCs derived from a young donor (8–12 weeks) and elderly donor rats (24–26 months) was assessed for their responsiveness to anoxia and assessment reparability of infarcted myocardium post engraftment in an experimental animal model of acute myocardial infarction (Jiang et al. 2008). The young donorderived cells were more resistant to apoptotic signals upon anoxic exposure than their counterparts. Similarly, the young donor-derived MSCs showed more cardiomyogenic differentiation after transplantation and better preserved the global cardiac function of the infarcted myocardium. Similar results have also been reported by Wang et al. while comparing young and old BM-derived MSCs in an experimental animal model for cardiac repair (Wang et al. 2008). BM-derived MSCs have been the most extensively studied and denoted as the “gold standard” of MSCs among MSCs derived from different tissue sources (Arutyunyan et al. 2016). From among the most important tissues sources of MSCs, we will discuss in this chapter BM-derived MSCs, Ad-derived MSCs, and Wharton’s jelly-derived MSCs (WJ-derived MSCs) with particular emphasis on the latter two as the most well-studied types of MSCs in regenerative medicine.

Bone Marrow-Derived MSCs BM-derived MSCs constitute a heterogeneous group of cells. They were the first MSCs to be isolated and reported in 1966 by Friedenstein and colleagues. They isolated a population of cells from guinea-pig BM with fibroblast-like plastic adherent phenotype in vitro with colony-forming abilities. These cells could selfrenew, express mesenchymal markers, such as CD29, CD44, CD73, CD90, and CD105, CD106, and CD166, and lacking in the expression of hematopoietic markers, such as CD14, CD34, and CD45, and capable of spontaneous bone formation in the diffusion chambers (in vitro), besides inducible osteogenesis in the presence of transitional epithelium (Friedenstein et al. 1970). Later on, in 1991, Caplan suggested changing the name to “mesenchymal stem cells” and ascribed these cells as responsible for bone and cartilage formation in the embryo and responsible for their repair and turnover in adult life (Caplan 1991). Caplan also described the role of some extrinsic factors and the inherent genomic potential to control the rate and phenotype of the cells in the emerging tissue from these cells and predicted their emergence as a novel therapeutic modality for the repair of skeletal tissues. While describing the mesengenic process, Caplan described a pivotal role of MSCs in terms of undergoing proliferation, lineage commitment, and terminal differentiation to adopt the desired phenotype, that is, osteogenic, chondrogenic, and adipogenic (Caplan 1994). Going further, in 2017, Caplan has now suggested to rename MSCs as “medicinal signaling cells” to reflect on the ability of MSCs to migrate to the site of injury and participate in the repair process (Caplan 2017). Although BM-derived MSCs are the most extensively studied, wellcharacterized, and widely used cells in clinical settings, MSCs represent only 0.001–0.01% of mononuclear cells in the BM. Due to this small fraction, BM-derived MSCs necessitate in vitro expansion to acquire sufficient numbers for

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in vivo use (Pittenger et al. 1999). In this regard, various in vitro strategies have been adopted that include culturing of cells under serum-free conditions to 3D-culturing that simulate the natural habitat of the cells (Bhat et al. 2021).

Advantages of BM-Derived MSCs BM-derived MSCs show multipotent differentiation potential toward different embryonic lineages. These lineages include the mesodermal lineage, that is, adipocytes, chondrocytes, osteocytes, endothelial cells, and skeletal muscles, ectodermal lineage, that is, neurons, retinal cells, and endodermal lineage, that is, hepatocytes and insulinproducing cells (Friedenstein et al. 1970; Polisetti et al. 2010). However, the ability of MSCs to adopt different lineage phenotypes is considered tissue source of MSCsdependent. For example, BM-derived MSCs show stronger osteogenic but lower adipogenic potential than Ad-MSCs, but they both offer similar chondrogenic potential (Xu et al. 2017). Similarly, a comparison of synovial membrane-derived MSCs and BM-derived MSCs showed that the latter have enhanced osteogenic differentiation potential, although their chondrogenic potential was similar (Gale et al. 2019). This multilineage differentiation enables BM-derived MSCs to provide vast therapeutic applications for different diseases in regenerative medicine (Haider and Ashraf 2005). BM-derived MSCs are still the most used MSCs in clinical settings (Wang et al. 2016a). Interestingly, BM-derived MSCs were the first “stem cell drug” to be registered by the Food and Drug Administration (FDA) as a drug (Prochymal™) against graft vs. host rejection disease (GvHD) (Prasad et al. 2011). Presently, BM-derived MSCs have more than 400 registered clinical trials with almost 160 completed trials involving a broad scope of immune, chronic, cardiovascular, and degenerative diseases, for instance, diabetes mellitus, neurodegenerative diseases, and heart diseases (ClinicalTrials.gov 2019). Limitations of BM-Derived MSCs Despite the advantages mentioned earlier, a few limitations have led to cautious advancement of BM-derived MSCs to the clinic in regenerative medicine. For example, the isolation of BM-derived MSCs is very complicated and more invasive than UCB, adipose tissue, and WJ. Harvesting of BM-derived MSCs can cause possible discomfort and morbidity due to the invasive BM aspiration process (Mazini et al. 2019). In addition, BM-derived MSCs isolation may require centrifugation to separate mononuclear cells from RBCs, platelets, and granulocytes (Naji et al. 2019). Moreover, the limited yield of cells necessitates in vitro expansion, which may result in altering the immunogenic status and differentiation potential of cells. However, several reports have reported that BM-derived MSCs exhibit signs of early senescence during in vitro culture expansion. All these problems directed researchers to search for alternative sources of MSCs.

Adipose Tissue-Derived MSCs Ad-MSCs constitute a heterogeneous population of cells isolated from adipose tissue of different body parts. They exhibit all the essential characteristics of the MSCs

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proposed by the ISCT, namely plastic adherence, expression of mesenchymal cellspecific surface membrane markers and lacking in hematopoietic stem cell-specific surface membrane markers, and the ability to undergo trilineage differentiation, that is, adipocytes, chondrocytes, and osteocytes. Like the other tissues-derived MSCs, Ad-MSCs also offer several unique advantages over other sources of MSCs (Bourin et al. 2013; Fathi and Farahzadi 2009). Besides the typical mesenchymal surface markers, Ad-MSCs also express primitive cell surface markers of core circuity of self-renewal, that is, OCT4, NANOG, SOX2, and the neurogenic lineage-specific genes, that is, NEUROD1, PAX6, and SOX3 (Gentile et al. 2019). Although similar in surface marker expression to BM-derived MSCs, the yield of Ad-MSCs is approximately 500 times more than the BM per gram of the respective tissue (Hall et al. 2010). They show higher proliferative potential without losing much of their stemness properties (Zhu et al. 2008). Another study comparing BM-derived MSCs and Ad-MSCs derived from patients with coronary artery disease has shown the superiority of Ad-MSCs in their response to low oxygen culturing that renders them more suitable for transplantation for cardiovascular therapy (Adolfsson et al. 2020).

Advantages of Adipose Tissue-Derived MSCs Ad-MSCs propose several advantages over BM-derived MSCs that make them of special interest in the clinical perspective. Firstly, the ease of availability in tissue harvesting and isolation of the Ad-MSCs from the harvested tissue constitutes a significant advantage of these cells. Similarly, Ad-MSCs can be isolated from subcutaneous fats of different parts of the body, including the abdomen, thighs, and buttocks, with far less invasive procedure than the BM-derived MSCs. This less invasive procedure is associated with lesser donor morbidity, pain, or complications than the BM-MSCs (De Ugarte et al. 2003). Second, the lipoaspirate showed a higher population of MSCs, reaching 10% compared to the scarce population of 0.01–0.001% of the mononuclear cells of BM-derived MSCs (Kern et al. 2006; Zhu et al. 2008). Ad-MSCs from lipoaspirate might reach 500 times that obtained from BM (Marigo and Dazzi 2011). Thirdly, and most importantly, Ad-MSCs showed better-culturing abilities, greater proliferation capacity, and they can maintain their phenotype longer in culture (Kunze et al. 2020). These properties are very significant in producing many “clinical grade” MSCs for clinical applications. Besides, several reports have shown that Ad-MSCs could differentiate into different cell types of the three developmental germ layers (endoderm, mesoderm, and ectoderm), including adipocytes, osteoblasts, chondrocytes, neurocytes, and hepatocyte (Chen et al. 2016; Dai et al. 2016). These properties render Ad-MSCs as a front-runner for clinical applications. Limitations of Adipose Tissue-Derived MSCs Although Ad-MSCs clinical applications have increased lately, several limitations must be overcome (Mazini et al. 2020). Ad-MSCs yield, culture properties, and differentiation capacities are dependent on several factors, including the harvesting/ isolation methods and the donor health conditions (Zhang et al. 2020a). Harvesting cells by excision has a better yield than lipoaspiration and directs the cells toward mesoderm and ectoderm differentiation rather than the endoderm associated with the

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lipoaspirate (Bian et al. 2016; Gnanasegaran et al. 2014). Besides, no single controlled procedure exists to isolate reproducible Ad-MSCs following Good Manufacturing Practice (GMP) (Kassem and Kamal 2020c; Kim et al. 2015; Koh et al. 2016). Moreover, Ad-MSCs properties are highly affected by the medical comorbidity of the patients. For example, obesity of the donor induces early senescence in the Ad-MSCs. The cells obtained from obese patients showed inadequate proliferative capacity due to upregulation of p16, p53, IL-6, and MCP1, but their emigrational capability was sustained (Conley et al. 2020). Similarly, results about the declining function of adipose tissue-derived stem cells have also been reported with extensive passaging (Zhu et al. 2008). It has also been reported that the differentiation capacity and the proliferative potential of Ad-MSCs are highly influenced by the donors’ conditions such as age, BMI, suffering from diabetes mellitus, exposure to radiotherapy, or endocrine therapy (Varghese et al. 2017). For example, the differentiation potential of Ad-MSCs from diabetic patients is significantly lost due to generalized inflammatory response in the patients (Barbagallo et al. 2017). However, further investigations are required to confirm the clinical significance of such differences (Zhang et al. 2020a).

Isolation of Adipose Tissue-Derived MSCs Generally, adipose tissue is harvested using different methods, that is, liposuction, resection, micro-fat harvesting, etc., and each one of the methods has advantages and limitations and significantly affect the yield and characteristics of the Ad-MSCs thus derived therefrom, as discussed elsewhere (Prantl et al. 2021). The tissue harvesting step is followed by subsequent washing of the harvested adipose tissue to remove the contaminating erythrocytes. The tissue-digestion step is next followed in a typical protocol that may be achieved either mechanically or enzymatically and centrifuged to separate the stromal vascular fraction (SVF). The cells of SVF are then suspended in serum-containing media for culture and expansion of Ad-MSCs. Coleman’s method is most commonly used to harvest adipose tissue from among the many methods reported in the literature to date for harvesting adipose tissue (Alstrup et al. 2020). The method of harvesting can influence the number of Ad-MSCs obtained, the stemness, the proliferative capacity, the multilineage potency, and the aging rate of the isolated cells (Bajek et al. 2017). For example, it was reported that surgical resection is associated with a large number of Ad-MSCs showing a higher rate of clonogenicity. On the other hand, cells isolated by lipoaspiration showed increased proliferation and the least aging rate. Some other methods for the isolation of Ad-MSCs, the process of adipose tissue harvesting may involve surgical resection, power-assisted lipoaspiration, laser-assisted lipoaspiration, etc. (Khazaei et al. 2021; Fontes et al. 2018). As for the digestion of the adipose tissue, mechanical digestion methods, such as shaking, centrifugation, or filtration, are cost-effective but these are time-intensive and harsh on the cells to inflict mechanical damage on the cells. This may also lead to a poor yield of the viable cells (Khazaei et al. 2021). Several enzymes were used for digestion of the adipose tissue, such as collagenase, trypsin, and dispase (Bourin et al.

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2013; Yang et al. 2011; Zhu et al. 2013), However, adipose tissue digestion with collagenase is the gold standard among the currently used methods (Banyard et al. 2015). However, as collagenase is of bacterial origin, using high-grade enzyme preparation and removing excess enzyme by centrifugation is essential to ensure safety and decrease toxic effects on the cells for subsequent clinical applications (Aguena et al. 2012). Still, the methods for adipose tissue harvesting and isolation of Ad-MSCs from the harvested tissue require more efforts to standardize and apply GMP to ensure the production of “clinical grade” cells for patient applications.

Clinical Applications of Adipose Tissue-Derived MSCs Several excellent reviews have been published discussing the clinical applications of Ad-MSCs for different diseases (Shukla et al. 2020; Zhang et al. 2020a). Zhang and colleagues have presented an excellent review of various applications of Ad-MSCs for wound healing and the treatment of bone, cartilage, nervous system, liver, heart, skin, and even trachea and gall bladder. These studies are either preclinical or clinical studies. Shukla and co-workers (2020) published a review to discuss extracellular vesicles with a focus on exosomes derived from Ad-MSCs for clinical applications. These reviews reflect an increasing interest of the researchers and the ever-growing applications of Ad-MSCs in regenerative medicine to treat various diseases.

Umbilical Cord-Derived MSCs Human Umbilical Cord Structure and Function During pregnancy, the placenta and growing fetus are connected by the UC, also called “the naval string” (Spurway et al. 2012) that prevents umbilical vessels from kinking, compression, or torsion during movement of the fetus, thus ensuring proper blood supply to the fetus (Kim et al. 2013). Anatomically, the human UC comprises an outer layer of amniotic epithelium enclosing a vein and two arteries embedded within a mucoid connective tissue. The mucoid connective tissue enclosing the three umbilical vessels or the UC matrix is known as “Wharton’s jelly.” Thomas Wharton first described WJ in 1656 (Wharton 1656). In the early 1970s, UCB was reported as a natural reservoir and a rich source of hematopoietic stem cells (Knudtzon 1974). Nowadays, UCB has many therapeutic applications for various hematopoietic and non-hematopoietic disorders in the clinics (Munoz et al. 2014; Roura et al. 2015). Given their extensive regenerative potential, the collection, and banking of UCB and its derivative stem cells as part of personalized medicine (Harris 2014), including very small embryonic-like stem cells (Bhartiya et al. 2012), is gaining popularity for use as an autologous source of cells for cell-based therapy (Badowski and Harris 2012; Um et al. 2020). The first report providing robust evidence that WJ-derived stromal cells can be classified as MSCs was published by Wang et al. (2004), which showed that the cells derived from WJ displayed a fibroblast-like phenotype when expanded in vitro (Wang et al. 2004). Also, these cells expressed high levels of MSCs markers

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CD29, CD44, CD73, and CD105 but lacked the expression of the hematopoieticspecific markers, that is, CD31, CD34, and CD45. Moreover, those cells could differentiate into osteogenic and adipogenic lineages under favorable culture conditions (Wang et al. 2004). More interestingly, treatment with 5-azacytidine or cardiomyocyte-conditioned medium, the cells expressed cardiomyocyte-specific markers, that is, N-cadherin, cardiac troponin-I. UCB is also a source of MSCs, but to a much lesser extent than WJ (Arutyunyan et al. 2016; Sibov et al. 2012). Compared with the success rate of harvesting MSCs, it has to be almost 100% from WJ compared to a meager 6% from UCB (Shetty et al. 2010). A direct comparison of various protocols of MSCs isolation from UCB and WJ tissue has been provided by Salehinejad et al. (2012). These protocols are developed to optimize the methodological aspects of the procedure. The latest advancement in this regard is the proteolytic enzyme-free method for the isolation of MSCs (Singh et al. 2019). It is noteworthy that the therapeutic potential of MSCs derived from these two UC compartments could differ significantly in preclinical and clinical settings (El-Demerdash et al. 2015; Kassem and Kamal 2020b).

Advantages of WJ-Derived MSCs WJ-derived MSCs possess exceptional stemness properties due to the presence of a primitive cell population (Troyer and Weiss 2008; Fong et al. 2011). WJ-derived MSCs have several advantages over other types of stem cells. First of all, they can be easily isolated from the readily available UC tissue discarded at labor as medical waste. Thus, unlike BM-derived MSCs, the isolation of WJ-derived MSCs is non-invasive, and unlike ESCs, the availability and use of WJ-derived MSCs are without any moral and ethical concerns (Hass et al. 2011). Second, WJ-derived MSCs isolated from neonatal tissue have more primitive characteristics than adult tissue-derived MSCs (Frausin et al. 2015). They are believed to represent an intermediate state between ESCs and adult stem cells (Marino et al. 2019). Interestingly, WJ-derived MSCs exhibit a mix of human ESCs and MSCs-specific markers and maintain stemness for several serial passages (Nekanti et al. 2009). Interestingly, while they possess several characteristics of ESCs, they do not form teratomas upon transplantation (Troyer and Weiss 2008). Third, WJ-derived MSCs are immune-privileged due to human leukocyte antigen-G (HLA-G) expression, besides lacking in the expression of human leukocyte antigen- antigen D-related (HLA-DR) like other MSCs types (La Rocca et al. 2009). These immunomodulatory properties were reported to prevent rejections even after xeno-transplantation of post-differentiated MSCs without immunosuppression (Moffett and Loke 2003). A direct comparison of MSCs derived from four different tissues, adipose tissue, BM, WJ, and UCB, have shown that WJ-derived MSCs possess the highest immunomodulatory effects (Li et al. 2014). Given their excellent immunomodulatory properties, WJ-derived MSCs are an attractive choice when immune cellular therapy is required (Najar et al. 2012). Finally, WJ-derived MSCs, and their counterparts isolated from UCB, both have an excellent potential for biobanking (Chatzistamatiou et al. 2014).

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Limitations of WJ-Derived MSCs Despite many advantages of WJ-derived MSCs over other stem cell types, several limitations have hampered their successful translation from the bench-to-bedside. For example, among these limitations is the requirement of in vitro expansion, which is significantly affected by culture conditions. For WJ-derived MSCs, the serum is an essential component of the culture as well as cryopreservation media; however, the use of culture-expanded cells causes serious immunological reactions due to the residual animal serum proteins from animal serum slowly settled on the cells’ surface during long-term culture. This adversely alters the immunological characteristics of the cells besides reducing their therapeutic benefits post engraftment (Li et al. 2015b). It is noteworthy that several alternatives of xenogeneic serum have been suggested to achieve humanized stem cell culture using human alternatives for the xenogenic protein (Bieback et al. 2009; Tekkatte et al. 2011). Given its unique growth factor and cytokine-rich composition, one proposed alternative is plateletrich plasma (PRP) (Kandoi et al. 2018; Haider 2017). The authors observed at least a twofold increase in the yield from the explant culture with significantly preserved immunomodulatory properties of the cells. Besides enhancing the safety aspects of the in vitro cultured cells, xeno-free culture expansion also improves their therapeutic functionality (Kang et al. 2020). Besides the xenoprotein-free culture system, Obradovic et al. have reported in vitro expansion protocol that closely mimics the natural habitat of the cells in terms of low oxygen presence (Obradovic et al. 2019). The authors reported that the cells cultured under 3% oxygen conditions retained their expression of OCT4A, OCT4B, NANOG, and SOX2 expression and showed excellent migratory capacity. Another limitation of WJ-derived MSCs is the lack of standardized protocols for their processing, cryopreservation, or banking. These include the isolation of WJ-derived MSCs from fresh tissue versus frozen ones (Fong et al. 2016). Additionally, during prolonged in vitro culture, WJ-derived MSCs have been reported to show changes in their transcriptome profile, compared to the early passage cells, where these cells might exhibit a progressive decline in their physiological properties; a phenomenon known as “Cellular aging” (Gatta et al. 2013). Regarding the clinical application of WJ-MSCs, similar to stem cells from other tissue sources, various parameters, such as the best route of administration, time of injection, dose of the cells, frequency of doses, as well as the time intervals between multiple injections, remain controversial issues (Kamal and Kassem 2020). Isolation Methods of WJ-Derived MSCs Various isolation protocols have been optimized and reported to generate WJ-derived MSCs from the UC tissue. Primarily, these protocols are based on tissue explant and chemical enzymatic digestion approaches, which are incidentally the two methods for WJ-derived MSCs isolation (Han et al. 2013; Xu et al. 2010). Sometimes a mix between enzymatic and mechanical digestion approach, using enzymes like collagenase with/without hyaluronidase or trypsin, followed

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by passing the treated WJ through an 18-G needle syringe (Azandeh et al. 2012). The explant method is based on the ability of the MSCs to move and migrate from the tissue and adhere to the plastic surface (Goyal et al. 2018). It is generally described as cutting the UC into shorter pieces, excision of the blood vessels, and fine chopping of the WJ sections of the cord tissue, followed by plating those fine fragments with complete media. Besides being cost-effective and reproducible, the primary advantage of the tissue explant method is that it does not affect the vitality of cells. It is noteworthy that enzymes may degrade the cell membrane during enzymatic digestion, resulting in further damage to the cells (La Rocca et al. 2009). Hua and co-workers concluded that the 10 mm size tissue explant method is the optimal protocol for isolating MSCs from UC (Hua et al. 2013).

Clinical Applications of WJ-Derived MSCs Over the last many years, the published data from the preclinical experimental studies have demonstrated the safety and highly promising therapeutic potential of WJ-derived MSCs. In vitro characterization reveals a primitive cell population positive for OCT4 and NANOG expressing cells (Pirjali et al. 2013; Shaer et al. 2014). Based on the hypothesis that tissue source may determine the biological characteristics and functionality of the derivation MSCs, a recent study by Laroye and colleagues have shown a better profile of WJ-derived MSCs as compared to the BM-derived MSCs in mice model of sepsis by caecal ligation and puncture (Laroye et al. 2019). Various clinical trials have been conducted to test the safety, feasibility, and efficacy of WJ-derived MSCs-based cell therapy for many diseases, including graft versus host disease (GvHD), bone/cartilage disease, immunological diseases, diabetes mellitus and its complications, cardiac disease, liver disease, neurological disorders, as well as cancer (Abbaszadeh et al. 2020). Many of these clinical trials have been completed and demonstrated the safety and efficacy of WJ-MSCs (Can et al. 2017). The latest application is the effective use of WJ-derived MSCs in patients suffering from COVID-19 pneumonia (Zhang et al. 2020b). Intravenous infusion of WJ-derived MSCs has shown promising results due to their strong immunomodulatory actions (Shi et al. 2021).

Therapeutic Strategies of Using MSCs MSCs have made their mark as a promising therapeutic option for many regenerative medicine applications. Originally, MSCs were thought to mediate their reparative potential by virtue of a multilineage differentiation capability that enabled them to replace damaged cells (Mahmood et al. 2003; Murphy et al. 2003). However, later on, a growing body of evidence has revealed that in response to tissue injury, MSCs home into the site of tissue damage and can mediate tissue repair via paracrine action through the release of soluble and insoluble secretome and immunomodulatory activities (Chen et al. 2008; Karp and Teo 2009). The various therapeutic strategies of MSCs have been summarized in Fig. 2.

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Fig. 2 Therapeutic strategies of MSCs including their ability to regenerate damaged tissue, either by direct mechanisms or indirectly via secretion of cytokines, growth factors, exosomes, and immune system modulating agents. (Created with Biorender.com)

Tissue Regeneration Irrespective of their source, MSCs have demonstrated remarkable capability to regenerate various tissue types, especially those of mesodermal origin, such as muscle, bones, and cartilage (Pittenger et al. 2019; Le et al. 2020), as well as cardiomyocytes, neurons, and pancreatic β-cells (Farini et al. 2014). For instance, MSCs have exhibited an excellent regenerative capacity for bones and cartilage repair, both in experimental as well as clinical settings (Kangari et al. 2020). Elucidating the cascade of events involved in the bone repair process, the transplanted cells caused the mobilization of macrophages, induced functional switch from pro-inflammatory to pro-resolving phenotype, and recruitment of endothelial progenitor cells and cells with osteogenic potential intrinsically from the BM (Tasso et al. 2013). Tadoeschi et al. proposed that transplanted MSCs modified the in vivo environment conducive to angiogenesis and bone regeneration (Todeschi et al. 2015). Additionally, the transplanted cells also undergo endothelial differentiation to participate in the formation of the new capillary network (Chen et al. 2009). Various strategies have been adopted to enhance the efficiency of bone repair. For example, Zhou et al. used in vitro osteogenically induced human MSCs for bone tissue engineering in mice to repair skull defects (Zhou et al. 2015). The latest advancement in this regard is the use of scaffold-based delivery of MSCs to enhance their efficacy. Li et al. used a composite scaffold of WJ and chondroitin sulfate loaded with human UC-derived MSCs knee repair in a rodent model (Li et al. 2021). At a molecular level, a variety of mediators with regenerative effects include,

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transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), indoleamine 2,3-dioxygenase (IDO), bone morphogenic proteins (BMPs), and others (Granero-Moltó et al. 2009; Qin et al. 2014) have a significant role therein. Interestingly, MSCs-based therapy also represents a novel efficient therapeutic strategy for cartilage repair owing to its outstanding chondrogenic differentiation potential (Le et al. 2020). MSCs secrete several bioactive factors, which help to restore the extracellular matrices (ECMs) that are essential for the recovery of cartilage functions (Lories and Luyten 2011). Finally, engineered scaffolds have also been used in combination with MSCs and exogenous biochemical stimuli, and demonstrated profound progress in cartilage regeneration (Smith and Grande 2015).

Immunomodulation of MSCs Although MSCs were initially used to exploit their regenerative capacity and reparability, however, the therapeutic potentials of MSCs have been attributed more to paracrine action and immunomodulation with partial contribution from multilineage differentiation (Ceccarelli et al. 2020). It is now well-established that MSCs exhibit a hypoimmunogenic phenotype as they lack the major histocompatibility class II (MHC-II) and release of immunomodulatory cytokines (Lacy et al. 2021). Thus, these characteristics enable them to evade recognition by the recipient immune system (McIntosh et al. 2006; Puissant et al. 2005). These immunemodulatory effects of MSCs collectively involve cell-to-cell contact-dependent mechanisms and paracrine effects by releasing a plethora of cytokines and growth factors that regulate the immune functions besides decreasing the secretion of pro-inflammatory cytokines (Sotiropoulou et al. 2006; Zhou et al. 2018). Interestingly, Ad-MSCs are superior in immune-modulatory effects than BM-derived MSCs in a matched donor (Melief et al. 2013). This superiority can be attributed to Ad-MSCs’ ability to suppress lymphocyte proliferation, inhibit monocytes-derived dendritic cells, natural killer (NK) cytotoxic activities, and the secretion of higher levels of cytokines (Melief et al. 2013; Russell et al. 2016; Valencia et al. 2016). MSCs influence innate immunity and adaptive immunity through the two axes mentioned earlier, the cell-to-cell contact and the paracrine action. MSCs affect almost all the immune cells through cell-to-cell contact, including T-cells, B-cells, NK cells, macrophages, and DC (Zhou et al. 2019). It has been documented that MSCs inhibit naïve T-cells and memory T-cell responses to communicate with the antigen-presenting cells (Krampera et al. 2003) and inhibit the proliferation of CD4+ and CD8+ T-cells through galectin-1 (Gieseke et al. 2010). Besides their effects on T-cells, MSCs also increase the survival of quiescent B-cells through cell-to-cell contact (Franquesa et al. 2015). It is important to mention that infused MSCs, such as UC-MSCs, reside in the lungs, are phagocytosed by the monocytes causing changes in the monocyte’s phenotype and function and modulate the adaptive immune system responses

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(de Witte et al. 2018). Moreover, MSCs suppress the cytotoxic NK cells’ granular polarization (Hu et al. 2019). In this regard, Ad-MSCs are known to switch activated inflammatory M1 macrophages to an M2 macrophage-like phenotype via prostaglandin E2 (PGE2) release (Manferdini et al. 2017). Regarding the second axis of cytokine secretion via paracrine mechanisms, the MSCs secretome consists of many cytokines, chemokines, and growth factors, such as transforming growth factor-β1 (TGF-β1), PGE2, IFN-γ, hepatocyte growth factor (HGF), indoleamine-pyrrole 2,3-dioxygenase (IDO), tumor necrosis factor-α (TNF-α), fibroblast growth factor (FGF), and nitric oxide, besides many others (Salgado et al. 2010; Li and Hua 2017; Zhou et al. 2019). Interestingly, these bioactive molecules are encapsulated in extracellular vesicles, such as exosomes, micro-vesicles (MVs), or apoptotic bodies, depending on the type of cells of their origin (Ferreira et al. 2018). MSCs inhibit T-helper 17 (Th17) differentiation by IL-10, PGE2 induction, and IL-17, IL-22, and IFN-γ inhibition (Ghannam et al. 2010). Moreover, MSCs can also inhibit CD4+ T-cells activation via the secretion of programmed death ligand-1 (PD-L1) and PD-L-2 (Davies et al. 2017). MSCs can also inhibit IL-2 induced NK cells activation by the secretion of IDO and PGE2 (Spaggiari et al. 2008). Additionally, MSCs-derived IL-6 protects neutrophils from apoptosis and preserves them in the BM niche (Raffaghello et al. 2008). IL-6 can also prevent the differentiation of monocytes toward an anti-inflammatory IL-10-producing phenotype (Melief et al. 2013). Also, MSCs-derived PGE2 empower MSCs to suppress the differentiation of monocytes to mature DCs (Spaggiari et al. 2009). Even the conditioned media (CM) of MSCs containing both soluble and insoluble factors (MSCs-derived exosomes) shows immune-modulatory effects emphasizing the involvement of paracrine actions of the cells. MSC-derived exosomes have been shown to augment neutrophil viability. In contrast, MSCs-derived CM increases neutrophil function, demonstrating that both MSCs-derived exosomes and CM are helpful for improving immunity by modulating neutrophils (Mahmoudi et al. 2019). Additionally, MSCs-derived EVs can attenuate DC maturation, as well as pro-inflammatory cytokines (IL-6 and IL-12p70) and upregulation of the anti-inflammatory cytokine TGF-β (Reis et al. 2018). Besides, MSCs-derived exosomes trigger macrophage polarization (Lo Sicco et al. 2017) by enhancing the formation of anti-inflammatory M2 macrophages over M1-like inflammatory macrophages via downregulation of IL-23 and IL-22 (Hyvarinen et al. 2018).

Extracellular Vesicles/Exosomes Derived from MSCs Interestingly, a growing body of research has revealed that MSCs mediate several therapeutic/beneficial effects via secreting extracellular-vesicles (EVs) (Newton et al. 2017). Generally, the secreted membrane-enclosed vesicles, collectively called EVs, include exosomes, ectosomes, microvesicles, apoptotic bodies, and other EV subsets (Lötvall et al. 2014).

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Initially considered the “garbage bags for the disposal of cellular waste,” MSCsderived exosomes are now emerging as important mediators of intercellular communication. They have a specific cargo of DNA, mRNA, miRNA, proteins, growth factors, etc. (Haider and Aramini 2020). The content profile of exosomes is cell-type dependent and constantly fluctuates with many determinant factors, including the microenvironmental factors in which the cells are present at a particular time point. They have notably exhibited therapeutic potential for various diseases and are fast emerging as innovative tools in nanomedicine (Kassem and Kamal 2020a; NorouziBarough et al. 2021; Wei et al. 2021). Based on these characteristics, exosomes are defined as nano-sized bioactive vesicles derived from the cell’s endosomal membrane system and secreted into surrounding body fluids (Théry et al. 2018). Exosomes can reprogram the recipient cells acting as “signalosomes” for controlling fundamental cellular functions by transferring their cargo to the recipient cells (Gangoda et al. 2015; Bjorge et al. 2018). Recently, exosomes have sparked great interest as a potential cell-free therapy alternative to the current cellular therapies (Haider and Aslam 2018). MSCs-derived exosomes are gaining special attention among the different cell types due to their unique cargo and ease of manipulation (Janockova et al. 2021). Like their parent cells, exosomes stimulate functional recovery and cellular regeneration in various disease conditions (Derkus et al. 2017). However, unlike their parent cells, they have no critical safety concerns; they pose no risk of tumor formation or any concern regarding the immune rejection by the recipient, and they can pass through biological barriers, that is, the bloodbrain barrier, more efficiently. Moreover, since exosomes are naturally equipped to mediate intercellular communication via the transfer of genetic information to recipient cells, they can be utilized as drug delivery systems for gene therapy applications (Colao et al. 2018). MSCs from various tissue sources, Ad-MSCs, BM-derived MSCs, and UCB-derived MSCs, are efficient and established mass producers of exosomes (Yeo et al. 2013; Janockova et al. 2021). Furthermore, MSCs-derived exosomes have been suggested to act through the protein-based mechanism of action as well as micro-RNAs and/or packaged metabolites (Luther et al. 2018; Showalter et al. 2019; Toh et al. 2018). Conclusively, exosomes derived from MSCs provide a novel platform for a wide array of therapeutic strategies for various disease conditions and eventually may develop into a standardized allogeneic off-the-shelf immunomodulatory and regenerative therapeutics. After extensive evaluation for safety and efficacy studies in the preclinical experimental animal models, they have moved to clinical assessment and www.clinicaltrials.gov shows 226 registered clinical studies involving evaluation of exosome delivery for various diseases, including cardiovascular diseases, stroke, cancer, and COVID-19 pneumonia.

MSCs Act as an Efficient Tool for Gene Therapy Over the past few years, due to their homing capacity (Karp and Teo 2009) and immunomodulatory activities (Song et al. 2020), MSCs have been nominated as an

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efficient tool for gene therapy to treat several pathological conditions, lying at the intersection of cell and gene therapy. Several techniques have been used to generate genetically modified MSCs leading to the up-regulation or downregulation of native genes related to their regenerative potential or immunomodulatory functions. Alternatively, genetic engineering has been used to modulate them to serve as carriers of transgenes by introducing foreign genes of interest. The genetically modulated cells serve as tiny little factories that continue to secrete the product of the overexpressing genes that significantly contribute to the therapeutic effects of the genetically modulated MSCs (Varkouhi et al. 2020). Such a novel approach using genetically-modified MSCs as gene delivery vehicles widely expand the spectrum of the possible applications of MSCs in various diseases (Oggu et al. 2017). Interestingly, several cell-therapy pre-clinical studies employing genetically modified MSCs in various critical illness conditions such as acute myocardial infarction, acute liver failure, or even acute lung injury revealed promising results (Varkouhi et al. 2020). Furthermore, genetically engineered MSCs have been used as a vehicle to deliver biological agents to various tumors in preclinical cancer models. In these studies, MSCs have been engineered to express prodrug-converting enzymes, cytokines, pro-apoptotic factors, or antiangiogenic agents either single or multiple transgene overexpression to promote their therapeutic benefits after transplantation (Jiang et al. 2006; Mohr and Zwacka 2018; Mosallaei et al. 2020). Simultaneous overexpression of Akt and angiopoietin-1 has been shown to enhance their endothelial commitment (Lai et al. 2012). MSCs have also been used as carriers of microRNAs, either by genetic modulation to improve their survival and reparability post engraftment (Kim et al. 2012). Transgene overexpression in MSCs has also been used for their reprogramming to pluripotency (Buccini et al. 2012). Conclusively, MSCs indeed represent an efficient tool for gene-therapy applications in regenerative medicine.

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Considerations for Clinical Use of Mesenchymal Stromal Cells Abdelkrim Hmadcha, Bernat Soria, Juan R. Tejedo, Francico J. Bedoya, Jose Miguel Sempere-Ortells, and Tarik Smani

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypic Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs’ Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Migration Toward Damaged Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSC-Based Therapy for Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs for Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Hmadcha (*) Department of Biotechnology, University of Alicante, Alicante, Spain University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain e-mail: [email protected] B. Soria University of Pablo de Olavide, Sevilla, Spain Department of Physiology, Institute of Bioengineering-Alicante Institute for Health and Biomedical Research (ISABIAL), University Miguel Hernández School of Medicine, Alicante, Spain e-mail: [email protected] J. R. Tejedo · F. J. Bedoya Department of Molecular Biology and Biochemical Engineering, University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_3

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The Anti-tumor Activity of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Pro-tumor Activity of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Imprint of Disease on MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considerations for Clinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Manufacturing for Clinical Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considerations for Cellular Medicament . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attempts to Improve the Therapeutic Outcomes of Cellular Medicament . . . . . . . . . . . . . . . . . . Concluding Remarks and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The clinical application of stem cells continues to fascinate the scientific and clinical communities. Despite the controversies surrounding this field, it is clear that stem cells have revolutionized regenerative medicine. Cell therapy is a progressively growing field that is moving fast from preclinical model development to clinical application. In this regard, outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy that deals with unmet medical treatment for several disorders with no therapeutic alternatives. The application of stem cells in regenerative medicine is addressing a wide range of clinical conditions using various types of stem cells. Mesenchymal stromal cells (MSCs) have been established as promising candidate sources of universal donor cells for cell therapy due to their contributions to tissue and organ homeostasis, repair, and support by self-renewal and multi-differentiation, as well as by their anti-inflammatory, anti-proliferative, immunomodulatory, trophic, and proangiogenic properties. Various diseases have been successfully treated by MSCs in animal models. Additionally, hundreds of clinical trials related to the potential benefits of MSCs are in progress or have concluded satisfactorily. However, although all MSCs are considered suitable to exert these functions, dissimilarities have been found among MSCs derived from different tissues. The same levels of efficacy and desired outcomes have not always been achieved in the diverse studies that have been performed thus far. Therefore, collecting information regarding the characteristics of MSCs obtained from different sources and the influence of other medical and physiological conditions on MSCs is important for J. M. Sempere-Ortells Department of Biotechnology, Immunology Division, University of Alicante, Alicante, Spain e-mail: [email protected] T. Smani Cardiovascular Pathophysiology, Institute of Biomedicine of Seville (IBiS), University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain Department of Medical Physiology and Biophysics, University of Seville, Seville, Spain Biomedical Research Networking Centers of Cardiovascular Diseases (CIBERCV), Madrid, Spain e-mail: [email protected]

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assuring the feasibility, safety, and efficacy of cell-based therapies. This chapter will update and discuss the state of the art in MSCs’ cell-based therapies and provide relevant information regarding factors to consider for the clinical application of MSCs. Keywords

Advanced therapy · Cell therapy · Clinical trial · Good manufacturing practice · Immunomodulation · Inflammation · Medicinal products · Mesenchymal stromal cells · Trophic factors Abbreviations

Ad-MSCs AGEs ATMPs bFGF BM-MSCs CAFs Cas9 CCL CFU-F CRISPR CXCL12 Dkk-1 ECs EMA ESCs EVs FBS FDA GM-CSF GMP GPS GVHD GVL HCELL HGF HLA-DR HLA-G5 HSCT IBMIR ICAM-2 IDO IFN-γ IGF-1

Adipose tissue-derived MSCs Advanced glycation end products Advanced therapy medicinal products Basic fibroblast growth factor Bone marrow-derived MSCs Cancer-associated fibroblasts CRISPR-associated protein 9 Chemokine (C-C motif) ligand Colony-forming unit fibroblast Clustered regularly interspaced short palindromic repeats C-X-C motif chemokine 12 (or SDF1) Dickkopf-1 Endothelial cells European Medicines Agency Embryonic stem cells Extracellular vesicles Fetal bovine serum Food and Drug Administration Granulocyte-macrophage colony-stimulating factor Good manufacturing practice Glycotransferase-programmed stereo substitution Graft-versus-host diseases Graft-versus-leukemia Hematopoietic cell E-selectin/L-selectin ligand Hepatocyte growth factor Human leukocyte antigen-DR isotype Human leukocyte antigen-G5 Hematopoietic stem cell transplantation Instant blood-mediated inflammatory reaction Intercellular adhesion molecule 2 Indoleamine-2,3-dioxygenase Interferon-gamma Insulin-like growth factor 1

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IL IL-1α IL-1β iPSCs ISCT ITP LFA-3 MCP-1 MHC-HLA MMP-2 MSCs PAI-1 PDGF PD-MSCs PGE-2 RA SDF-1 SLE SSc STC1 TALENs TbRIII TGF-β TNF-α tPA TRAIL Trx1 TSG-6 UCB-MSCs VCAM-1 VEGF ZFNs

Interleukin Interleukin-1 alpha Interleukin-1 beta Induced pluripotent stem cells International Society for Cellular Therapy Immune thrombocytopenic purpura Lymphocyte function-associated antigen 3 (or CD58) Monocyte chemoattractant protein 1 Major histocompatibility complex-human leukocyte antigen Matrix metalloproteinase 2 Mesenchymal stromal cells Plasminogen activator inhibitor-1 Platelet-derived growth factor Placenta-derived MSCs Prostaglandin-E2 Rheumatoid arthritis Stromal cell-derived factor 1 Systemic lupus erythematosus Systemic sclerosis Stanniocalcin-1 Transcription activator nucleases Type III TGF-β receptor Transforming growth factor beta Tumor necrosis factor alpha Tissue plasminogen activator TNF-related apoptosis-inducing ligand Thioredoxin-1 Tumor necrosis factor-stimulated gene-6 Umbilical cord-derived MSCs Vascular cell adhesion protein 1 Vascular endothelial growth factor Zinc finger nucleases

Introduction Regenerative medicine is a novel emerging medical approach that drives the current understanding of biological and medical processes and suggests new treatments. As defined by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA), advanced therapies include cell and gene therapy and tissue engineering (Iglesias-López et al. 2019). Advanced therapies open up a broad set of translational fields and targets in areas of unmet medical need. In this regard, cellbased therapy through the application of cells, either alone or engineered, as a pharmacologically active substance seeks to restore the functioning of damaged

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tissues or organs through the protection of cellular integrity, the replacement of damaged cells, and the promotion of trophic, anti-inflammatory, and immunomodulatory effects among others. However, while the progression of cell-based therapy in early-phase clinical trials with patients has progressed promisingly, the translation from laboratory to bedside to late-phase clinical trials has not been as rapid as expected. It is necessary to consider that these new therapeutic alternatives also involve unknown side effects that must be detected and characterized in-depth to improve and ensure safety, feasibility, and efficacy of cell application (García-Bernal et al. 2021; Hmadcha et al. 2020; Soria-Juan et al. 2019; Escacena et al. 2015; Gálvez et al. 2013). In this regard, mesenchymal stromal cells (MSCs) are the most common cell type used in cell-based therapy due to their unique biological properties, including easy expansion and culture. The MSC-Committee of the International Society for Cellular Therapy (ISCT-MSC) first proposed that plastic-adherent cells of the bone marrow (BM) generally described as “mesenchymal stem cells” should be defined as “multipotent mesenchymal stromal cells.” In contrast, the term “mesenchymal stem cells” should be reticent for a subset of these cells that show stem cell activity by clearly stated criteria. As the acronym MSCs may be used to define both cell populations, the combined definition “mesenchymal stem/stromal cells” is probably more appropriate, especially when the “stemness” of the whole MSC population is not demonstrated (Horwitz et al. 2005). Recently, this committee offers a position statement to clarify the nomenclature of “mesenchymal stem/stromal cells.” The ISCT-MSC committee continues to support the use of the acronym “mesenchymal stromal cells” but recommends that this should be complemented by the tissue origin from which the cells were derived, which would highlight tissue-specific properties: that they be referred to as “stromal” unless there are rigorous in vitro and in vivo evidence of their stemness supplemented by a robust matrix of functional assays to demonstrate the “mesenchymal stromal cells” properties. Thus, they should not be defined generically, but based on the intended therapeutic mode of action (Viswanathan et al. 2019). The MSCs are now considered as “cellular medicament” but are widely accepted to represent a heterogeneous population of multipotent non-hematopoietic progenitor cells with varying degrees of stemness, which mean that they have self-renewal and multi-differentiation abilities, the capability to differentiate into multiple cell types, including adipocytes, chondrocytes, and osteoblasts, depending on in vitro culture conditions (Soria-Juan et al. 2019). The MSCs reside in almost all tissues, are found in virtually all post-natal organs and tissues, and are derived from the mesodermal germ layer. Furthermore, MSCs can be obtained from easily accessible sources by minimally invasive methods (e.g., peripheral blood, adipose tissue) and can be rapidly expanded in large scale for clinical use (Escacena et al. 2015). This allows producing a patient-specific cellular medicament (e.g., autologous medicinal product) within a therapeutic time window. In addition, the possibility of obtaining MSCs from adult tissue circumvents the ethical issues associated with the use of embryonic source (Lo and Parham 2009;

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Ramos-Zúñiga et al. 2012). MSCs are inexpensively isolated and are easily expanded in vitro due to their fibroblastic characteristic and high adherence to plastic. MSCs are characterized by a specific pattern of membrane markers, consisting of the expression of CD73, D90, and CD105 and the absence of expression of CD14, CD34, CD45, and human leukocyte antigen-DR (HLA-DR), making them promising candidate sources of donor cells for use in cell-based therapy and transplantation (Horwitz et al. 2005). MSCs function in tissue repair and support, contributing to tissue homeostasis. Even though the exact origin of MSCs remains elusive, there is strong evidence that MSC progenitors are found in the perivascular zone (Escacena et al. 2015) in an environment that promotes a quiescent state, ensuring the maintenance of homeostasis. Upon tissue damage, MSCs enter the bloodstream and are attracted to pro-inflammatory cytokines in the areas of injury. Therefore, MSCs have been termed “guardians of inflammation” (Prockop and Oh 2012). The cytoskeleton, extracellular matrix molecules, cell-cell contacts, adhesion ligands, and receptors are involved in the repair process. While the exact mechanisms related to MSCs’ migration to specific sites and through the endothelial cell layer are still unknown, chemokines and their receptors may play a role in this process (Hmadcha et al. 2020; Petrie et al. 2009). Furthermore, MSCs’ survival, permanent engraftment, and differentiation into resident cells were thought, initially, to be necessary to obtain the beneficial effects of these cells, and clinical experience and several experiments have shown that one of the primary functions of MSCs, most likely their critical function, is to secrete several bioactive molecules related to the microenvironment “niche” in which these cells are located. Consequently, the secretome reproduces most of the effects of MSCs transiently; in this sense, MSCs secrete a wide variety of pro-inflammatory and anti-inflammatory cytokines, chemokines, growth factors, and prostaglandins under resting and inflammatory conditions (Hmadcha et al. 2009). These molecules are associated with immunomodulation (indoleamine-2,3dioxygenase (IDO), prostaglandin-E2 (PGE-2), transforming growth factor beta (TGF-β), human leukocyte antigen-G5 (HLA-G5), and hepatocyte growth factor (HGF)), anti-apoptosis (vascular endothelial growth factor (VEGF), granulocytemacrophage colony-stimulating factor (GM-CSF), TGF-β, stanniocalcin-1 (STC1), and insulin-like growth factor 1 (IGF-1)), angiogenesis (VEGF, monocyte chemoattractant protein 1 (MCP-1), and IGF-1), local stem and progenitor cell growth and differentiation support (CSF complex, angiopoietin-1, and stromal cell-derived factor 1 (SDF-1)), anti-fibrosis (HGF and basic fibroblast growth factor (bFGF)), and chemoattraction (chemokine (C-C motif) ligands 2 and 4 (CCL2, CCL4) and C-X-C motif chemokine 12 (CXCL12 also called SDF1)) (Meirelles Lda et al. 2009). MSCs display a low expression of major histocompatibility complex class I human leukocyte antigen (MHC-HLA class I), while they are constitutively negative for HLA-class II; likewise, they do not express costimulatory molecules such as CD80, CD86, CD40, and CD40L. However, MSCs share the expression of surface markers, such as vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 2 (ICAM-2), and lymphocyte function-associated antigen 3 (LFA-3 or CD58) with the thymic epithelium, which is crucial for the interaction with T cells

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(Hmadcha et al. 2009; Le Blanc 2003). Whereas MSCs remain in a quiescent state showing anti-apoptotic properties and contributing to homeostasis, in an inflammatory environment (presence of IFN-γ, TNF-α, IL-1α, and IL-1β), they begin to exercise their immunomodulation abilities, inhibiting the proliferation of effector cells and their cytokine production. In the same way, MSCs can block various immune cell functions (Hmadcha et al. 2009; Cagliani et al. 2017). There is a complex “cross-talk” interaction between MSCs and endothelial cells (ECs). MSCs increase the proliferation and migration of the ECs, promoting early events of angiogenesis and decreasing the permeability of the monolayer of the ECs. In direct co-cultures of MSCs and ECs, MSCs increased the persistence of pre-existing blood vessels in a dose-dependent manner (Duffy et al. 2009). Moreover, beneficial therapeutic effects of the use of conditioned media of MSCs have been reported; even it is therapeutically better than the cells themselves (Burlacu et al. 2013; Shrestha et al. 2013) and to stimulate the proliferation of local ECs (Potapova et al. 2007). Likewise, in addition to direct “cell-cell” contact, speculation has been made with a possible transfer of mitochondria or vesicular components (secretome) that contain mRNA, microRNA, and proteins (Tan et al. 2021). Not only have this, the exosomes, secretory extracellular vesicles (EVs) from MSCs, also been identified to produce the same immunomodulatory activity as MSCs (Haider and Aramini 2020). Targeting the MSCs’ secretome as an acellular therapeutic agent could provide several advantages over the use of cell-based therapies for various diseases paving the way for cell-free therapy (Haider and Aslam 2018; Bari et al. 2019). Altogether, these features constitute an area of research in expansion in the last decade and make MSCs an eligible therapeutic candidate to be evaluated within clinical trials for a plethora of diseases such as diabetes and diabetes complication and cardiovascular and neurological diseases; in immune-mediated disorders, such as graft-versus-host diseases (GVHD), multiple sclerosis (MS), Crohn’s disease (CD), and osteoarthritis (OA); and even in immune-dysregulating infectious diseases such as the novel coronavirus disease 2019 (COVID-19) (see ▶ Chap. 6, “Mesenchymal Stromal Cells for COVID-19 Critical Care Patients,” of this book for review on COVID-19). When writing this chapter (May 2021), 1.276  103 publicly and privately funded clinical studies worldwide in which MSCs have been used have been reported and registered in the US National Library of Medicine database (NIH-ClinicalTrials.gov). Although the therapeutic efficacy of MSCs has been demonstrated in different disease animal models and numerous human phase 1/2 clinical trials and generally communicated, only very few (84 studies) phase 3/4 clinical trials using MSCs are registered (Table 1) and have demonstrated the expected potential therapeutic benefit. Almost all registered clinical trials are early phase 1/2 with safety as the primary objective. For efficacy and effectiveness issues, other advanced phases are mandatory. In all cases, one cannot consider these issues (efficacy nor effectiveness) unless phase 3 clinical trials are developed (García-Bernal et al. 2021) (Fig. 1). Even though MSCs and their EVs have been shown to have high potential benefits in regenerative medicine and cell-free-based therapy, their clinical application remains controversial; thus, considerations and determination of possible side

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Table 1 Public registry list of phase 3/4 clinical trials using MSCs as cell-based therapy (NIHClinicalTrial.gov) NCT number NCT03106662

NCT04351932

NCT02755922

NCT04224207

NCT01854125

NCT02218437

NCT01716481

NCT00366145

NCT03766217

NCT04689152

NCT01676441

Title Mesenchymal Stem Cell Infusion in Haploidentical Hematopoietic Stem Cell Transplantation in Patients with Hematological Malignancies Bone Marrow Versus Adipose Autologous Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis Bone Regeneration with Mesenchymal Stem Cells

Status Completed

Phases Phase 3

URL https:// ClinicalTrials. gov/show/ NCT03106662

Not yet recruiting

Phase 3

Completed

Phase 3

Management of Retinitis Pigmentosa by Mesenchymal Stem Cells by Wharton’s Jelly Derived Mesenchymal Stem Cells Autologous Mesenchymal Stem Cell Transplantation in Cirrhosis Patients with Refractory Ascites

Completed

Phase 3

https:// ClinicalTrials. gov/show/ NCT04351932 https:// ClinicalTrials. gov/show/ NCT02755922 https:// ClinicalTrials. gov/show/ NCT04224207

Unknown status

Phase 3

Treatment Protocol of Child SAA with the Injection of Mesenchymal Stem Cells (Umbilical Cord Derived) The STem Cell Application Researches and Trials In NeuroloGy-2 (STARTING-2) Study Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease Bone Tissue Engineering with Dental Pulp Stem Cells for Alveolar Cleft Repair

Unknown status

Phase 4

Unknown status

Phase 3

Completed

Phase 3

Completed

Phase 3

Clinical Trial to Evaluate the Efficacy and Safety of CellgramLC Administration in Patients with Alcoholic Cirrhosis Safety and Efficacy of Autologous Mesenchymal Stem Cells in Chronic Spinal Cord Injury

Not yet recruiting

Phase 3

Terminated

Phase 2| Phase 3

https:// ClinicalTrials. gov/show/ NCT01854125 https:// ClinicalTrials. gov/show/ NCT02218437 https:// ClinicalTrials. gov/show/ NCT01716481 https:// ClinicalTrials. gov/show/ NCT00366145 https:// ClinicalTrials. gov/show/ NCT03766217 https:// ClinicalTrials. gov/show/ NCT04689152 https:// ClinicalTrials. gov/show/ NCT01676441 (continued)

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Table 1 (continued) NCT number NCT01157403

NCT03325504

NCT01652209

NCT01873625

NCT01392105

NCT00543374

NCT01394432

NCT02442817

NCT00482092

NCT01526850

NCT04421274

Title Autologous Transplantation of Mesenchymal Stem Cells for Treatment of Patients with Onset of Type 1 Diabetes A Comparative Study of 2 Doses of BM Autologous H-MSC +Biomaterial vs Iliac Crest AutoGraft for Bone Healing in Non-Union To Evaluate the Efficacy and Safety of Hearticelgram ®-AMI in Patients with Acute Myocardial Infarction Transplantation of Bone Marrow Derived Mesenchymal Stem Cells in Affected Knee Osteoarthritis by Rheumatoid Arthritis Safety and Efficacy of Intracoronary Adult Human Mesenchymal Stem Cells After Acute Myocardial Infarction Extended Evaluation of PROCHYMAL ® Adult Human Stem Cells for TreatmentResistant Moderate-to-Severe Crohn’s Disease ESTIMATION Study for Endocardial Mesenchymal Stem Cells Implantation in Patients After Acute Myocardial Infarction Linagliptin and Mesenchymal Stem Cells: A Pilot Study

Status Unknown status

Phases Phase 2| Phase 3

URL https:// ClinicalTrials. gov/show/ NCT01157403 https:// ClinicalTrials. gov/show/ NCT03325504

Recruiting

Phase 3

Recruiting

Phase 3

Completed

Phase 2| Phase 3

Completed

Phase 2| Phase 3

Completed

Phase 3

Unknown status

Phase 3

https:// ClinicalTrials. gov/show/ NCT01394432

Completed

Phase 4

Evaluation of PROCHYMAL ® Adult Human Stem Cells for Treatment-Resistant Moderateto-Severe Crohn’s Disease Efficacy and Safety Study of Allogenic Mesenchymal Stem Cells for Patients with Chronic Graft Versus Host Disease Bone Marrow Mesenchymal Stem Cells Transfer in Patients with ST-Segment Elevation Myocardial Infarction

Completed

Phase 3

Unknown status

Phase 2| Phase 3

Completed

Phase 2| Phase 3

https:// ClinicalTrials. gov/show/ NCT02442817 https:// ClinicalTrials. gov/show/ NCT00482092 https:// ClinicalTrials. gov/show/ NCT01526850 https:// ClinicalTrials. gov/show/ NCT04421274

https:// ClinicalTrials. gov/show/ NCT01652209 https:// ClinicalTrials. gov/show/ NCT01873625 https:// ClinicalTrials. gov/show/ NCT01392105 https:// ClinicalTrials. gov/show/ NCT00543374

(continued)

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Table 1 (continued) NCT number NCT03818737

Title Multicenter Trial of Stem Cell Therapy for Osteoarthritis (MILES)

Status Active, not recruiting

Phases Phase 3

NCT02223897

Mesenchymal Stem Cells Transplantation for IschemicType Biliary Lesions

Unknown status

Phase 2| Phase 3

NCT00891501

The Use of Autologous Bone Marrow Mesenchymal Stem Cells in the Treatment of Articular Cartilage Defects Different Efficacy Between Rehabilitation Therapy and Stem Cells Transplantation in Patients with SCI in China Parkinson’s Disease Therapy Using Cell Technology

Unknown status

Phase 2| Phase 3

Completed

Phase 3

Recruiting

Phase 2| Phase 3

Combination of Autologous MSC and HSC Infusion in Patients with Decompensated Cirrhosis Efficacy in Alveolar Bone Regeneration with Autologous MSCs and Biomaterial in Comparison to Autologous Bone Grafting Stem Cell Therapy for Treatment of Female Stress Urinary Incontinence

Completed

Phase 4

Recruiting

Phase 3

Completed

Phase 3

Left Ventricular Assist Device Combined with Allogeneic Mesenchymal Stem Cells Implantation in Patients with End-Stage Heart Failure Therapy of Toxic Optic Neuropathy via Combination of Stem Cells with Electromagnetic Stimulation Mesenchymal Stem Cell Therapy for SARS-CoV-2Related Acute Respiratory Distress Syndrome A Phase 3 Study to Evaluate the Efficacy and Safety of JointStem in Treatment of Osteoarthritis

Active, not recruiting

Phase 2| Phase 3

Completed

Phase 3

Recruiting

Phase 2| Phase 3

Completed

Phase 3

NCT01873547

NCT04146519

NCT04243681

NCT04297813

NCT02334878

NCT01759212

NCT04877067

NCT04366063

NCT03990805

URL https:// ClinicalTrials. gov/show/ NCT03818737 https:// ClinicalTrials. gov/show/ NCT02223897 https:// ClinicalTrials. gov/show/ NCT00891501 https:// ClinicalTrials. gov/show/ NCT01873547 https:// ClinicalTrials. gov/show/ NCT04146519 https:// ClinicalTrials. gov/show/ NCT04243681 https:// ClinicalTrials. gov/show/ NCT04297813 https:// ClinicalTrials. gov/show/ NCT02334878 https:// ClinicalTrials. gov/show/ NCT01759212 https:// ClinicalTrials. gov/show/ NCT04877067 https:// ClinicalTrials. gov/show/ NCT04366063 https:// ClinicalTrials. gov/show/ NCT03990805 (continued)

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Table 1 (continued) NCT number NCT02672267

Title A Study of Allogeneic Low Oxygen Mesenchymal Bone Marrow Cells in Subjects with Myocardial Infarction Using Mesenchymal Stem Cells to Fill Bone Void Defects in Patients with Benign Bone Lesions Clinical Trial to Evaluate the Efficacy and Safety of Stem Cells

Status Completed

Phases Phase 3

Withdrawn

Phase 2| Phase 3

Completed

Phase 3

Clinical Study to Evaluate Efficacy and Safety of ASC and Fibringlue or Fibringlue in Patients with Crohn’s Fistula MSCs Combined with CD25 Monoclonal Antibody and Calcineurin Inhibitors for Treatment of Steroid-Resistant aGVHD Follow-Up Study for Participants of JointStem Phase 3 Clinical Trial

Recruiting

Phase 3

Unknown status

Phase 2| Phase 3

Enrolling by invitation

Phase 3

NCT03633565

Comparative Study of Strategies for Management of Duchenne Myopathy (DM)

Not yet recruiting

Phase 4

NCT03389919

Intraosseous Administration of Mesenchymal Stromal Cells for Patients with Graft Failure After Allo-HSCT A Phase 2b/3a Study to Evaluate the Efficacy and Safety of JointStem in Patients Diagnosed as Knee Osteoarthritis Clinical Study to Evaluate Efficacy and Safety of ALLOASC-DFU in Patients with Diabetic Foot Ulcers MSC for Severe aGVHD

Recruiting

Phase 3

Not yet recruiting

Phase 2| Phase 3

Active, not recruiting

Phase 3

Recruiting

Phase 2| Phase 3

Safety and Efficacy of Repeated Administrations of NurOwn ® in ALS Patients

Completed

Phase 3

NCT00851162

NCT01803347

NCT04612465

NCT02241018

NCT04427930

NCT04368806

NCT03370874

NCT03631589

NCT03280056

URL https:// ClinicalTrials. gov/show/ NCT02672267 https:// ClinicalTrials. gov/show/ NCT00851162 https:// ClinicalTrials. gov/show/ NCT01803347 https:// ClinicalTrials. gov/show/ NCT04612465 https:// ClinicalTrials. gov/show/ NCT02241018 https:// ClinicalTrials. gov/show/ NCT04427930 https:// ClinicalTrials. gov/show/ NCT03633565 https:// ClinicalTrials. gov/show/ NCT03389919 https:// ClinicalTrials. gov/show/ NCT04368806 https:// ClinicalTrials. gov/show/ NCT03370874 https:// ClinicalTrials. gov/show/ NCT03631589 https:// ClinicalTrials. gov/show/ NCT03280056 (continued)

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Table 1 (continued) NCT number NCT04738981

NCT02809781

NCT01041001

NCT02240992

NCT01541579

NCT03905824

NCT01233960

NCT02291770

NCT01929434

NCT01759823

NCT01626677

Title Efficacy and Safety of UC-MSCs for the Treatment of Steroid-Resistant aGVHD Following Allo-HSCT A Pilot Study of MSCs Iufusion and Etanercept to Treat Ankylosing Spondylitis

Status Not yet recruiting

Phases Phase 3

Unknown status

Phase 2| Phase 3

Study to Compare Efficacy and Safety of Cartistem and Microfracture in Patients with Knee Articular Cartilage Injury MSCs With or Without Peripheral Blood Stem Cell for Treatment of Poor Graft Function and Delayed Platelet Engraftment Adipose Derived Mesenchymal Stem Cells for Induction of Remission in Perianal Fistulizing Crohn’s Disease The Effectiveness of Adding Allogenic Stem Cells After Traditional Treatment of Osteochondral Lesions of the Talus Evaluation of PROCHYMAL ® for Treatment-Refractory Moderate-to-Severe Crohn’s Disease Treatment of Chronic GraftVersus-Host Disease with Mesenchymal Stromal Cells

Completed

Phase 3

Unknown status

Phase 2| Phase 3

Completed

Phase 3

Recruiting

Phase 3

Completed

Phase 3

Unknown status

Phase 3

Efficacy of Stem Cell Transplantation Compared to Rehabilitation Treatment of Patients with Cerebral Paralysis Bone Marrow Derived Stem Cell Transplantation in T2DM

Completed

Phase 3

Completed

Phase 2| Phase 3

Follow-Up Study of CARTISTEM ® Versus Microfracture for the Treatment of Knee Articular Cartilage Injury or Defect

Completed

Phase 3

URL https:// ClinicalTrials. gov/show/ NCT04738981 https:// ClinicalTrials. gov/show/ NCT02809781 https:// ClinicalTrials. gov/show/ NCT01041001 https:// ClinicalTrials. gov/show/ NCT02240992 https:// ClinicalTrials. gov/show/ NCT01541579 https:// ClinicalTrials. gov/show/ NCT03905824 https:// ClinicalTrials. gov/show/ NCT01233960 https:// ClinicalTrials. gov/show/ NCT02291770 https:// ClinicalTrials. gov/show/ NCT01929434 https:// ClinicalTrials. gov/show/ NCT01759823 https:// ClinicalTrials. gov/show/ NCT01626677 (continued)

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Table 1 (continued) NCT number NCT03404063

Title Cardiovascular Clinical Project to Evaluate the Regenerative Capacity of CardioCell in Patients with Acute Myocardial Infarction (AMI) Clinical Extension Study for Safety and Efficacy Evaluation of Cellavita-HD Administration in Huntington’s Patients Cell Therapy Associated with Endobronchial Valve

Status Completed

Phases Phase 2| Phase 3

URL https:// ClinicalTrials. gov/show/ NCT03404063

Not yet recruiting

Phase 2| Phase 3

Not yet recruiting

Phase 2| Phase 3

NCT04230902

Effects of α MAT Versus Steroid Injection in Knee Osteoarthritis (STα MAT-Knee Study)

Recruiting

Phase 3

NCT03418233

Randomized Clinical Trial to Evaluate the Regenerative Capacity of CardioCell in Patients with Chronic Ischaemic Heart Failure (CIHF) Cardiovascular Clinical Project to Evaluate the Regenerative Capacity of CardioCell in Patients with No-option Critical Limb Ischemia (N-O CLI) Study for the Treatment of the Bone Marrow Edema:Core Decompression VS Bone Marrow Concentrate (BMC) VS Bone Substitute Effect of Microvesicles and Exosomes Therapy on β-Cell Mass in Type I Diabetes Mellitus (T1DM) Allogeneic Mesenchymal Stromal Cells for Angiogenesis and Neovascularization in No-option Ischemic Limbs Co-transplantation of MSC in the Setting of Allo-HSCT

Completed

Phase 2| Phase 3

https:// ClinicalTrials. gov/show/ NCT04219241 https:// ClinicalTrials. gov/show/ NCT04018729 https:// ClinicalTrials. gov/show/ NCT04230902 https:// ClinicalTrials. gov/show/ NCT03418233

Active, not recruiting

Phase 2| Phase 3

https:// ClinicalTrials. gov/show/ NCT03423732

Terminated

Phase 4

https:// ClinicalTrials. gov/show/ NCT03112122

Unknown status

Phase 2| Phase 3

Not yet recruiting

Phase 2| Phase 3

Recruiting

Phase 2| Phase 3

Evaluation the Efficacy and Safety of Mutiple Lenzumestrocel (Neuronata-R ® Inj.) Treatment in patients with ALS

Recruiting

Phase 3

https:// ClinicalTrials. gov/show/ NCT02138331 https:// ClinicalTrials. gov/show/ NCT03042572 https:// ClinicalTrials. gov/show/ NCT04247945 https:// ClinicalTrials. gov/show/ NCT04745299

NCT04219241

NCT04018729

NCT03423732

NCT03112122

NCT02138331

NCT03042572

NCT04247945

NCT04745299

(continued)

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Table 1 (continued) NCT number NCT04126603

Title Impact of Semaglutide on CD34 + EPC and Fat Derived MSC

Status Recruiting

Phases Phase 3

NCT04371393

MSCs in COVID-19 ARDS

Active, not recruiting

Phase 3

NCT04310215

Efficacy and Safety of Allogenic Stem Cell Product (CARTISTEM ®) for Osteochondral Lesion of Talus ViviGen Cellular Bone Matrix for Hindfoot or Ankle Arthrodesis

Enrolling by invitation

Phase 3

Enrolling by invitation

Phase 4

Evaluation of Soft Tissue Profile Changes Following Autogenous Fat or Onlay Polyetheretherketone (PEEK) Augmentation Versus Sliding Genioplasty for Correction of Deficient Chin Clinical Study to Evaluate Efficacy and Safety of ALLOASC-DFU in Patients with Diabetic Wagner Grade 2 Foot Ulcers The Effect of Platelet-Rich Plasma in Patients with Osteoarthritis of the Knee

Unknown status

Phase 3

Recruiting

Phase 3

https:// ClinicalTrials. gov/show/ NCT04569409

Completed

Phase 3

NCT04864509

The Effects of Melatonin Treatment on Bone, Marrow, Sleep and Blood Pressure

Not yet recruiting

Phase 4

NCT02448849

Autologous BM-MSC Transplantation in Combination with Platelet Lysate (PL) for Nonunion Treatment Nintedanib for the Treatment of SARS-Cov-2 Induced Pulmonary Fibrosis

Unknown status

Phase 2| Phase 3

Recruiting

Phase 3

Study on Autologous Osteoblastic Cells Implantation to Early Stage Osteonecrosis of the Femoral Head

Terminated

Phase 3

https:// ClinicalTrials. gov/show/ NCT01926327 https:// ClinicalTrials. gov/show/ NCT04864509 https:// ClinicalTrials. gov/show/ NCT02448849 https:// ClinicalTrials. gov/show/ NCT04541680 https:// ClinicalTrials. gov/show/ NCT01529008

NCT04138017

NCT03747822

NCT04569409

NCT01926327

NCT04541680

NCT01529008

URL https:// ClinicalTrials. gov/show/ NCT04126603 https:// ClinicalTrials. gov/show/ NCT04371393 https:// ClinicalTrials. gov/show/ NCT04310215 https:// ClinicalTrials. gov/show/ NCT04138017 https:// ClinicalTrials. gov/show/ NCT03747822

(continued)

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Table 1 (continued) NCT number NCT00562497

NCT02849613

NCT04629833

NCT02336230

NCT02032004

Title Efficacy and Safety of Prochymal™ Infusion in Combination with Corticosteroids for the Treatment of Newly Diagnosed Acute Graft Versus Host Disease (GVHD) Regenerative Stem Cell Therapy for Stroke in Europe

Status Completed

Phases Phase 3

URL https:// ClinicalTrials. gov/show/ NCT00562497

Withdrawn

Phase 2| Phase 3

Treatment of Steroid-Refractory Acute Graft-Versus-Host Disease with Mesenchymal Stromal Cells Versus Best Available Therapy A Prospective Study of Remestemcel-L, Ex-Vivo Cultured Adult Human Mesenchymal Stromal Cells, for the Treatment of Pediatric Patients Who Have Failed to Respond to Steroid Treatment for Acute GVHD Efficacy and Safety of Allogeneic Mesenchymal Precursor Cells (Rexlemestrocel-L) for the Treatment of Heart Failure

Not yet recruiting

Phase 3

https:// ClinicalTrials. gov/show/ NCT02849613 https:// ClinicalTrials. gov/show/ NCT04629833

Completed

Phase 3

https:// ClinicalTrials. gov/show/ NCT02336230

Completed

Phase 3

https:// ClinicalTrials. gov/show/ NCT02032004

effects need to be addressed to optimize the clinical application of this double-edged sword cellular medicament. This chapter updates and discusses the state of the art in MSCs’ cell-based therapies and provides relevant information regarding factors to consider for the clinical application of MSCs.

Biological Characteristics Phenotypic Profile Since Friedenstein and colleagues first isolated a colony-forming unit fibroblast (CFU-F) from BM, BM has been widely used as a source of MSCs for many investigations and clinical trials. In addition to BM (BM-derived MSCs), MSCs have been isolated from different tissues, such as adipose tissue (Ad-MSCs), umbilical cord (UCB-MSCs), dental pulp, synovial liquid, and amniotic fluid. All these tissues vary in their cellular components, signals, and factors secreted, resulting in different immediate microenvironment conditions, thus developing

Amniotic fluid

Muscle

Bone marrow

Blood

Teeth

Do not express CD45, CD34, CD14 or CD11b, CD79or CD19 and HLA-DR

Do express CD73, CD90, CD105

Rapidly expanded

Fast growth

Plastic adherence

MSCs

Large-scale production

Myocyte

Neuron

Osteocyte

Chondrocyte

Adipocyte

Multilineage differentiation

Treg T cell B cell NK

Angiogenic

VEGF, TGFb, bFGF, IL6, IGF1, MCP1, EPO, CXCL12, MIP

Anti-fibrotic

KGF, bFGF, TF, TIMP, HGF, MMP

Anti-apoptotic

TGFb, VEGF, IGF1, bFGF, GM-CSF,HGF, TRAIL, STC1, SFRP2

Anti-inflammatory

IL4 TGFb iNOS IDO IFNg IL12 TNFa IL10

Inmmunomodulatory

MN iDC

MSC

Pleiotropic effects

Skin problem

Diabetes

Cardiac ischemia

Liver disorder

Neurological damage

Clinical application

Fig. 1 The MSCs have multiple potential advantages for their clinical application. Among other advantages, MSCs can be isolated from various sources, are produced on a large scale, differentiate into various cell types, and have pleiotropic effects. Such advantages make MSCs suitable for clinical application in different disease conditions, such as neurological damage, liver disorders, cardiac ischemia, diabetes, or skin problems. (Re-published from Hmadcha 2020,

Placenta

Umbilical cord

Synovia

Adipose tissue

Liver

Hair follicles

Multiple sources

66 A. Hmadcha et al.

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several physiological niches (Hmadcha et al. 2009; Escacena et al. 2015). Although isolated and long-term cultured MSCs of most tissues show similar immunophenotypic characteristics, some differences have been found among MSCs of different tissue origins according to data obtained by in vitro experiments. In 2006, the International Society for Cellular Therapy (ISCT) published the minimal criteria to define MSCs by nomenclature and by biological characteristics to allow studies from different groups to be compared and contrasted. These criteria include the co-expression of markers such as CD73, CD90, and CD105, a lack of expression of hematopoietic markers (CD45, CD34, and CD14) and HLA-DR, multipotent differentiation potential, and adherence to plastic (Horwitz et al. 2005). However, several researchers have noted that Ad-MSCs express CD34 and CD54 in early passages and have lower expression of CD106 and that umbilical cord blood-derived MSCs (UCB-MSCs) express CD90 and CD105. Other markers have been used in different studies, and other differences have emerged, such as VEGFR-2 (Flk-1) expression, which was significantly higher in periosteum-derived cells than in adipose tissue- and muscle-derived cells, or the rate of NGFR positivity, which was much higher in muscle-derived cells than in other mesenchymal tissue-derived cells (Escacena et al. 2015). Although some immunophenotypic differences have been documented, many researchers consider that these differences could be due to distinct extraction methods and different culture methodologies, resulting in variations of MSC surface markers. Therefore, this chapter aimed to investigate markers and characteristics that are more specific to select better sources of MSCs for clinical applications. Likewise, expanding the cells in vitro is necessary to obtain the desired numbers for therapeutic approaches. Changes in the proteomic phenotype of MSCs have been observed during high passages, although no proper approaches to examine the state of cells continuously during long-term in vitro culture have been established (Capra et al. 2012). Some researchers ascribe these variations to the adaptation of cells to the environment; thus, determining the biomolecular markers that are involved in these variations is essential for obtaining a better phenotypic characterization of these cells and thus for achieving more effective cell therapy in the future (Escacena et al. 2015).

ä Fig. 1 (continued) article published under CC-BY terms). Abbreviations: bFGF basic fibroblast growth factor, CXCL12 C-X-C motif chemokine 12, EPO erythropoietin, GM-CSF granulocytemacrophage colony-stimulating factor, HGF hepatocyte growth factor, HLA-DR major histocompatibility complex class II DR, iDC immature dendritic cell, IDO indoleamine-2,3-dioxygenase, IGF1 insulin-like growth factor 1, IL-10 interleukin-10, IL-12 interleukin-12, IL-4 interleukin-4, IL-6 interleukin-6, INF-γ interferon-γ, iNOS inducible nitric oxide synthase, KGF keratinocyte growth factor, MCP1 monocyte chemoattractant protein 1, MIP macrophage inflammatory protein, MMP matrix metalloproteinases, MN monocyte, NK natural killer cell, SFRP2 secreted frizzledrelated protein 2, STC1 stanniocalcin 1, TF tissue factor, TGF-β transforming growth factor beta, TIMP tissue inhibitor of metalloproteinases, TNF-α tumor necrosis factor α, TRAIL TNF-related apoptosis-inducing ligand, Treg regulatory T cell, VEGF vascular endothelial growth factor

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MSCs’ Proliferation The proliferative activity of MSCs is another feature that may be affected by the different origins of MSCs. The rate and persistence of MSC proliferation appear to vary between source tissues. MSCs are considered adult stem cells, and unlike embryonic stem cells (ESCs), these cells have a limited proliferative capacity. Physiological niches maintain adult stem cells in an undifferentiated state; however, when MSCs are cultured in vitro, they age, which affects their therapeutic properties, such as alterations in phenotype, differentiation potential, global gene expression patterns, miRNA profiles, and even chromosomal abnormalities, particularly after long-term culture or when cells of multiple doublings are used (Escacena et al. 2015). Large numbers of MSCs are needed for therapeutic applications, and in vitro expansion is required to produce the desired MSC numbers. In vivo, MSCs represent 0.0001% of nucleated BM cells, and their number decreases with the donor’s age. The quantity of MSCs (CFU-Fs) among nucleated BM cells decreases with age from one MSC in 104 BM cells in newborn to one MSC in 105 cells in teenagers and one MSC in 106 cells in older individuals (Caplan 2009). Furthermore, MSCs from older human donors differ significantly from younger donors in morphology, replicative lifespan, doubling time, healing capacity, and differentiation potential. Sufficient evidence has indicated that MSCs from older donors have limited therapeutic efficacy. Some studies have suggested that the difference between preclinical and clinical findings is due to the donor age (Stenderup et al. 2003; Escacena et al. 2015). Therefore, considering that several age-related diseases exist and that elderly patients are potential users of cell therapy, understanding the molecular and biological effects of aging on MSCs is essential for developing safe and effective MSC-based autologous cell therapy. Meanwhile, the use of allogeneic MSCs may be a treatment option for these specific patients. As commented below, MSCs elude allogeneic rejection, and their infusion is feasible and well-tolerated, with no adverse effects (McAuley et al. 2014; Liang et al. 2010).

Differentiation Capacity MSCs can differentiate in vitro into several mesenchymal lineages, including adipose tissue, bone, cartilage, and muscle (Pittenger et al. 1999; Prockop 1997; Bruder et al. 1997). Furthermore, MSCs can differentiate into ECs, neurons, and glial cells because MSCs express genes related to specific lineages rather than those of the mesenchymal lineage (Woodbury et al. 2002). Although multilineage differentiation is another minimal criterion advised by the ISCT and undoubtedly represents a fundamental property of MSCs, this ability depends primarily on the source tissue from which these cells are derived. As such, Sakaguchi and colleagues (Sakaguchi et al. 2005) compared human MSCs isolated from BM, synovium, periosteum, skeletal muscle, and adipose tissue. The cells were expanded by similar processes; synovium-derived cells had the most remarkable ability for chondrogenesis; adipose- and synovium-derived cells, for adipogenesis; and BM-, synovium-, and

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periosteum-derived cells, for osteogenesis. In another comparative analysis, UCB-MSCs showed no adipogenic differentiation capacity compared to BM- and Ad-MSCs (Kern et al. 2006). As discussed by Horwitz (Horwitz et al. 2002), who used differentiated MSCs in a study to test the regeneration of damaged tissues, BM-derived MSCs can engraft after transplantation, differentiate to functional osteoblasts, and contribute to the formation of new dense bone in children with osteogenesis imperfecta. Most likely, the microenvironment in which MSCs are transplanted directly influences in their distinct differentiation pathways. New insights into the biological characteristics of MSCs are needed to achieve future therapies.

Cellular Transformation In general, successive passages or long-term cultures induce genetic instability and cell transformation. Several authors have described that MSCs cultivated in vitro can be expanded multiple times without an apparent loss of differentiation potential or chromosomal alterations and even that long-term MSC cultures can develop chromosomal abnormalities but without an obvious potential for transformation (Koç et al. 2000; Le Blanc et al. 2004; Ringdén et al. 2006; Fang et al. 2006; Ning et al. 2008). Although no tumor formation in humans has been reported after the administration of MSCs, several factors must be considered that can contribute significantly to the induction of cytogenetic abnormalities, such as aspects related to the manufacturing process of the cellular medicine (e.g., culture conditions and duration of cell expansion) and heterogeneity of the MSC population (e.g., cells in different stages of duplication). The tumorigenic potential of a cell therapy medicament may depend on intrinsic and extrinsic factors, such as the administration site in the patient (due to the receptor’s microenvironment) and/or the manipulation of the culture ex vivo.

Mechanism of Action Cell Migration Toward Damaged Tissues The success of an advanced therapy medicinal product initially depends on its ability to reach target tissues. MSCs possess inherent tropism toward damaged sites controlled by many factors and mechanisms, including chemoattractant signals. For instance, the C-X-C motif chemokine ligand 12 (CXCL12) is a frequent triggering factor at the injury site. It has been demonstrated that a subpopulation of MSCs expresses the C-X-C chemokine receptor type 4 (CXCR4) that binds to its ligand, the CXCL12, to mediate cell migration (Wynn et al. 2004; Ma et al. 2015). Aside from CXCR4, MSCs express other chemokine receptors, such as CCR1, CCR2, CCR4, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CX3CR1 (Sordi et al. 2005; Von Lüttichau et al. 2005; Honczarenko

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et al. 2006; Ringe et al. 2007). These receptors are essential to respond to triggering factors at the site of injury. In addition, MSCs also express cell adhesion molecules, including CD49d, CD44, CD54, CD102, and CD106 (De Ugarte et al. 2003). These chemokines and cell adhesion molecules orchestrate the mobilization of MSCs’ injury sites in a similar manner to white blood cells (Kolaczkowska and Kubes 2013). MSC mobilization is a multistep process that encompasses the attachment of free circulating MSCs in the bloodstream to transmigrate between ECs with the ultimate goal of migrating and engrafting to the target tissue.

Tissue Repair Once recruited in the injured site, MSCs contribute to tissue repair and regeneration by activating several mechanisms. A growing body of research has demonstrated that MSCs display pleiotropic effects, which give them enormous therapeutic potential. MSCs secrete various mediators of tissue repair in response to injury signals, including anti-apoptotic, anti-inflammatory, immunomodulatory, anti-fibrotic, and angiogenic agents (Caplan and Dennis 2006; Meirelles Lda et al. 2009; Maltman et al. 2011; Escacena et al. 2015). Among pleiotropic effects, antiinflammatory and immunomodulatory properties are mainly responsible for the therapeutic benefits of MSCs. As sensors of inflammation, MSCs release soluble factors, such as TGF-β, IDO, TNF-α, IL-10, and INF-γ, which interfere with the immune system and modify the inflammatory landscape (Prockop and Oh 2012). Pivotal studies showed that MSCs inhibit the proliferation of T and B cells (Di Nicola et al. 2002; Corcione et al. 2006; Song et al. 2019), suppress the activation of natural killer cells (Sotiropoulou et al. 2006), and prevent the generation and maturation of monocyte-derived dendritic cells (English et al. 2008; Spaggiari et al. 2009). Furthermore, MSCs can promote the generation of regulatory T cells (Maccario et al. 2005), which exert immunosuppressive effects. Although soluble factors play a key role in the immunosuppressive activity of MSCs, cell-to-cell contact also influences immune responses (Ren et al. 2010; Li et al. 2019). For instance, direct contact between MSCs and pro-inflammatory macrophages has been shown to induce immune tolerance by inducing tumor necrosis factor-stimulated gene-6 (TSG-6) production (Li et al. 2019). MSC mediated modulations of the immune response set in motion essential inflammatory processes that significantly promote tissue repair and regeneration by driving healing, scarring, and fibrosis (Julier et al. 2017).

Immunomodulatory Potential The immunomodulatory properties of MSCs and their immune-privileged condition make these cells good candidates for use in several clinical trials related to chronic, inflammatory, and autoimmune diseases. MSCs interact with cells of the innate or

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adaptive immune system (T cells, B cells, NK cells, monocyte-derived dendritic cells, and neutrophils) (Di Nicola et al. 2002; Raffaghello et al. 2008). For a cell to be recognized by the immune system, the expression of major histocompatibility complex (MHC) and co-stimulatory molecules is necessary. MHC class I and class II human leukocyte antigens (HLAs) are master triggers of robust immunological rejection of grafts because they present antigens to cytolytic T lymphocytes (CTL). Human mesenchymal stem cells (hMSCs) are characterized by low expression of MHC class I HLAs but are constitutively negative for class II HLCs; these cells do not express co-stimulatory molecules such as B7-1, B7-2, CD80, CD86, CD40, and CD40L (Hmadcha et al. 2009; Le Blanc 2003). However, similar to the thymic epithelium, MSCs express the surface markers VCAM-1, ICAM-2, and LFA-3 (Le Blanc 2003; Conget and Minguell 1999), which are crucial for T-cell interactions. Although a T-cell response should be expected, hMSCs can modulate the activation and proliferation of both CD4+ and CD8+ cells in vitro by arresting T cells in G0/G1 phase (Glennie et al. 2005; Benvenuto et al. 2007). Different studies have suggested that cell-cell interactions and certain soluble factors are the mechanisms used by MSCs to mediate the immune response. Factors, such as IDO, TGF-β1, IFN-γ, IL-1β, TNF-α, IL-6, IL-10, PGE-2, HGF, HLA-G5, and others, are secreted by MSCs or released after interactions with target cells. As mentioned above, MSCs remain in a resting state, display anti-apoptotic properties, and maintain different cells such as hematopoietic stem cells (HSCs), thus contributing to tissue homeostasis. However, in an inflammatory environment such as that created by cytokines such as IFN-γ, TNF-α, IL-1α, and IL-1β, MSCs begin to exert their immunosuppressive effects and polarize, inhibiting the proliferation of effector cells and their production of cytokines. In this regard, IFN-γ is postulated as a “licensing” agent for MSC anti-proliferative action. MSCs may also acquire behavior as antigenpresenting cells (APCs) under specific concentrations of IFN-γ (Stagg et al. 2006; Uccelli et al. 2008). However, no consensus regarding what concentration of IFN-γ is more necessary for MSCs to show inhibitory or APC functions exists. Likewise, TNF-α is another pro-inflammatory cytokine involved in the MSC immune response, and TNF-α enhances the effect of IFN-γ. IFN-γ, with or without the help of TNF-α, stimulates the production of IDO by MSCs, inhibiting the proliferation of activated T or NK cells and thus enhancing the homing potential and reparative properties of these cells; however, some potential risks are associated with the role of IFN-γ (Krampera et al. 2006; Sivanathan et al. 2014). Some authors have maintained that the immunomodulatory properties of MSCs are comparable, while others have argued that MSCs of different tissue origins or species cannot have equivalent and comparable immunomodulatory properties (Najar et al. 2010; Yoo et al. 2009; Ricciardi et al. 2012; Krampera 2011). For example, MSCs from perinatal sources (umbilical cord and amniotic membrane) show a higher immunomodulatory capacity, differential gene expression profiles, and paracrine factor secretion compared to BM-MSCs (Wegmeyer et al. 2013). Lee and colleagues found that HLA-G, a specific MHC-I antigen that is critical for maintaining the

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immune-tolerant state of pregnancy and that is a contributing factor to the induction of more substantial immunosuppression, is strongly positive only in placentaderived MSCs (PD-MSCs) (Lee et al. 2012a). This is in contrast to BM-derived MSCs and Ad-MSCs and suggests that the immunophenotype of PD-MSCs may be superior to other MSCs in terms of their immunosuppressive function (Hunt et al. 2005). Nonetheless, some authors claimed that BM-derived MSCs were more immunomodulatory than PD-MSCs (Fazekasova et al. 2011). And others concluded that the immunomodulatory capacities of BM-derived MSCs and Ad-MSCs are similar but that differences in cytokine secretion cause Ad-MSCs to have more potent immunomodulatory effects than BM-derived MSCs (Melief et al. 2013). Bartholomew and colleagues (Bartholomew et al. 2002) showed that allogeneic MSCs prolonged skin graft survival in baboons. Mouse MSCs have been used in related experiments; these cells use inducible nitric oxide synthase (iNOS) for immunosuppression instead of IDO. These findings indicate that MSCs differ between species (Ren et al. 2009). Since then, several preclinical models have been used to analyze the biological effects of MSCs and their ability to modulate immune responses, considering that not all animal models mimic human diseases. Once more, these differences could be due to isolation procedures, to culture methodology, or, more likely, to differences in the microenvironments where cells reside. These and other findings lead us to conclude that determining whether these differences may be relevant for clinical applications and whether MSCs of a particular tissue type are more appropriate for specific therapies or diseases.

Preclinical Applications Preclinical models are essential for clinicians, researchers, and both national and international regulatory agencies to demonstrate the safety and efficacy of MSC-based therapies (Krampera et al. 2013). Because MSCs can exert immunomodulatory properties and act on different immune cells in vitro and in vivo, these cells have begun to be used against autoimmune diseases based on multiple autoimmune experimental models. Pioneer studies in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, reported that MSCs derived from numerous tissue origins show efficacy against neurodegenerative disorders (Zappia et al. 2005; Rafei et al. 2009; Constantin et al. 2009; Bai et al. 2009; Zhang et al. 2005). BM-MSC and UCB-MSC treatments have improved clinical and laboratory parameters in systemic lupus erythematosus (SLE) (Sun et al. 2010). Furthermore, ameliorating effects have been observed in experimental mouse models of rheumatoid arthritis (RA) (González et al. 2009). Diabetes is another autoimmune disorder in which MSCs have been employed (Jurewicz et al. 2010; Lee et al. 2006). Although promising results and progress have been observed in this field, the interspecies differences, and contradictory experimental outcomes, and the inability to recreate the complete pathophysiology of some diseases make it necessary to search for new animal models for comparable results.

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MSC-Based Therapy for Autoimmune Diseases The MSCs are being used to facilitate the engraftment of transplanted HSCs and treat graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) based on their immunomodulatory properties and provide appropriate conditions. However, preclinical and clinical experiments with MSCs do not always show similar results for the prevention and treatment of GvHD. In a study using a mouse model of GvHD (Sudres et al. 2006), MSCs suppressed alloantigen-induced T-cell proliferation in vitro in a dose-dependent manner but yielded no clinical benefit regarding the incidence or severity of GvHD. Instead, when UCB-MSCs were administered in weekly doses in a xenogenic model of GvHD, a marked decrease in human T-cell proliferation was observed, and none of the mice developed GvHD. No therapeutic effect was obtained when UCB-MSCs were administered at the onset of GvHD (Tisato et al. 2007). In the same line of research, serial infusions of mouse AD-MSCs could efficiently control the lethal GvHD that occurred in recipients transplanted with haploidentical hematopoietic grafts (Yañez et al. 2006). Mixed results have also been achieved in human patients. One study found that the co-transplantation of culture-expanded MSCs and HSCs from HLA-identical sibling donors after myeloablative therapy accelerated hematopoietic engraftment (Lazarus et al. 2005); however, a significant reduction of GvHD symptoms was not shown, although the incidence or severity of GvHD did not increase. Koç and colleagues (Koç et al. 2000) reported a positive impact of MSCs on hematopoiesis; rapid hematopoietic recovery was observed in a clinical study with breast cancer patients who received autologous HSCT together with autologous MSCs. Therapeutic effects have also been reported at the onset of GvHD, such as the case of a 9-year-old boy with severe treatment-resistant GvHD after allogeneic HSCT for acute lymphocytic leukemia who received haploidentical MSCs derived from his mother. He showed improvement after two administrations of MSCs (Le Blanc et al. 2004). Similar results have been obtained in steroid-refractory GvHD pilot studies with BM-MSCs and AD-MSCs (Ringdén et al. 2006; Fang et al. 2006). Several infusions appear to be required to maintain the level of active immunomodulation by MSCs. Similarly, the expression of pro-inflammatory cytokines such as IFN-γ in the environment at the time of MSC administration is required by these cells to exert their immunosuppressive effect. A lack of MSC “licensing” can result in the absence of the desired therapeutic effect. While evidence that MSCs are effective in combination or after HSCT in specific hematological and non-hematological diseases has been shown, adverse reactions and risk factors intrinsic to this practice have been reported. In a pilot study, HLA-identical sibling-matched HSCs were transplanted with or without MSCs in hematological malignancy patients. Although MSCs were well-tolerated and this treatment effectively prevented GVHD, six patients (60%) in the MSC group and three (20%) in the non-MSC group had 3-year disease-free survival rates of 30 and 66.7%, respectively. The relapse rate in the experimental group was higher than that

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in the control group, suggesting that MSCs may impair the therapeutic graft-versusleukemia (GVL) effect (Ning et al. 2008). In vitro and in vivo studies regarding the relationship between the immunosuppressive properties of MSCs and the stimulation of cancer growth have been performed. Mouse MSCs from the BM, spleen, and thymus injected together with a genetically modified tumor cell vaccine could equally prevent the onset of an anti-tumor memory immune response, thus leading to tumor growth in normally resistant mice (Krampera et al. 2007). In another in vivo experiment with a murine melanoma tumor model, the authors observed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were co-injected (Djouad et al. 2003). The functions of MSCs can be influenced by the existing microenvironment, making them acquire supportive properties toward cancer cells and decrease immune reactions (Galiè et al. 2008). Therefore, potential risks related to the growth support and enhancement of undetected or “resident” cancer exist, and the administration of MSCs in these patients must be thoroughly evaluated.

MSCs for Cancer Treatment The therapeutic benefits of MSCs have prompt their use in cell-based strategies to treat different diseases, including cancer (Hmadcha et al. 2020). Similar to damaged tissues, tumors exert chemoattractant effects on MSCs that influence their recruitment to tumor sites. The CXCL12/CXCR4 axis is one of the most frequently studied signaling pathways in mobilizing MSCs to the tumor microenvironment (Gao et al. 2009; Xu et al. 2009; Lourenco et al. 2015; Wobus et al. 2015; Kalimuthu et al. 2017). However, the ability of MSCs to migrate toward cancerous tissue is also controlled by other agents, including diffusible cytokines, such as IL-8, growth factors such as TGF-β1 or platelet-derived growth factor (PDGF), and extracellular matrix molecules, such as matrix metalloproteinase 2 (MMP-2) (Nakamizo et al. 2005; Birnbaum et al. 2007; Bhoopathi et al. 2011). Once the tumor niche is reached, MSCs interact with cancer cells via direct and indirect mechanisms that affect tumor development. The paracrine action of MSCs is one of the main mechanisms involved in cancer regulation and is mediated by multiple factors, including growth factors and cytokines. These paracrine factors affect cellular processes involving the tumor cell cycle (e.g., cell proliferation), cell survival, angiogenesis, and immunosuppression/immunomodulation, allowing MSCs to regulate cancer. The paracrine agents can be directly secreted into the extracellular space or packaged into EVs for spreading in the tumor milieus (Rani et al. 2015). The interaction of MSCs with the tumor cell cycle is the most commonly accepted process by which MSCs exert their therapeutic effects (Fathi et al. 2019). By inhibiting proliferation-related signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), MSCs can induce cell cycle arrest and reduce cancer growth (Lu et al. 2019). In addition, MSCs can undergo differentiation into other cell types, such as cancer-associated fibroblasts (CAFs), to directly

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contribute to cancer progression (Jotzu et al. 2011; Barcellos-de-Souza et al. 2016; Aoto et al. 2018). Accumulating evidence indicates that the cross-talk between MSCs and tumor cells results in both pro-tumor and anti-tumor effects, raising safety concerns for clinical application in oncology (Barkholt et al. 2013). The discrepancies in the ability of MSCs to promote or suppress tumor development may be attributable to differences in experimental tumor models, MSC tissue source, dose or timing of the MSC treatment, cell delivery method, control group chosen, and other experimental conditions (Bortolotti et al. 2015; Bajetto et al. 2017). In this regard, a study demonstrated that direct (cell-to-cell contact) or indirect (released soluble factors) interaction between umbilical cord MSCs and glioblastoma stem cells produces divergent effects on cell growth, invasion, and migration (Bajetto et al. 2017). Moreover, the application of MSCs for cancer patients is a more complex situation in which other factors have to be taken into consideration. For instance, the pathological conditions of each patient may induce cellular and molecular changes in MSCs that interfere with their therapeutic effects (Capilla-González et al. 2018; Pérez et al. 2018; Rivera et al. 2019). Therefore, it is important to be cautious while drawing conclusions from a single study regarding the therapeutic effects of MSCs in cancer.

The Anti-tumor Activity of MSCs Although compelling evidence shows a pro-tumorigenic role of MSCs, these cells also have potent tumor-suppressive effects that have been exploited as cancer therapeutics. Previous studies have demonstrated that MSCs release cytotoxic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) that selectively induces apoptosis in different types of cancer (Wiley et al. 1995; Hao et al. 2001; Takeda et al. 2001; Akimoto et al. 2013). Recently, a report indicated that BM-derived MSCs promote apoptosis and suppress the growth of glioma U251 cells through downregulation of the PI3K/AKT signaling pathway (Lu et al. 2019). Likewise, intravenously transplanted MSCs were found to suppress tumor growth by blocking AKT activation in a Kaposi sarcoma mouse model (Khakoo et al. 2006). In mammary carcinomas, umbilical cord MSCs attenuated cell growth and triggered apoptosis through inhibiting ERK1/2 and AKT activation (Ganta et al. 2009). The Wnt signaling pathway has also been involved in the ability of MSCs to inhibit tumor cell proliferation (Qiao et al. 2008a, b). A mechanistic study of the inhibitory effect of MSCs on breast cancer cells demonstrated that the protein dickkopf-1 (Dkk-1) released from MSCs blocks tumor growth via depression of Wnt signaling (Qiao et al. 2008a). In contrast to investigations describing the pro-angiogenic effect of MSCs (Zhang et al. 2013; Li et al. 2016), the anti-tumor activity of MSCs via the inhibition of tumor angiogenesis has also been documented. A study reported that BM-derived MSCs restrict vascular growth in 1Gli36 glioma xenograft through the downregulation of the PDGF/PDGFR axis (Ho et al. 2013). In particular, the expression of PDGF-BB protein was significantly reduced in tumor lysates when

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treated with MSCs, which correlated with reduced levels of activated PDGFR-b and the active isoform of its downstream target AKT (Ho et al. 2013). In a melanoma mouse model, transplanted MSCs inhibited angiogenesis in a concentration-dependent manner, leading to reduced tumor growth (Otsu et al. 2009). Confirmatory in vitro studies suggested that the anti-angiogenic effect was due to MSC-induced capillary degeneration (Otsu et al. 2009). Furthermore, MSCs have elicited anti-tumor immune responses through released inflammatory mediators, such as the multifunctional cytokine TGF-β. Like several signaling molecules, TGF-β plays a dual role in cancer development (Bierie and Moses 2006). Besides the aforementioned pro-tumor functions, TGF-β signaling exhibits suppressive effects in cancer (Dong et al. 2007; Guasch et al. 2007). While the expression of type III TGF-β receptor (TbRIII) decreases during breast cancer progression, restoring TbRIII expression suppresses tumorigenicity (Dong et al. 2007).

The Pro-tumor Activity of MSCs The pleiotropic effects of MSCs that promote tissue repair and regeneration may also confer pro-tumor functions to these cells. For instance, metastatic human breast carcinoma cells were found to induce the secretion of the chemokine (C-C motif) ligand 5 (CCL5) from MSCs, which enhanced tumor invasion (Karnoub et al. 2007). Seminal reports demonstrated that MSCs could also inhibit apoptosis in tumor cells by secreting pro-survival factors such as VEGF and bFGF (König et al. 1997; Dias et al. 2002). Numerous studies converged on the finding that MSCs contribute to cancer pathogenesis by releasing inflammatory factors that promote immunosuppressive effects. For example, an in vitro study showed that MSCs isolated from gastric tumors mediate cancer progression through the secretion of IL-8 (Li et al. 2015). This pro-inflammatory chemokine favors the recruitment of leukocytes. It is known that recruited leukocytes, such as macrophages and neutrophils, facilitate cancer initiation and progression (Guo et al. 2017; Powell et al. 2018). Similarly, MSCs are able to secrete TGF-β that promotes macrophage infiltration at the tumor site and facilitates tumor escape from immune surveillance (Kim et al. 2006; Byrne et al. 2008). Compelling evidence indicates that MSCs can also support tumor angiogenesis, an essential process in cancer progression that supplies tumors with oxygen and nutrients. For instance, MSCs recruited in breast and prostate tumors were found to increase the expression of angiogenic factors, including TGF-β, VEGF, and IL-6, which contribute to tumor growth and vascularization (Zhang et al. 2013). Similarly, a correlation between increased expression of TGF-β and higher microvessel density was observed in hepatocellular carcinomas of mice receiving intravenous injections of human MSCs (Li et al. 2016), which further supports that MSCs may enhance tumor angiogenesis via TGF-β. Furthermore, MSCs can also respond to soluble factors secreted from cancer cells and differentiate into CAFs, a cell type within the tumor microenvironment capable of promoting tumorigenesis (Mishra et al. 2008). In particular, TGF-β secreted from cancer cells plays a critical

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role in the differentiation of MSCs into CAFs (Jotzu et al. 2011; Barcellos-de-Souza et al. 2016; Aoto et al. 2018). It is known that the transition of MSCs into CAFs contributes to tumor progression in part by their active secretome. Profiling of the secretome shows that it is rich in many bioactive molecules, including immune-modulating agents (CXCL12, granulocyte-macrophage colony-stimulating factor), pro-angiogenic factors (VEGF, TGF-β, PDGF), pro-survival factors (hepatocyte growth factor, insulinlike growth factor 1, interleukin 6), and extracellular matrix modulators (MMP, tissue inhibitor of metalloproteinases) among others (Kalluri 2016). Cell engulfment has also been identified as an interacting process between MSCs and cancer cells that enhances tumor aggressiveness. A recent report demonstrated that breast cancer cell engulfment of MSCs leads to changes in the transcriptome profile of tumor cells. These changes are mainly associated with oncogenic pathways. This MSC engulfment enhances epithelial-to-mesenchymal transition, stemness, invasion, and metastasis of breast cancer (Chen et al. 2019).

The Imprint of Disease on MSCs One of the strategies to obtain MSCs for therapeutic purposes is an autologous approach. These cells are collected from patients by more or less invasive methods, isolated, seeded in culture under good manufacturing practice (GMP) quality standards, and re-injected into the patient. Nevertheless, when body’s repair mechanisms are insufficient or ineffective, this treatment results in a homeostatic imbalance in the organism, producing degradation and disease and compromising the pool of endogenous cells, thus resulting in low efficacy. Some conditions/diseases provoke changes in the BM microenvironment, which is one of the primary sources of MSCs, thus producing changes in the endogenous pool of MSCs and altering their biological features (Mazzanti et al. 2008). MSCs from patients with acute myeloid leukemia showed abnormal biological properties, including morphological heterogeneity, limited proliferation capacity, and impaired differentiation and hematopoiesis supportability (Zhao et al. 2007). MSCs derived from patients with multiple myeloma showed impaired immuneinhibitory effects on T cells, decreasing their osteogenic potential (Li et al. 2010). Poor proliferation, differentiation potential, and cytokine release defect were found in BM-derived MSCs derived from patients with aplastic anemia, another hematopoietic disorder (Chao et al. 2010; Bacigalupo et al. 2005). Although the mechanisms remain unknown, MSCs appear to be involved in autoimmune pathologies. For instance, MSCs derived from patients with autoimmune diseases display the following altered functions; MSCs from rheumatoid arthritis (RA) patients have an impaired ability to support hematopoiesis and lower proliferative and clonogenic potentials (Papadaki et al. 2002; Kastrinaki et al. 2008). MSCs from immune thrombocytopenic purpura (ITP) patients have a reduced proliferative capacity and a lower inhibitory effect on T-cell proliferation than MSCs from healthy donors (Pérez-Simón et al. 2009). MSCs from systemic lupus erythematous (SLE) patients

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display deficient growth, abnormal morphology, and upregulated telomerase activity (Nie et al. 2010; Sun et al. 2007). MSCs from systemic sclerosis (SSc) patients display early senescence (Cipriani et al. 2007). In metabolic diseases such as diabetes, alterations in autologous MSCs have also been documented. A study using MSCs from type 2 diabetic mice showed that the number of these cells was diminished and their proliferation and survival abilities were impaired in vitro. Moreover, diabetic MSC engraftment produced only limited improvement in the diabetic subjects and could not produce the same therapeutic outcomes as in their nondiabetic counterparts in vivo (Shin and Peterson 2012). Advanced glycation end products (AGEs) accumulate in the tissues of aged people, and these products are involved in diabetes and musculoskeletal diseases. In 2005, Kume and colleagues (Kume et al. 2005) investigated the effect of AGEs on MSCs. They showed that AGEs inhibited MSC proliferation, induced MSC apoptosis, and interfered with MSCs’ differentiation into adipose tissue, cartilage, and bone. Type 2 diabetesderived Ad-MSCs have been found to have functional impairments in their multilineage potential and proliferative capacity because of prolonged exposure to high glucose concentrations (Cramer et al. 2010). Diabetic-derived Ad-MSCs have an altered phenotype related to plasminogen activator inhibitor-1 (PAI-1) expression levels and display reduced fibrinolytic activity (Acosta et al. 2013), which suggests that the immunogenicity of MSCs could have associated effects on the coagulation system (Wang et al. 2012; Moll et al. 2012). Thus, MSC-based therapy could lead to thrombotic events in particular recipients. Although the possibility of healing with autologous cells is desirable, little is known regarding the influence of different disease states and concomitant medications on MSCs (Benvenuti et al. 2007; Lee et al. 2009). Thus, although the use of autologous MSCs for cell therapy is widespread, their use in humans must be handled with extreme caution. Researching and analyzing both the risks and benefits of this therapy in individual patients and for each disease are necessary.

Considerations for Clinical Applications Several clinical trials are in progress to ensure the safety and efficacy of MSCs used as medicaments. For cell-based products, it must be considered that cells are living products and that their interactions with body fluids remain unclear (Acosta et al. 2013; Moll et al. 2014). Phase 1 clinical trials are the first step in investigating a new drug. They include pharmacokinetic and pharmacodynamic studies in which the patient’s safety plays an essential role in the development of medicaments. The primary goal of phase 2 clinical trials is to provide preliminary information regarding the drug efficacy and safety supplement data obtained in phase 1 trials. For efficacy and effectiveness issues, other advanced phases are mandatory. In all cases, one cannot consider these issues (efficacy nor effectiveness) unless phase 3 clinical trials are developed (García-Bernal et al. 2021; Hmadcha et al. 2020; Escacena et al. 2015). Usually, safety evaluations are based on possible complications derived from the procedure in a time-dependent manner after administering the cells. Efficacy

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parameters focus on the improvement of clinical effects at a given time. MSC-based cell therapy is a relatively new therapeutic option for certain diseases, and data regarding the long-term monitoring of patients remain lacking. Nevertheless, the administration of MSCs is considered a feasible and safe procedure with no adverse events reported. However, the risks associated with stem cell therapy (Herberts et al. 2011) must be considered because these risks increase the probability of an adverse event. The cell source, donor origin, product manufacturing, and recipient disease status are important factors related to the safety and efficacy of MSCs. In this regard, the use of bovine proteins in the medium used to culture these cells (Horwitz et al. 2002) and the observed formation of ectopic tissue in animal models (Breitbach et al. 2007; Kunter et al. 2007), as well as malignant transformation (Wang et al. 2005; Røsland et al. 2009) and immune responses, must be evaluated before wider clinical applications and registration are accepted.

Safety Concerns Cell therapy is incredibly complex due to the nature of the product. The mode of action is not always clear, and the potency tests are imprecise, by which it might not be possible to predict the risks thoroughly. When considering the use of expanded MSCs ex vivo for clinical applications, it is necessary to consider a series of potential risks that could affect the cellular product. The administration of stem cells could affect the host’s immune system. These cells could directly influence the immune system (e.g., pro-inflammatory environment) or have an immunomodulatory effect. Although MSCs have been considered immune-privileged in this regard, long-term exposure to the culture medium can make them more immunogenic by positively regulating the normal set of histocompatibility molecules (Moll et al. 2011, 2014). On the one hand, the allogeneic use of the cells entails a greater risk of rejection by the immune system. This rejection could lead to a loss of the function of the administered cells, and consequently, their therapeutic activity could be compromised. The use of immunosuppressants could limit these risks, but, in turn, could cause adverse reactions due to immunosuppressive medication. On the other hand, MSCs isolated from healthy donors have shown uniform and consistent properties, while patients with some degenerative and inflammatory disease differ in their biological and functional characteristics (Capilla-González et al. 2018; Rennert et al. 2014). In this regard, studies with MSCs from diabetic patients suggest that the hyperglycemic environment and other metabolic disorders associated with diabetes affect the endogenous cellular reserve and their proliferation, differentiation, and angiogenic capacity, among other cellular characteristics (Minteer et al. 2015; Rennert et al. 2014; George et al. 2018; Moll et al. 2019). Once infused in the recipient, the cells come into direct contact with the tissues, bloodstream, and other host cells; the cell-recipient interaction process still needs a thorough investigation and characterization.

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Physiologically, MSCs reside in the perivascular compartment of almost every tissue (Bianco et al. 2008; Crisan et al. 2008); however, one of the hurdles to the sustained success of their therapeutic effect is early cell loss. This is primarily due to the incompatibility responses after systemic infusion of cells, a reaction termed as instant blood-mediated inflammatory reaction (IBMIR) suggesting that the immune and inflammatory system reacts to cells that generally are not in contact with the blood circulation (Gupta et al. 2014; Moll et al. 2011, 2014, 2019, 2020; Bianco et al. 2008; Crisan et al. 2008; Nilsson et al. 2014). Even more, it has been further shown that different MSC products display varying levels of highly pro-coagulant tissue factor, a decrease in tissue plasminogen activator (tPA), or an increase in PAI-1 and may adversely trigger the IBMIR or microthrombosis in the target tissue (Acosta et al. 2013; Moll et al. 2019). Although MSCs are considered to be safe, they can promote fibrinolysis (Hashi et al. 2007; Neuss et al. 2010; Moll et al. 2020). Safety and efficacy are the basic pillars that support the viability of clinical application to treat any disease. Except for hematopoietic stem cell transplants, stem cell therapies used to treat any disease are considered medicinal products; therefore, their development, approval, and use must be per the specific standards established nationally and internationally for such medicines. Thus, regulatory authorities guarantee the safety of the studies (Fig. 2).

Cell Manufacturing for Clinical Use Except for hematopoietic stem cell transplants, stem cell therapies used to treat any disease are considered drugs; therefore, their development, approval, and use must be per the specific standards established for such medicines nationally and internationally. In this context, MSCs are now considered as “cellular medicament” and are called advanced therapy medicinal products (ATMPs) and are under regulation No. 1394/2007 (Escacena et al. 2015; Gálvez et al. 2013). Relating production processes and development staff, clinicians and researchers must achieve GMP procedures under European regulations (Sensebé et al. 2013; Gálvez et al. 2014). Currently, no standardized manufacturing platform exists, although most facilities employ standard release criteria to measure sterility, viability, and chromosomal stability to meet European or FDA regulations (Phinney 2012; Iglesias-López et al. 2019). Although regulation establishes common parameters to follow, different protocols are used to isolate these cells, and the processes, plating densities, and reagents used cause the results to differ from each other. Donor selection in terms of age and disease status is another variable to consider due to known MSC donor-to-donor heterogeneity (Phinney et al. 1999). The cell source is another important factor related to the efficacy of the product. As reported previously, MSCs derived from different tissues do not consistently achieve the same level of efficacy. Additionally, culture media used for the production of MSCs could affect the basic characteristics of cells; thus, designing a fully defined medium free of animal and human origins is crucial.

Considerations for Clinical Use of Mesenchymal Stromal Cells

Fig. 2 The MSCs possess a wide range of paracrine effects, including anti-inflammatory, immunomodulatory, trophic, anti-apoptotic, and anti-fibrotic properties. These are mostly mediated by molecules released by MSCs, but also by direct cell-cell contacts. For cell-based therapy, the paracrine properties of MSCs have beneficial effects for the patient. However, the interaction between MSCs and the host can lead to adverse side effects. (Re-published from

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Thus far, no MSC-based medicine product has marketing authorization in the European Union, although four gene and cell-based products have a valid marketing authorization awarded by the EMA. However, since 2011, three MSC products have received marketing approval in other regions (Ancans 2012). The MSCs’ field continues its upward progression, with a growing number of established companies established and ongoing clinical trials, but remaining challenges must be overcome. Bottlenecks exist regarding donor selection, cell sources, isolation protocols, culture media used, open-culture systems, bioreactors, and recipient disease status. Establishing a standardized and comparable process is also crucial to ensure biological and functional equivalence between product lots.

Considerations for Cellular Medicament General Considerations The cell expansion and culture protocol are not standardized, although the regulatory agencies (e.g., EMA, FDA) recommend a set of standards to be followed to produce cellular drugs. Currently, there is no protocol or universal definition for stem cell culture and expansion. The different sources of origin, and the different methodologies for obtaining tissue cells, make it very difficult to compare research groups in search of the fastest, most effective, economical, high-yielding, efficient, and clinical-grade quality method. Cell viability after the infusion is poor; in this regard, it is known that very few cells survive after infusion. Although the in vivo follow-up is ethically and technically complicated, it is necessary to continue investigating this line to understand the intrinsic mechanisms of integrating the infused cells in the concrete microenvironment. The cellular dose to obtain the desired effects is also unknown. Investigations with HSCs have revealed that the administration of sufficient cells promotes faster cell recovery and reduces hospitalizations (Mohty et al. 2011). Preclinical studies using murine animal models have established a minimum dose of 1  106 cells/kg of weight, a quantity necessary to obtain quantifiable but weak benefits (Shabbir et al. 2009; Mastri et al. 2012). The dose for cellular treatment is probably influenced by the patient’s body weight and the biodistribution of paracrine factors secreted by MSCs in the human body; however, most clinical trials use a similar cell dose (Tan et al. 2012; Jiang et al. 2011). The doses used have been insufficient in most cases to show clear therapeutic benefits. This fact leads us to design future trials to test different cell doses. Likewise, the frequency of administration is currently unknown. ä Fig. 2 (continued) Soria-Juan 2019, article published under CC-BY terms). Abbreviations: B-cell B lymphocyte, CXCL C-X-C motif chemokine ligand, DC dendritic cell, G-CSF granulocyte colony-stimulating factor, HGF hepatocyte growth factor, IL interleukin, INF-γ interferon-γ, MSC mesenchymal stem cells, NK natural killer cells, T-cell T lymphocyte, TGF-α transforming growth factor α, Treg regulatory T cell

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The effectiveness of cell therapy is probably related to the number of others applications (Cobellis et al. 2008; Teraa et al. 2015; Molavi et al. 2016), similar to that established with conventionally used medications. The timing and the ideal number of cellular applications are still unknown. Since conventional medicines are depending on the dose, cellular therapy may need to be adjusted accordingly. The most suitable cell type remains a challenge for regenerative medicine. Knowing which cell type is most appropriate for each particular pathology or if a combination of these would be more recommended is another big issue in cell therapy. The method for the cellular administration continues without giving conclusive results because cell viability must be preserved as much as possible, and compromised tissue is often associated with ischemia, fibrosis and inflammation, which could impair cell survival, therapeutic delivery of stem cells in the distal areas to the damaged tissue appear to offer some advantage. There are no conclusive findings of a more significant benefit within the existing modes of administration, so this is another variable to have into account for future clinical trials. The desired therapeutic effect depends on many factors since mechanism of action of stem cells in tissue regeneration is likely to be multifaceted. Cellular competition can be dictated by the ability of injected cells to migrate, survive, integrate, differentiate, and produce functional paracrine mediators (“cell-cell interactions”). It is known that many diseases (e.g., diabetes, cancer, etc.) affect the phenotypic and therapeutic properties of stem cells. Finally, for the therapy to be effective, the recipient tissue must respond favorably to the injected cells, which would result in the activation of endogenous regeneration mechanisms (Lee 2010). Understanding integration of the exogenous mechanisms (injected cells) with the endogenous (host) will play a decisive role in the future clinical use of adult stem cells (Acosta et al. 2013; Moll et al. 2019).

Attempts to Improve the Therapeutic Outcomes of Cellular Medicament Advances in the production compliance under good manufacturing practices (GMP) standards of more sophisticated cellular products are now opening up the way for the second generation of cell therapy clinical trials. One of the reasons why unmodified MSCs have not shown the therapeutic efficacy expected in human clinical trials is that, after their systemic infusion (intravenous), these cells become trapped in the vascular filters (fundamentally the liver and lung) and only a small percentage reach the target tissues. Therefore, strategies must be designed that favor migration, nesting, and localization in the inflammatory and/or infectious focus to increase their effectiveness. Biodistribution and long-term follow-up of these cells in animal models show that only a few cells remain after long periods. This will support the idea that most of the effects of MSCs are based on a “hit and run effect.” To increase the concentration of ATMPs in the injured tissue, the CD44 antigen on MSCs’ cell membrane by enzymatic fucosylation has been converted into hematopoietic cell E-selectin/L-selectin ligand (HCELL) glycoform (Dimitroff et al. 2001;

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Pachón-Peña et al. 2017). This molecular change favored the migration of the MSCs to the inflamed tissues (Sackstein et al. 2008; García-Bernal et al. 2020). This method, called glycosyltransferase-programmed stereo substitution (GPS) of cell surface glycans, has been optimized for its clinical application so that the reagents used (glycosyltransferases and buffers) have been specifically formulated to preserve cell viability and phenotype (García-Bernal et al. 2021). Moreover, this modification not only increases the adhesion of the MSCs to the endothelium, but it also enhances their transmigration through it by activating the alfa4/beta1 integrin in the absence of chemokine stimulation (López-Lucas et al. 2018). Therefore, this modification by fucosylation could improve the efficacy of the treatment with MSCs by increasing the migratory capacity of the cells to the inflamed tissues after being administered systemically (García-Bernal et al. 2020). Other strategies may include expressing CXCR4. These strategies will help to engineer new generation of MSCs for use when both increased migration and targeting and an increased power are required. Expression of the CXCR4 receptor will increase the migration of the MSCs toward the inflammatory focus (Zhu et al. 2021). On the other hand, the co-expression of the antiinflammatory cytokine IL-10 and/or the anti-infectious cytokine IL-7 will increase the anti-inflammatory effect (IL-10) and even the anti-infective effect (IL-7) (Mao et al. 2017). Furthermore, the extensive use of fetal bovine serum (FBS) in the MSC-expansion media represents an explicit limitation for the introduction of ATMP at the clinical level. Currently, cell expansion is carried out in culture media supplemented with FBS (Gottipamula et al. 2013). The SFB used must be a clinical-grade (free of animal pathogens). Associated with the growing demand for MSCs, this has led to a series of technical and ethical conditions of production (using a high number of bovine fetuses) and geographic (zones free of prion diseases), which have had an impact on their price (Kinzebach and Bieback 2013; Wessman and Levings 1999). The substitution of FBS by human serum and platelet lysate also represents technical limitations mainly related to the supply of human material and the absence of uniformity of the lots. All these considerations force the development of robust processes of production of MSC in chemically defined culture media free of animal and human components. These media are supplemented with recombinant proteins (albumin, insulin, TGF-β, and bFGF), iron, selenium, and an antioxidant system (2-mercaptoethanol) (Badenes et al. 2016; Jayme and Smith 2000). Although several serum-free media are found in the literature and market (Chase et al. 2010; Ishikawa et al. 2009), there is still no effective means of functioning. The therapeutic efficacy of MSCs has been further optimized by genetically modifying MSCs to produce trophic cytokines or other beneficial gene products in numerous preclinical models by transfecting MSCs with viral or non-viral vectors (Jiang et al. 2006; Haider et al. 2008; Kim et al. 2012a, b). These MSCs have been successfully modified to express therapeutic peptides and proteins to express therapeutic peptides and proteins in animal models (Zhou et al. 2021). For example, MSCs expressing thioredoxin-1 (Trx1, a potent antioxidant, transcription factor, and growth factor regulator) improved cardiac function in post-myocardial infarction rat models (Suresh et al. 2015). Simultaneous overexpression of Akt and Ang-1 in

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BM-derived MSC not only enhanced their reparability of the infarcted myocardium with sustained beneficial effect (Jiang et al. 2006, 2008), but it also led to non-hypoxic stabilization of HIF-1 to enhance their endothelial commitment (Lai et al. 2012a) and increased their proliferation potential via the involvement of miR-143 (Lai et al. 2012b). The MSCs expressing IL-12 showed potent anticancer activity against melanoma, breast cancer, and hepatoma (Gao et al. 2010; Han et al. 2014). In addition, interferon-γ-expressing MSCs inhibited tumor growth in mouse models of neuroblastoma and lung carcinoma (Relation et al. 2018; Seo et al. 2011). Similar to these advances achieved in animal models, several MSC-based therapies are under clinical development. Both viral and non-viral vectors, however, have some limitations (Kim and Haider 2001). Non-viral vectors exhibit transient gene expression and low transfection efficiency. In contrast, viral transduction is associated with an increased risk of chromosomal instability, insertional mutagenesis, and proto-oncogene activation, despite the inherent high transfection efficiency (Cheng et al. 2019). It has been reported that adverse immune reactions induced by viral transduction impair transgene stability (Mingozzi and High 2013; Wang et al. 2018). Thus, limitations and adverse responses must be assessed when modifying MSCs by transfection. Several studies have sought to use MSCs derived from induced pluripotent stem cells (iPSCs) to obtain better expansion capacity. In fact, therapeutic transgenes could be inserted into iPSC-derived MSCs before MSC derivation. Such a strategy could eliminate insertional mutations and ensure stable expression of transgenes during a prolonged expansion (Zhao et al. 2015). Therefore, MSCs derived from iPSCs may be a renewable source of MSCs for theranostic applications. It is pertinent to mention that BM-derived MSCs have also been successfully reprogrammed to pluripotent status and used for the efficient repair of infarcted myocardium in an experimental animal model (Buccini et al. 2012). Interestedly, CRISPR-Cas9 technology was used to obtain highly homogeneous MSCs. Genetic modifications of MSCs can be performed with greater efficiency and specificity using CRISPR/Cas9 technology (Gerace et al. 2017). This is faster, costefficient, and easier to use compared to alternatives such as transcription activator nucleases (TALENs) and zinc finger nucleases (ZFNs) (Faulkner et al. 2020). CRISPR/Cas9 has been widely employed in the stem cell field, particularly in MSC research, including knock-in, knock-out, gene activation, or gene silencing. In this regard, the application of CRISPR/Cas9 in MSCs has demonstrated its efficacy in treating diseases, such as myocardial infarction (Golchin et al. 2020). Targeting gene knock-in further promoted the differentiation capacity of MSCs and, in turn, improved the insufficiency of functional cells at local sites (Miwa and Era 2018). Genetically modified MSCs have been evaluated in clinical trials, such as the “TREAT-ME-1” clinical trial, an open-label, multicenter, first-in-human phase 1/2 trial, which aimed to evaluate the safety, tolerability, and efficacy of the application of genetically modified autologous MSCs-apceth-101 in patients with advanced gastrointestinal adenocarcinoma (von Einem et al. 2019). Despite promising advances in this field, further research is still needed to obtain solid evidence on the differentiation and regenerative potentials of MSCs in vivo. Undoubtedly, the

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next-generation sequencing and genotyping techniques could serve as valuable tools to improve the efficacy of targeting specific cell types for personalized medicine. Besides, priming MSCs with exogenous small molecules has been found to boost their therapeutic function. Since current MSC manufacturing cannot meet the requirements of clinical trials in terms of production scale, the alternative is to enhance the function of limited cells by priming MSCs. Cell priming, or cell preconditioning, is a commonly used concept in the field of immunology and has been adapted to the stem cell arena (Lu et al. 2010; Haider and Ashraf 2012; Carvalho et al. 2013; Noronha et al. 2019) by ex vivo addition to MSCs of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-1α, and IL-1β. More priming approaches are currently being proposed and optimized to improve MSC function, proliferation, survival, and therapeutic efficacy (Afzal et al. 2010; Kim et al. 2012a, b; Lu et al. 2012; Kim et al. 2018; Mead et al. 2020). In this regard and as mentioned before, other approaches are focused on enhancing the therapeutic effects of cell therapy products regulating their biological characteristics (Mangi et al. 2003; Mei et al. 2007; Lee et al. 2012b; Liao et al. 2017). The beneficial effects of PDGF-BB to restore the defective phenotype of therapeutic MSCs derived from type 2 diabetic patients have been demonstrated. The pretreatment with PDGF-BB potentiates proliferation, migration, and homing of defective MSCs and recovers their impaired fibrinolytic ability. Furthermore, PDGF-BB has been found to exert its beneficial effects through the ERK-SMAD pathway. Therefore, the pretreatment with PDGF-BB represents a suitable strategy to produce more effective MSCs for autologous therapies (Capilla-González et al. 2018).

Concluding Remarks and Future Perspective Treatments based on the use of human stem cells are novel and promising therapeutic alternatives for some diseases. Currently, the use of living cells as a medicinal product is becoming realistic. Cell therapy should be safe, pure, stable, and efficient. Cell-based products are more complex and depend on the physiological and genetic heterogeneity of the patient. Obtaining as much information as possible with the appropriate and available technology at our disposal is essential for ensuring the safety, reliability, quality, and effectiveness of the manufactured product. MSCs are leading the way into a new era of regenerative medicine, and their multifaceted features make them powerful candidates to become tools to treat several diseases. However, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share some common properties, they markedly differ in terms of their differentiation abilities, growth rates, healing capacity, and gene expression profile. Similarly, the disease status of donors and recipients is a critical factor to consider when using MSCs as therapeutic agents because factors such as the MSC behavior with body fluids and specific disease environments remain unclear. Available data suggest that some tissue-specific MSCs are more appropriate than others according

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to particular pathologies. Although no severe adverse effects related to the application and testing of MSCs in humans have been reported to date, some evidence has indicated that specific patient profiles are not suitable to be treated with these therapies. Thus, multiple bottlenecks for the standardization of therapeutic protocols exist. Future well-designed clinical trials, advanced-phase clinical trials (phase 3/4), and long-term monitoring of patients are crucial for obtaining additional information regarding the therapeutic use of MSCs.

Cross-References ▶ Mesenchymal Stromal Cells for COVID-19 Critical Care Patients

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Von Lüttichau I, Notohamiprodjo M, Wechselberger A, Peters C, Henger A, Seliger C, Djafarzadeh R et al (2005) Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev 14(3): 329–336. https://doi.org/10.1089/scd.2005.14.329 Wang J, Liu X, Qiu Y, Shi Y, Cai J, Wang B, Wei X et al (2018) Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells. J Hematol Oncol 11(1):11. https://doi.org/10.1186/ s13045-018-0554-z Wang Y, Huso DL, Harrington J, Kellner J, Jeong DK, Turney J, McNiece IK (2005) Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 7(6):509–519. https://doi.org/10.1080/14653240500363216 Wang B, Wu SM, Wang T, Liu K, Zhang G, Zhang XQ, Yu JH et al (2012) Pre-treatment with bone marrow-derived mesenchymal stem cells inhibits systemic intravascular coagulation and attenuates organ dysfunction in lipopolysaccharide-induced disseminated intravascular coagulation rat model. Chin Med J 125(10):1753–1759 Wegmeyer H, Bröske AM, Leddin M, Kuentzer K, Nisslbeck AK, Hupfeld J, Wiechmann K et al (2013) Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 22(19):2606–2618. https://doi.org/10.1089/scd.2013.0016 Wessman SJ, Levings RL (1999) Benefits and risks due to animal serum used in cell culture production. Dev Biol Stand 99:3–8 Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR et al (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3(6):673–682. https://doi.org/10.1016/1074-7613(95)90057-8 Wobus M, List C, Dittrich T, Dhawan A, Duryagina R, Arabanian LS, Kast K et al (2015) Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12. Int J Cancer 136(1):44–54. https://doi.org/10. 1002/ijc.28960 Woodbury D, Reynolds K, Black IB (2002) Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 69(6):908–917. https://doi.org/10.1002/jnr.10365 Wynn RF, Hart CA, Corradi-Perini C, O’Neill L, Evans CA, Wraith JE, Fairbairn LJ et al (2004) A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 104(9):2643–2645. https://doi. org/10.1182/blood-2004-02-0526 Xu WT, Bian ZY, Fan QM, Li G, Tang TT (2009) Human mesenchymal stem cells (hMSCs) target osteosarcoma and promote its growth and pulmonary metastasis. Cancer Lett 281(1):32–41. https://doi.org/10.1016/j.canlet.2009.02.022 Yañez R, Lamana ML, García-Castro J, Colmenero I, Ramírez M, Bueren JA (2006) Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells 24(11):2582–2591. https://doi.org/ 10.1634/stemcells.2006-0228 Yoo KH, Jang IK, Lee MW, Kim HE, Yang MS, Eom Y, Lee JE et al (2009) Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol 259(2):150–156. https://doi.org/10.1016/j.cellimm.2009.06.010 Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, Giunti D et al (2005) Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106(5):1755–1761. https://doi.org/10.1182/blood-2005-04-1496 Zhang J, Li Y, Chen J, Cui Y, Lu M, Elias SB, Mitchell JB et al (2005) Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol 195(1): 16–26. https://doi.org/10.1016/j.expneurol.2005.03.018 Zhang T, Lee YW, Rui YF, Cheng TY, Jiang XH, Li G (2013) Bone marrow-derived mesenchymal stem cells promote growth and angiogenesis of breast and prostate tumors. Stem Cell Res Ther 4(3):70. https://doi.org/10.1186/scrt221

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4

Stem Cells in Wound Healing and Scarring Roohi Vinaik and Marc G. Jeschke

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wound-Healing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impaired Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excessive Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cell Subtypes in Skin Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embryonic Stem Sells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epithelial (Epidermal) Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications in Defective Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells in Overscarring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cell Therapy in Pathological Scarring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Stem cells are cells with the ability for self-renewal and differentiation into a myriad of cellular lineages. Here, we discuss their potential in skin regeneration, focusing on traumatic and nontraumatic healing and scarring. We identify and R. Vinaik Sunnybrook Research Institute, Toronto, ON, Canada e-mail: [email protected] M. G. Jeschke (*) Sunnybrook Research Institute, Toronto, ON, Canada Division of Plastic Surgery, Department of Surgery, University of Toronto, Toronto, ON, Canada Department of Immunology, University of Toronto, Toronto, ON, Canada Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_4

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elaborate on the various types involved, including embryonic stem cells (ESCs) and ESC-like cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). We discuss the role of iPSCs and MSCs in attenuating inflammation and fibrosis, thus promoting wound closure in models of defective wound healing and reducing both normal and aberrant scarring (i.e., keloids). In particular, we focus on MSCs and fibrotic changes, detailing their inhibitory function in TGFb/Smad signaling, and thus postinjury scar formation. Furthermore, we elaborate on ESCs and ESCs-like populations, discussing applications in normal skin appendage regeneration and recovery of nonhealing wounds, while ESCs-like cells function as a potential source of profibrotic keloid myofibroblasts. Although ESCs-like populations are implicated in scarring, the discussed studies posit that harnessing certain stem cell subpopulations could be an attractive strategy for rapid, scarless wound healing. This has implications in conditions of chronic inflammation and impaired healing and vascularity (e.g., diabetes) as well as traumatic conditions that necessitate rapid skin regeneration, such as burns. Keywords

Mesenchymal stem cells · Embryonic stem cells · Stem cells · Therapy · Transplantation · Wound Abbreviations

ADMSCs ADSCs AT BMP-2 CM ECM EGF EMT EPCs ESCs GDF HIF-1α IFN IL iPSCs LIF MMP MSCs NK PDGF PGE2 SSEA

Adipose tissue-derived MSCs Adipose-derived stem cells Adipose tissue Bone-morphogenetic protein-2 Conditioned medium Extracellular matrix Epidermal growth factor Endothelial-to-mesenchymal transition Endothelial progenitor cells Embryonic stem cells Growth differentiation factor Hypoxia-inducible factor Interferon Interleukins Human-induced pluripotent stem cells Leukemia inhibitor factor Matrix metalloproteinase Mesenchymal stem cells Natural killer Platelet-derived growth factor Prostaglandin E2 Stage-specific embryonic antigen

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TGF-b TNF-a VEGF

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Transforming growth factor Tumor necrosis factor-a Vascular endothelial growth factor

Introduction The integumentary system serves as a protective barrier against pathogens, dehydration, and fluid loss (Lee et al. 2006). Damage to skin integrity can occur due to nontraumatic (e.g., diabetic ulcers) or traumatic (e.g., burns) conditions and results in a carefully coordinated wound healing process comprised of overlapping phases of cellular migration, proliferation, and extracellular matrix deposition (Eming et al. 2007). However, disruptions or alterations in cellular signaling may culminate in a spectrum of poor wound healing, from chronic nonhealing wounds to excessive scarring (Eming et al. 2007). Either of the outcomes necessitates extensive, costly medical interventions, impairs patients’ quality of life, increases trauma, and enhances mortality. Therefore, significant efforts have been devoted to the development of rapid, scarless wound healing therapies. Regenerative medicine has emerged as an alternative to traditional wound care and focuses on stem cell therapy. Stem cells are an endogenous reservoir of cells that can self-renew and differentiate into a myriad of cell types. Poor wound healing and chronic wounds are correlated with either depletion in resident stem cells or an insufficient response in extensive injuries such as full-thickness burns (Nijnik et al. 2007; Zouboulis et al. 2008; Van Zant and Liang 2003). Therefore, exogenous administration is an attractive potential therapeutic strategy. In this chapter, we discuss the role of stem cells during normal, impaired wound healing, and “overhealing” (i.e., hyperproliferative scars such as keloids and hypertrophic scars). We elaborate on the key endogenous stem cell subtypes involved in skin regeneration, including embryonic, mesenchymal, epithelial, and melanocytic stem cells. Furthermore, we discuss the role of harnessing stem cells in therapy for patients with defective healing or excessive scarring, underscoring the broad application of stem cell therapy in wound-healing aberrancies.

Wound-Healing Process Normal Wound Healing Physiologic cutaneous healing consists of an inflammatory, proliferative, and remodeling phase (Fig. 1) (Gonzalez et al. 2016). Wound healing begins with hemostasis, or coagulation and fibrin clot formation. The fibrin clot serves as a scaffold to recruit key mediators involved in healing, including fibroblasts, keratinocytes, endothelial cells, and leukocytes (i.e., neutrophils and monocytes) (Broughton et al. 2006; Witte and Barbul 1997). In particular, activation of macrophages plays a significant role in the inflammatory phase, and macrophages regulate

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Fig. 1 Stages of post-trauma wound healing

the production of cytokines, angiogenesis, and recruitment of fibroblasts to the wound bed (Rodero and Khosrotehrani 2010). Fibroblast recruitment prompts type III collagen, proteoglycan, and elastin secretion during the proliferative phase, which results in granulation tissue formation (Tracy et al. 2016). This is coupled with the recruitment of vascular endothelial cells by vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF-b) secretion and angiogenesis (Corliss et al. 2016). This phase is followed by a tissue-remodeling phase after wound closure during which macrophages, fibroblasts, and endothelial cells are removed via apoptosis (Larouche et al. 2018). At this point, the wound site contains a primarily acellular collagenous matrix with predominantly type III collagen, which is replaced with type I collagen over time (Xue and Jackson 2015). In addition to the cells mentioned above, wound healing is in part mediated by stem cells, which secrete paracrine factors to recruit various cells and are present in all skin layers: epidermis, dermis, and hypodermis (Wong et al. 2012; Ito et al. 2005; Levy et al. 2005). In the epidermis, endogenous stem cells are comprised of three key populations – the basal layer of the inter-follicular epidermis, sebaceous glands, and the hair follicle bulge or lower region of the outer root sheath (Fig. 2) (Fuchs 2008). These stem cells have several key features in common, such as expression of K5, K14, and p63 and association with the basement membrane (Fuchs 2008). Under physiological conditions, differentiation of stem cells consistently replenishes the relevant skin components as mentioned above. Interestingly, cells from these components are capable of replenishing each other during injury. For example, reepithelialization in partial thickness injuries relies on the migration of cells from sebaceous glands and the hair follicle bulge (Rittié 2016). While these cells are not essential for wound closure and are eventually replaced with interfollicular epidermal stem cells during recovery, they significantly expedite wound closure (Ito and Cotsarelis 2008). Delayed healing is a risk factor for increased infection,

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Fig. 2 Anatomy of the hair follicle

hypertrophic scarring, and mortality, rendering these stem cell sources potential targets in regenerative therapy (Finnerty et al. 2016). Interestingly, dermal signaling (e.g., expression of BMP2 and BMP4) can also contribute to epithelial regeneration (Plikus et al. 2008). An example of this is the contribution of dermal sheath cells to melanocyte maturation. Melanocytic stem cells, which reside in the outer root sheath of the lower permanent portion of hair follicles, are responsible for pigmentation (Lee and Fisher 2014). However, dermal sheath stem cells additionally can differentiate into epidermal melanocytes, likely via mesenchymal-epithelial transition (Li et al. 2011). These cells reside within dermal papillae of hair follicles and are subdivided into three distinct populations based on the transcription factor Sox2. Sox2-expressing cells are involved with Wnt, BMP, and FGF signaling, while Sox2-negative cells exploit Shh, insulin growth factor (IGF), Notch, and integrin signaling (Kellner and Coulombe 2009; Driskell

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et al. 2009; Biernaski et al. 2009). Importantly, dermal sheath stem cells can differentiate into a wound-healing fibroblast (myofibroblast) phenotype, which has implications in scarring that will be discussed later (Jahoda and Reynolds 2001). Another dermal stem cells’ source is dermal perivascular regions, which function as a niche for mesenchymal stem cells (MSCs) (Jackson et al. 2012). These cells protect the integrity of the local wound-healing matrix via inhibition of matrix metalloproteinase (MMP) pathways and promotion of neovascularization (Jackson et al. 2012). MSCs can also be isolated from various sources, including bone marrow and adipose tissue (AT), and are later recruited to the site of injury in response to factors such as hypoxia-inducible factor (HIF)-1α (Martinez et al. 2017). MSCs, in turn, promote endothelial progenitor cell (EPC) migration, which facilitates angiogenesis and wound closure (Li et al. 2018). AT is another key source of multipotent skin stem cells (adipose-derived stem cells, ADSCs) that maintain the multilineage differentiation potential (e.g., smooth muscle, endothelium, and bone) (Frese et al. 2016). These ADSCs are associated with perivascular cells and promote vascular stability under normal conditions (Cherubino et al. 2011). Additionally, evidence suggests that ADSCs can enhance macrophage recruitment and anti-inflammatory polarization, fibroblast migration, VEGF-mediated angiogenesis, fibroblast growth factor-2 (FGF2), bone morphogenetic protein-2 (BMP2), and MMPs after injury (Lee et al. 2009). This culminates in neovascularization and deposition, and maintenance of a collagen matrix. While current studies suggest that ADSCs are intimately associated with vascular regeneration, additional research is needed to elucidate their role in the maintenance of skin integrity in normal wound healing (Hutchings et al. 2020; Yu et al. 2018; Lu et al. 2018). Given that AT is enriched in stem cells, ease of isolation, and the plasticity of ADSCs, these cells could potentially be harnessed for wound-healing applications in conditions of impaired healing.

Impaired Wound Healing Cutaneous wounds are of two subtypes: acute wounds (e.g., lacerations, abrasions, and surgical wounds), which generally heal uneventfully, and persistent, chronic wounds (e.g., diabetic ulcers). The latter occur secondary to interruptions in the healing process as a result of underlying medical conditions, including metabolic diseases (diabetes, obesity) and compromised blood supply (vascular disease, radiation injury) (Ojeh et al. 2015). Impaired circulation and enhanced tissue hypoxemia compromise wound healing by decreasing nutrient and oxygen supply, and hence the metabolic activity of cells involved in the repair, including keratinocytes and fibroblasts (MacKay 2003; Gottrup 2004). Additionally, patients with chronic wounds show an inadequate cellular response to relative hypoxia at the injury site. Under normal wound-healing conditions, hypoxia results in the activation of HIF-1a and recruitment of MSCs to the wound bed (Hong et al. 2014). As indicated earlier, these cells, in turn, promote EPC migration and neovascularization to enhance oxygen delivery to the site of injury (Li et al. 2018). However, EPCs obtained from patients with chronic wounds such as

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diabetes show decreased hypoxia-induced adhesion, migration, and proliferation (Vasa et al. 2001; Tepper et al. 2002; Loomans et al. 2004). This culminates in impaired angiogenesis and prolonged ischemia, further compromising wound healing (Capla et al. 2007). As discussed in the context of routine wound healing, ASCs also play a key role in improving vascularization and maintaining the collagen matrix. Complementarily, diabetic wounds exhibit functionally impaired ASCs, which results in diminished growth factor production (Cianfarani et al. 2013). Combined with abnormal cytokine signaling, decreased growth factor production detrimentally impacts wound-healing outcomes (Galkowska et al. 2006). In addition to promoting revascularization, factors such as FGF also contribute to the recruitment and replication of stem cells (i.e., MSCs), further enhancing growth factor production (Rodrigues et al. 2010). Lack of growth factors is associated with impaired recruitment and function of vascular endothelial cells and multiple stem cell lineages. The source of these factors is macrophages, and the altered macrophage activity (e.g., in diabetic ulcers) is a responsible cause and result of altered signaling pathways (Maruyuma et al. 2007). Macrophages obtained from chronic diabetic wounds release fewer growth factors relative to healthy tissue macrophages, functioning as a significant cause of decreased angiogenesis and impaired healing in these patients (Toma et al. 2005; Yamanishi et al. 2012). Taken together, growth factors and stem cells have a complementary relationship, and aberrancies in either can result in impaired or excessive healing.

Excessive Wound Healing On the opposite spectrum of healing is overhealing or pathological scarring, which includes hypertrophic scars and keloids. The scarring has significant detrimental physical, social, and psychological consequences, providing an impetus for further research regarding tissue regeneration. Many studies focus on differentiating mechanisms underlying adult versus fetal wound healing, which is scar-free. Interestingly, midgestational fetal wounds heal rapidly and are characterized by regeneration of all skin components (including skin appendages) and maintenance of identical collagen patterns to uninjured tissue (Hu et al. 2014). This contrasts to adult wounds, which lack skin appendages and heal by a fibroproliferative response characterized by a disorganized collagen network, resulting in scar tissue that vastly differs from intact skin (Fig. 3) (Hu et al. 2014). We compare mechanisms in the fetal healing process to adult skin repair to understand the causative factors underlying pathological scarring. Fetal wounds differ from adult wounds regarding to inflammatory responses, extracellular matrix (ECM) components, and differential growth factor expression (Mast 1992). For example, fetal wounds exhibit a paucity of immune cells, lower levels of the proinflammatory cytokine IL6 and elevated anti-inflammatory IL10, immediate collagen deposition with a higher Type III to Type 1 collagen ratio, higher VEGF and HIF-1a production, and a lack of profibrotic myofibroblasts compared to postnatal wounds to list a few distinctions (Table 1) (Lo et al. 2012). In addition to

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Fig. 3 Anatomy of (a) normal skin, (b) hypertrophic scar, and (c) keloid

Table 1 Comparison between fetal versus adult wound healing. (Modified from Lo et al. (2012)) Characteristics ECM collagen Deposition rate Histology Type III/type I ratio Cross-linking Inflammatory response Inflammatory cells IL6 and IL8 IL10 Growth factors VEGF TGF-b1 and b2 HIF-1a Fibroblasts and stem cells Myofibroblasts (day 14) MSCs presence

Fetal

Adult

Immediate Fine, reticular, and basket-weave Higher Low levels

Delayed Dense parallel bundles Lower High levels

Few Low levels High levels

Many High levels Low levels

Higher Low levels Higher

Lower High levels Lower

Absent Higher levels at injury site

Present Lower levels at injury site

HIF hypoxia-inducible factor-1a, IL interleukin, MSCs mesenchymal stem cells, TGF-b transforming growth factor-b, and VEGF vascular endothelial growth factor

the aforementioned factors, fetal wounds also exhibit differences in epidermal and dermal stem cell location and function. For example, postnatal epidermal stem cells reside in the hair follicle bulge, which fetal skin lacks (Nowak et al. 2008). Additionally, as skin development progresses, these epidermal stem cells change in the pattern of cell division. Cell divisions are predominantly symmetric and parallel to the basement membrane initially in embryonic development. In later stages, stem cells begin to undergo asymmetric cell division, which correlates with

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a transition from a scar-free to scarring healing phase (Hu et al. 2014). While a significant amount of research is focused on epidermal stem cells, differential responses in dermal stem cells likely contribute to aberrant scarring (Hu et al. 2014). Hereto, we identified stem cells as contributors to skin regeneration and a promising therapeutic option in impaired healing. However, some studies have provided conflicting results, demonstrating that specific subpopulations of dermal stem cells (MSCs) may increase inflammation and risk of hypertrophic scarring (Zhang et al. 2009a; van der Veer et al. 2009; Ding et al. 2013). Furthermore, increasing evidence suggests the involvement of stem cells in keloid pathogenesis. For example, aberrant fibroblasts and profibrotic myofibroblasts may originate via epithelial-to-mesenchymal transition from MSCs, which in turn are derived from embryonic stem cells (ESCs) (Lee et al. 2015). In turn, keloid dermis-derived stem cells that express ESCs-specific markers OCT4, NANOG, Sox2, and pSTAT3 (ESC-like cells) promote aberrant fibroblasts and profibrotic myofibroblasts through an MSCs intermediate (Lim et al. 2019; Grant et al. 2016; Bagabir et al. 2012; Zhang et al. 2009b). Likely, establishment of a local inflammatory environment increases the number of keloid-derived precursor stem cells with a distinct MSCs-specific marker expression profile from normal skin precursor cells. The inflammatory niche induces TGFb, epidermal growth factor (EGF), FGF, and insulin-like growth factor (IGF) secretion by keloid-associated stem cells, which in turn results in excessive collagen production and ECM deposition, further recruitment of inflammatory cells and production of inflammatory mediator interleukins (IL) (e.g., IL1a, IL6), and scar formation (Smith et al. 1998; van der Veer et al. 2009). Taken together, studies suggest that the immune system and post-trauma-inflammatory period have an integral role in scarring, which likely occurs via local stem cell dysregulation. In the subsequent section, we will discuss the specifics of the various stem cell populations involved in wound healing.

Stem Cell Subtypes in Skin Regeneration Stem cells have an integral role in physiologic, impaired, and excessive healing. Given their general prohealing effect, stem cells can putatively be harnessed therapeutically. Here, we discuss the various stem cell subtypes involved in skin regeneration.

Embryonic Stem Sells ESCs, which are isolated from the inner cell mass of blastocysts, express the cell surface antigens, including stage-specific embryonic antigen (SSEA) SSEA3, SSEA4, T cell receptor alpha locus (TRA) TRA-1-60, and TRA-1-81 and transcription factors OCT4, NANOG, and Sox2 (Thompson et al. 1998; Reubinoff et al. 2000; Clark et al. 2004). The latter transcription factors promote self-renewal genes

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while inhibiting differentiation genes, establishing ESCs’ pluripotency and the potential to differentiate into any germ layers – endoderm, mesoderm, or ectoderm from which skin develops (Thomas et al. 1998). ESCs can form all somatic tissues and express high levels of telomerase, hence preventing cellular senescence. Depending on the growth factor milieu, ESCs can differentiate into keratinocytes and generate epidermal layers, besides showing proangiogenic properties, thus making them an attractive option for bioengineered skin substitutes (Aberdam 2004; Rufaihah et al. 2010). However, further research is needed before an efficient, controlled expansion and differentiation to particular cell types is feasible. Additionally, studies are required on how to deliver cells in a manner in which they can survive and integrate effectively in patients (de Wert and Mummery 2003). While these challenges occur in ESCs and adult stem cells, ESCs have additional considerations to adult stem cells. For example, ESCs use is still controversial due to ethical concerns regarding harvesting cells from live embryos (de Wert and Mummery 2003). There are also concerns for potential immune rejection and teratoma formation (Hentze et al. 2009). The latter is likely since ESCs harbor qualities reminiscent of cancerous cells, including self-renewal, persistent proliferation, lack of contact inhibition, and proangiogenic features (Burdon et al. 2002). ESCs also have reduced CpG island methylation in specific genes, allowing for increased gene expression compared to differentiated cells (Altun et al. 2010). This is coupled with hypermethylation and silencing of tumor-suppressor genes, a phenomenon also seen in cancerous cells (Altun et al. 2010). Given that ESCs are potentially tumorigenic, further work is needed before they can be implemented clinically. In the meantime, adult stem cells have garnered greater interest as an alternative source of stem cells for the treatment of diseases.

Induced Pluripotent Stem Cells While not an endogenous stem cell population, induced pluripotent stem cells (iPSCs) produced from adult-derived cells have significant therapeutic potential and merit an in-depth discussion (Ibrahim et al. 2016). iPSCs are programmed in vitro via induction of Oct4/Sox2/c-Myc/KLF4 or Oct4/Sox2/NANOG/LIN28 (Takahashi et al. 2007; Takahashi and Yamanaka 2006; Yu et al. 2007). They are pluripotent cells with the capacity for self-renewal and the ability to differentiate into any adult cell type, similar to ESCs (Ibrahim et al. 2016). As they are derived from adult somatic cells, iPSCs circumvent potential ethical issues faced with ESCs. Furthermore, iPSCs can easily be derived from the adult tissue harvested from multiple sources, including, bone marrow, skeletal muscle, and skin, thus providing a renewable source of pluripotent stem cells (Ahmed et al. 2011a; Buccini et al. 2012; Gorecka et al. 2019). They are also being used for disease modeling (Cagavi et al. 2018). Due to this ability, iPSCs-derived cells are potentially able to target each phase of wound healing. During the inflammatory phase, they can ameliorate impaired growth factor and cytokine secretion, promoting macrophage, fibroblast, and

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keratinocyte secretions lacking in chronic wounds (Casqueiro et al. 2012; Clayton et al. 2018; Kim et al. 2013; Açikgoz et al. 2004). During the proliferative phase, iPSC-derived cells promote angiogenesis and collagen deposition and include endothelial cells, smooth muscle cells, fibroblasts, keratinocytes, etc. (Tepper et al. 2005). Finally, the recruitment of cells such as fibroblasts and myofibroblasts during the proliferative phase impacts the remodeling phase of healing (Grenier et al. 2007). Interestingly, iPSCs can be induced to produce other stem cell populations such as MSCs, which are multipotent stem cells capable of differentiating into various cell types (discussed in the subsequent section). MSCs obtained from models of poor healing (e.g., diabetic wounds) exhibit impaired proliferation, differentiation, and production of proangiogenic factors (Aasen et al. 2008). IPSCs-derived MSCs may have a better wound-healing potential, and we later discuss iPSCs-based treatment in conditions of poor healing. However, while iPSCs may have a prohealing advantage over MSCs, several potential issues are associated with their implementation. Like ESCs, iPSCs exhibit tumorigenic potential and can form teratomas when undifferentiated (Ahmed et al. 2011b; Gledhill et al. 2015; Krause et al. 2001). Currently, there are strategies to minimize teratoma risk, including differentiation before cell transplantation (Bedel et al. 2017). Another potential issue is that initial production required retroviral transfection with the risk of viral integration into the host genome and insertional mutagenesis. However, more recent techniques are nonintegrative, circumventing potential safety issues (Haridhasapavalan et al. 2019; Malik et al. 2013; Deng et al. 2015). Taken together, iPSCs have a potential application in wound healing, although further studies regarding their safety profile and improvements in iPSCs’ generation are needed at this point.

Mesenchymal Stem Cells MSCs are progenitor cells for connective tissue found in multiple sites, including AT, bone marrow, and nerves (Danisovic et al. 2009). To classify as an MSC, cells should exhibit plastic adherence, express specific cell surface markers (CD73, CD90, and CD105), and lack CD14, CD34, CD45, and HLA-DR, and be able to differentiate in vitro into either adipocytes, chondrocytes, or osteoblasts (Dominici et al. 2006). While the aforementioned characteristics apply to all MSCs, slight variations are depending on the tissue of isolation. Here, we focus on adipose tissuederived MSCs (ADMSCs), which express the previous factors plus CD29, CD44, CD71, CD13, CD166, and STRO-1 (Ullah et al. 2015). Akin to ESCs, MSCs have beneficial features for skin regeneration applications, including self-renewal, the ability to home toward wounds, rapid proliferation, and the capacity to differentiate into a myriad of cell types (Sackstein 2004). Their prohealing effects can be attributed in part to the release of growth, cell recruitment, and immunoregulatory factors in response to inflammatory mediators that accumulate at the site of injury (Prockop and Oh 2012). An added advantage over ESCs is that MSCs are not immunologically active due to low MHC1 and lack of MHCII and costimulatory CD80, CD40, and CD86, which protects MSCs from natural killer

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(NK) cell lysis (Rasmusson et al. 2003). Furthermore, MSCs can inhibit NK and cytotoxic T-cells via various pathways, such as secretion of human leukocyte antigen G5, leukemia inhibitor factor (LIF), and interferon (IFN) (Selmani et al. 2008; Nasef et al. 2008; Sheng et al. 2008). More specifically, MSCs induce T-cell apoptosis, which enables macrophages to produce TGFb, thus promoting the generation of regulatory T-cells and macrophage phenotype switching to anti-inflammatory subtypes (Ohe et al. 2015; Barrandon and Green 1987; Green 2008). These immunomodulatory effects depend on the quantity and type of cytokines present and diminish the risk of immune rejection, making MSCs a viable option in inflammatory conditions and other clinical applications (Mansilla et al. 2005; Falanga et al. 2007). However, the intensity of inflammation regulates MSC-mediated immunomodulation, necessitating a healthy patient inflammatory status for optimal efficacy (Wang et al. 2014). Recent research identified ADMSCs within subcutaneous tissue, purporting a skin regeneration role (Marfia et al. 2015). In this context, ADMSCs have several advantages, including ease of harvesting and enhanced proliferative and immunosuppressive properties (Jacobs et al. 2013; Pachón-Peña et al. 2011). The ability for ADMSCs to proliferate, differentiate, and migrate is inherently dependent on the local milieu. ADMSCs home in on the injury site via enhanced expression of CXCR4, which modifies local immune cell (i.e., macrophages, T-cells, B-cells, and dendritic cells) inflammatory phenotypes to a healing, anti-inflammatory one likely via TGFb or growth differentiation factor (GDF) GDF11-mediated activation (Baharlou et al. 2017; Hyldig et al. 2017; Mazini et al. 2019). Production of TGF-b and GDF11, growth factors (i.e., FGF, VEGF), and cytokines, i.e., IL6, tumor necrosis factor-a (TNF-a), is likely induced by local IL6 production during the inflammatory phase (Mazini et al. 2020). Taken together, ADMSCs interact with dermal fibroblasts via cytokine and growth factor production to regulate their microenvironment. ADMSC secretome stimulates the migration and proliferation of dermal fibroblasts and keratinocytes, besides collagen and elastin deposition (Choi et al. 2018; Ferreira and Gomes 2018). Thus, preconditioning ADMSCs with a particular cytokine and growth factor combinations can enhance therapeutic benefits, which is an essential consideration for clinical applications.

Epithelial (Epidermal) Stem Cells Studies suggest that the epidermis comprises a basal layer containing 2–7% of stem cells (Potten et al. 1979). Although integrins, which are responsible for attaching the epidermal basal layer to the basement membrane, may be a potential candidate, these stem cells lack specific markers. Evidence suggests that epithelial stem cells, including epidermal interfollicular, sebaceous gland, and hair follicle bulge stem cells, have slightly different characteristics. For example, interfollicular stem cells are less potent than bulge stem cells, possibly suggesting that they are the progeny of bulge cells (Alonso and Fuchs 2003). Interfollicular cells lack a distinct niche and are more easily induced to proliferate, although injuries that destroy the interfollicular

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epidermis (i.e., superficial burns) but leave hair follicles intact can regrow epithelium and do not need grafting (Alonso and Fuchs 2003; Green 1991). Hair follicle bulge cells are multipotent cells capable of differentiating into all skin epithelial lineages (e.g., hair follicles, keratinocytes) and forming a stratified epidermis in vitro (Rochat et al. 1994). Taken together, this suggests that epithelial stem cells have different functions and importance, which has implications in skin regeneration and necessitates a greater understanding of the underlying signaling pathways. In particular, Wnt/beta-catenin signaling is associated with the ability of epithelial cells to exhibit multipotency features (Zhou et al. 1995; Gat et al. 1998; Andl et al. 2002). While only bulge cells are considered multipotent, stabilization of skin betacatenin induces adult interfollicular epidermal cells to behave similarly to embryonic skin. That is, they can differentiate into epidermal cells or hair follicles. Wnt signaling in embryonic skin epithelial cells results in beta-catenin-mediated activation of the DNA-binding protein family Lef/Tcf (Nusse 1999). When skin MSCs inhibit the BMP pathway, multipotent epithelial stem cells express Lef1 and commit to hair follicle formation (Jamora et al. 2003). The extent of Lef1/Tcf activation is essential in stem cell lineage commitment, and overexpression of Lef1 in skin epithelium results in inappropriate hair follicle formation, while interfering with Lef1/beta-catenin association results in hair follicles adopting a sebaceous cell fate (Zhou et al. 1995; Merrill et al. 2001; Niemann et al. 2001). Interestingly, despite consistently elevated stabilized beta-catenin levels and, hence, epithelial bulge stem cell commitment to hair follicle morphogenesis, the overall size of the stem cell niche does not change (Lowry et al. 2005). This suggests a balance between betacatenin-mediated self-renewal of bulge stem cells and their efflux from the niche. Under physiologic conditions, hair follicle and other epidermal cells function as distinct lineage niches. However, injury to the epidermis results in the recruitment of stem cells from different epidermal compartments, allowing for repopulation of cells in other epidermal compartments and expediting wound closure. Given endogenous stem cells’ role in wound healing, we subsequently assessed stem cells in defective and excessive healing, highlighting the pros and cons of different stem cell types.

Applications in Defective Wound Healing Chronic wounds, frequently associated with impaired stem cell function, can be managed with clinical approaches involving stem cell application. Several stem cell subtypes have potential in wound-healing management, including ESCs, EPCs, iPSCs, and MSCs (bone marrow and AD). Initially, ESCs were identified for their ability to differentiate into any of the three primary germ layers and, thus, their potential application in wound healing; however, ESCs can generate tumors and are controversial, as discussed earlier (Kanji and Das 2017). Therefore, focus has been shifted to adult stem cells as alternative treatment options. Here, we discuss current stem cell treatments and their effect on various physiologic events involved in wound healing: inflammation, angiogenesis, and fibrosis.

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Endogenous bone marrow-derived EPCs have a crucial role in promoting wound healing via angiogenesis. However, migration to the injury site occurs in response to hypoxic factors, which are impaired in conditions such as diabetes (Tepper et al. 2005; Ceradini et al. 2004). Exogenous stem cell transplant is an alternative strategy to circumvent this issue. A study utilizing diabetic murine models demonstrated that injecting CD34+ EPCs into ischemic wounds enhances vascular density and minimizes healing time (Sivan-Loukianova et al. 2003). Furthermore, injection of CD34+ hematopoietic progenitor cells into human chronic sacral ulcers decreased wound volume by 60% within 3 weeks of administration (Wettstein et al. 2014). Although the sample size was small (three patients), this study provided evidence of efficacy with stem cell treatment and, in this case, no tumorigenicity, paving the way for additional human clinical studies utilizing stem cells. An alternate source of stem cells is differentiated adult somatic cells, which can be de-differentiated to pluripotent iPSCs. For example, murine fibroblasts or keratinocytes treated with a combination of Oct3/4, Sox2, c-Myc, and Klf4 can revert to iPSCs, which in turn differentiate into tissue from all three germ layers (Takahashi and Yamanaka 2006; Aasen et al. 2008). In addition to the ability to induce pluripotency, directing differentiation into particular cell types is critical. In this regard, iPSCs can be selectively differentiated into fibroblasts and keratinocytes and, as an added advantage, elicit an anti-inflammatory response (Lu et al. 2014). Taken together, these characteristics render iPSCs an attractive option for treating chronic wounds and skin conditions (Zhang et al. 2015a). Indeed, iPSCs-derived cells obtained from patients promote healing and enhance collagen secretion (Gorecka et al. 2019). In murine models, iPSCs-derived MSCs injected into the wound site secrete type-VII collagen at the dermal-epidermal junction and improve epithelialization (Nakayama et al. 2018). These iPSCs can be obtained from donors with preexisting conditions (e.g., diabetes, epidermolysis bullosa), allowing autologous treatment and minimizing rejection (Tolar et al. 2011). This is coupled with a proangiogenic potential, allowing for potential applications in ischemic injury (e.g., myocardial infarction, peripheral arterial disease, and retinopathy). However, further studies regarding the safety-enhancement of iPSCs’ reprogramming protocols are needed to assure minimal risk of mutagenesis and teratogenicity (Okita et al. 2007). Similar to iPSCs, MSCs also lack immunological reactivity and are capable of rapidly proliferating and differentiating into a wide range of cell types (Nakagawa et al. 2005; Krause et al. 2001). MSCs express high levels of VEGF and angiopoietin1 or may function as pericytes, indicating that prohealing effects are due in part to enhanced angiogenesis and stabilization of blood vessels in normal and impaired healing models (Dai et al. 2007; Shumakov et al. 2003; Kwon et al. 2008). Clinically, MSCs-based therapy has also shown to improve healing by improving angiogenesis and dermal vascularity and thickness, increasing reepithelialization and granulation tissue formation, and, more importantly, modulating the immune response (Badiavas et al. 2003; Vojtassak et al. 2006; Cha and Falanga 2007). Irrespective of the source, MSCs-based cell therapy lowers inflammatory cell numbers and proinflammatory cytokines (e.g., IL1, TNFa) and enhances IL10 at the site of injury in animal wound-healing models (Liu et al. 2014a). Furthermore, MSC-based treatment influences macrophage polarization, promoting an anti-inflammatory M2 macrophage

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profile in murine wounds (Zhang et al. 2010). Since macrophage profile and inflammation also regulate fibrosis, the immunomodulatory effect putatively attenuates excessive ECM deposition and abnormal scarring, which will be discussed in-depth in the subsequent section.

Stem Cells in Overscarring MSCs administration increased the tensile strength of wounds and reduces wound contracture and scarring due to (1) anti-inflammatory effects, (2) paracrine signaling, and (3) modified collagen deposition and ECM remodeling (Hu et al. 2018). Prolonged inflammation can induce fibrosis, and the anti-inflammatory effect of MSCs in part accounts for reduced fibrosis. MSCs upregulate prostaglandin E2 (PGE2) that inhibits IL2 expression and diminishes T cell proliferation in the wound (Németh et al. 2009; Jarvinen et al. 2008; Djouad et al. 2007). PGE2 also facilitates the transition from TH1 to TH2 cells, which corresponds with a reduction in IFN expression and upregulation of IL4 (Zanone et al. 2010; Aggarwal and Pittenger 2005). Decreased IFN relative to IL4 favors anti-inflammatory M2 macrophages, which results in decreased proinflammatory signaling (Stout 2010; Varin and Gordon 2009). This is accompanied by the secretion of various factors that promote tissue regeneration. MSCs secrete various antifibrotic cytokines and growth factors including hepatocyte growth factor (HGF), FGF2, VEGF, IL10, and adrenomedullin (Li et al. 2008, 2009; Chen et al. 2008; Du et al. 2016). In particular, fibroblasts respond to HGF secretion by downregulating collagen I/III and TGFb1 expression via nuclear exclusion of SMAD3, a transcriptional factor associated with several profibrotic genes (Mou et al. 2009; Schievenbusch et al. 2009; Inagaki et al. 2008). In addition to enhanced HGF and VEGF, TGFb1/b2 neutralization or addition of TGFb3 is also associated with the scar-free repair, and MSCs maintain a higher ratio of TGFb3/ TGFb1 protecting against abnormal scar formation (Ono et al. 2004; Shah et al. 1995). MSCs contribute to collagen type III secretion, and a higher type III: Type I ratio is characteristic of scar-free fetal tissue healing (Longaker et al. 1990; Fathke et al. 2004). In addition to targeting collagen deposition, MSCs-secreted factors, such as HGF, upregulate the matrix metalloproteinases MMP-1, MMP-3, and MMP-13 in fibroblasts, and promoting ECM turnover (Kanemura et al. 2008). Interestingly, MSCs may inhibit profibrotic myofibroblast differentiation via HGF secretion (Shukla et al. 2009; Abe et al. 2001). While myofibroblast differentiation may be needed in normal healing, these cells produce excessive ECM compared to dermal fibroblast and may contribute to scar formation (Bucala et al. 1994). Elevated TGFb1, observed in conditions characterized by a prolonged acute inflammatory response, mediates myofibroblast differentiation and, as a result, contributes to the excessive ECM deposition, the formation of tight collagen bundles, and wound contraction resulting in scarring. Downstream effects of MSCs-mediated inhibition of myofibroblasts yield less scar tissue, as witnessed in oral epithelial healing that is characterized by high HGF levels and lack of significant myofibroblast differentiation (Shannon et al. 2006).

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Interestingly, ESC-like populations are implicated in fibrosis in models of aberrant healing (i.e., keloids). While MSCs attenuate inflammation and collagen remodeling, MSC intermediates may differentiate into keloid myofibroblasts (Lee et al. 2015). Keloid dermis contains a population of cells that express elevated ESCs-specific markers OCT4, NANOG, SOX2, and pSTAT3 in perivascular cells within keloid-associated lymphoid tissue, serving as a source of aberrant fibroblasts (Lee et al. 2015). This potentially occurs via the previously mentioned MSC intermediate, generated by the endothelial-to-mesenchymal transition (EMT). Interestingly, while MSCs-based treatment may have an actual clinical application in wound healing and scar prevention, endogenous dermal keloid-associated stem cells contribute to keloid pathogenesis. Thus, further studies regarding the complex signaling pathways involved in wound healing and the contribution of stem cells in the process are needed at this time to further enhance our understanding of the molecular mechanisms involved therein.

Stem Cell Therapy in Pathological Scarring Several studies demonstrated the use of stem cell-based therapy for hypertrophic scar and keloid treatment. For example, iPSCs-conditioned medium (CM) may suppress hypertrophic fibroblast activation, collagen I, as well as, alpha-smooth muscle actin, a marker for myofibroblasts (Ren et al. 2015). Furthermore, iPSCs-CM-secreted factors block inflammatory cell recruitment, decrease adhesion, and mitigate the contractile ability of dermal fibroblasts (Ren et al. 2015). In addition to multipotent iPSCs, MSCs also could be harnessed for scar treatment. An example of this is Wharton’s jelly MSCs (WJ-MSCs), which exhibit antifibrotic properties, and no teratoma formation or rejection (Gauthaman et al. 2012; Bongso and Fong 2013). In addition to preventing fibrosis, WJ-MSCs enhance healing in immunodeficient mice and diminish scarring in other animal models (Sabapathy et al. 2014; Azari et al. 2011). Bone marrow-derived MSCs similarly have been used to treat hypertrophic scars in rabbit models and murine skin fibrosis, facilitating the formation of uniform, basket-weave collagen organization analogous to normal skin with minimal inflammatory cells (Liu et al. 2014b; Wu et al. 2014). This is combined with a marked decrease in profibrotic TGFb1 and upregulation of MMPs (e.g., MMP-2/9/13) (Wu et al. 2014). Alternatively, injection of ADSCs in a rabbit-hypertrophic scar model led to the normal-appearing scars with a reduced scar elevation index (Zhang et al. 2015b). Likely, ADSCs modify the local host microenvironment via the production of antioxidants, free radical scavengers, and heat shock proteins, promoting normal wound healing (Gimble et al. 2007).

Conclusion In this chapter, we highlight the mechanisms underlying normal and aberrant posttrauma healing and the role of stem cells in these processes. In particular, we discuss how endogenous stem cells can impact all the phases of wound healing:

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inflammatory, proliferative, and remodeling. Currently, the studies suggest that application of stem cells may benefit dermal wound healing by accelerating reepithelialization, improving the tensile strength of new tissue, promote healing in chronic wounds, and minimize excessive scarring. However, further work regarding safety and regulation, clinical translatability, ability to scale up production, and efficient means of stem cell delivery is needed before widespread clinical use.

Cross-References ▶ Induced Pluripotent Stem Cells ▶ Vascular Functional Recovery and Reparation by Human Endothelial Progenitor Cells

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Mesenchymal Stem Cells From Identification and Characterization to Clinical Applications Maria Alvarez-Viejo and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells: Fist Description in Bone Marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone Marrow-Derived MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of MSCs from Different Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adipose Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Umbilical Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Umbilical Cord Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wharton’s Jelly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization MSCs Before Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STRO-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD271 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SSEA-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD146 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs in the Clinical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Based Therapy for Cardiovascular Pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Based Therapy for Acute Graft-Versus-Host Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Based Therapy for Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Based Therapy for Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Based Therapy for Rheumatoid Arthritis and Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . .

129 129 130 131 131 132 132 133 134 135 135 136 137 137 138 138 141 142 143 144

Maria Alvarez-Viejo and Khawaja Husnain Haider contributed equally with all other contributors. M. Alvarez-Viejo (*) Unidad de Terapia Celular y Medicina Regenerativa, Servicio de Hematología y Hemoterapia, Hospital Universitario Central de Asturias HUCA, Instituto de Investigación Biosanitaria del Principado de Asturias, Universidad de Oviedo, Asturias, Spain e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_6

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Exosomes-Based Therapeutic Intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Derived Exosome for Therapy “Without Cells” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Derived Exosomes and Acute Graft-Versus-Host Disease . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Derived Exosomes for Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Derived Exosomes for Rheumatoid Arthritis and Knee Osteoarthritis . . . . . . . . . . . . . MSCs-Derived Exosomes for the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Since Friedenstein and co-workers first reported bone marrow-derived mesenchymal stem cells (MSCs) during the early 1970s, several researchers have focused on their biology and in vitro and in vivo characterization in experimental animal models. After their initial isolation from the bone marrow, MSCs have also been isolated from almost every tissue including the adipose tissue, liver, skeletal muscle, amniotic fluid, umbilical cord blood, and dental pulp. Following the minimum criteria recommended by the International Society for Cellular Therapy, human MSCs show preferred plastic adherence in culture, can undergo trilineage differentiation (i.e., adipogenic, chondrogenic, and adipogenic), besides the expression of CD44, CD71, CD90, CD105, and lack of CD34 and CD45 expression. Other important markers include Stro-1, SSEA-4, CD146, and CD271. This identification criterion has gone a long way in comparing the findings from independent research labs involved in MSCs research. MSCs show remarkable immunomodulatory and anti-inflammatory properties besides their ability to undergo transdifferentiation and paracrine activity. These characteristics make them ideal candidates for cell therapy and for this reason; there are numerous clinical trials worldwide that use MSCs to treat various pathologies including myocardial infarction for which they have entered into Phase III trials. They also secrete exosomes as part of their paracrine activity, which has led to the emergence of “cell therapy without cells” approach. Several studies have suggested that exosomes, small extracellular vesicles, derived from MSCs could serve as a novel therapeutic tool in the field of regenerative medicine. This book chapter discusses in-depth the advancements in the field of MSCsbased cell therapy and cell-free therapy using their derivative exosomes with a special focus on the clinical perspective. Keywords

Bone marrow · Cell-free therapy · Clinical · Exosome · Mesenchymal · MSCs · Regenerative · Transplantation List of Abbreviations

AMI ASC BM

Acute myocardial infarction acute myocardial infarction Adipose stem cell Bone Marrow

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Mesenchymal Stem Cells

FAK F-CFU GvHD HSCs IBD LNGFR MSCs NGFR NTR UC UCB TNF WJ WJ-MSCs

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Focal adhesion kinase Fibroblast-colony forming unit Graft-versus-host disease Hematopoietic stem cells Inflammatory bowel disease Low-affinity nerve growth factor receptor Mesenchymal stem cells Nerve growth factor receptor Neurotropin receptor Umbilical cord Umbilical cord blood Tumor necrosis factor Wharton’s Jelly Wharton’s jelly derived MSCs

Introduction Mesenchymal Stem Cells: Fist Description in Bone Marrow The term mesenchymal stem cell (MSCs) is widely accepted today. The existence of cells in various tissues with the ability to differentiate into diverse cell types is assumed. But, when does this concept arise? We go back to the end of the nineteenth century when Goujon (1869) demonstrated that transplanted bone marrow (BM) in a heterotopic site generated bone and marrow de novo (Goujon 1869). A century later, Tavassoli and Crosby advanced knowledge of the potential of BM with their experiments published in a prestigious scientific journal (Tavassoli and Crosby 1968). These authors observed that autologous transplanted marrow fragments survived in various extramedullary sites in the rat, rabbit, and dog. These pioneering works revealed some cellular elements with proliferation and differentiation capacity in the BM. Based on their work, Friedenstein and collaborators would be the first to suggest a cell type in the BM with the ability for differentiation; it would be a different cell type to hematopoietic progenitors. They demonstrated that BM contains a population of cells with a high proliferative capacity that adhered to plastic and that presented a morphology characteristic fibroblast-like. These authors were also the first to propose the ability of these cells to form colonies from a single cell (the fibroblast-colony forming unit (F-CFU)) (Friedenstein et al. 1970). But it would not be until the beginning of the 1990s when the term MSCs was coined. Caplan proposes this name in analogy with “hematopoietic stem cells (HSCs)” (Caplan 1991). The concept of non-HSCs and their presence in BM was reinforced by Pittenger and co-workers’ pioneering data published in 1999 (Pittenger et al. 1999). The abstract published in a prestigious journal says, Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal

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stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

This text has already summarized two decades ago what has now been widely accepted as MSCs. Numerous laboratories worldwide became interested in this cell type, and many works began to be published from here onwards. However, there was no defined approach to characterize MSCs due to the lack of specific surface markers. Moreover, they constitute a heterogeneous group of subpopulations and possess a distinct expression of surface proteins and differentiation potential besides showing source-specific transcriptome under a given set of culture conditions (Elahi et al. 2016). Hence, various methods of cell isolation, expansion, and characterization protocols have been reported in the literature to ensure a uniform/homogeneous population of cells. Their low propensity in the BM, lack of surface markers, and diversity in the methods and protocols of isolation, purification, and expansion protocols rendered it difficult to compare the findings originating from independent research laboratories worldwide. To address this critical issue and standardize MSCs preparations for experimental and clinical usage, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has established standard criteria to fully define and characterize human MSCs preparations (Haider 2018). The minimal criteria include plastic-adherence when maintained in standard culture conditions; they must express specific surface antigens, that is, CD44, CD73, CD90, and CD105 and absence of CD11b, CD14, CD19, CD34, and CD45. Besides, they should undergo trilineage (i.e., adipogenic, chondrogenic, and osteogenic) differentiation in vitro (Dominici et al. 2006). Some additional surface markers expressed by MSCs include, CD9, Cd10, CD13, CD29, CD51, CD54, CD117, CD166, and Stro-1. These criteria facilitated the work of many groups involved in experimental and clinical research with this cell type. The favorable characteristics of MSCs, availability without any moral and ethical issues, ease of availability, simple isolation and undifferentiated in vitro expansion protocols, multilineage differentiation potential, inherent paracrine activity, and robust nature to carry exogenous genetic material for genetic therapy, etc., have made them attractive candidates for clinical application (Haider and Ashraf 2005).

Bone Marrow-Derived MSCs Currently, obtaining MSCs from BM is well-established in various laboratories around the world. Usually, spinal aspirates are obtained by aspiration of the iliac crest. Mononuclear cells are isolated from the BM aspirate on a Ficoll-density gradient by centrifuging. After washing twice the cells are re-suspended in culture medium and finally seeded into adequate flasks. Thereafter, the medium must be changed every 2–3 days and wait a time that may vary, but it may be around 2 weeks

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to obtain the confluence of the cells. To check the quality of the culture-expanded cells, the three parameters indicated above must be fulfilled – adherence to plastic, presence of specific surface antigens, and the ability to undergo multilineage differentiation (Dominici et al. 2006). Although BM remains the most used, and well-studied and characterized tissue source of MSCs, obtaining MSCs from BM has many drawbacks such as invasiveness of the process, pain and discomfort for the donor, morbidity, due to high proliferative capacity and resistance to apoptosis (Musiał-Wysocka et al. 2019). Moreover, availability in low cell numbers (only 0.001–0.00001 percent of the total cell population) necessitates in vitro expansion, making them immunogenic and may induce genetic instability and chromosomal abrasions (Haider 2006). This searches for alternative tissues to obtain this cell type with the capacity for selfrenewal and differentiation.

Isolation of MSCs from Different Sources Until recently, MSCs have been isolated from almost every tissue in the body. Some tissue sources and their derivative MSCs have been discussed in the following sections. Our focus will be only on some of these tissue sources for MSCs due to their interest in clinical applications.

Adipose Tissue Adipose tissue, like BM, is derived from the mesenchyme. It is a connective tissue present in all mammals that serve as simple protection of the viscera. It is currently well-supported by the published data that adipose tissue has an endocrine function. It is responsible for controlling of energy metabolism through the storage of lipids (Scheja and Heeren 2019). This endocrine function is attributed to the adipocytes, which are a rich source of endocrine hormones, that is, adipokines and lipokines released in response to metabolic stresses and physiological cues and have their specific targets in the biological system for action (Booth et al. 2016). Since the publication of the early reports showing the presence of stem cells and their characterization as MSCs by Zuk and colleagues (Zuk et al. 2001), adipose tissuederived stem cells (ASCs) have been extensively characterized and studied for differentiation capacity (Bunnell et al. 2008; Mizuno et al. 2012). The protocol for harvesting adipose tissue is accessing a concentrated pool of MSCs in the tissue, simple, minimally invasive, and can be performed under local anesthesia that makes adipose tissue ideal for obtaining cells for clinical use in the cellular therapy context (Vallée et al. 2009). ASCs are isolated either by the suction of adipose tissue (SVF) or from the excised human fat by enzymatic digestion followed by purification (Minteer et al. 2013; Alstrup et al. 2019). The quality attributes of the cell preparation depend upon the isolation and purification technique (enzymatic digestion and nonenzymatic) (Gentile et al. 2019). Similarly, the

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harvested cells’ yield is significantly determined by whether these methods were used singly or combined (Alstrup et al. 2020). Adipose tissue-derived stromal vascular fraction SVF is an attractive therapeutic product obtained in an operating room using an automated device in 60–90 min with prospective clinical utility and efficacy (Han et al. 2015). It is pertinent to mention that collagen digestion requires more time, technical and procedural know-how of culturing the derived cells, a qualified clean room, and working under good manufacturing practices (GMP). In contrast, while SVF requires a less restrictive regulatory pathway. Studies are underway to ascertain whether the cells obtained without enzymatic digestion are identical to those subjected to this process (Chaput et al. 2016; Kokai and Rubin 2016). Researchers are awaiting advances in nonenzymatic separation due to the advantages that this would bring, including eliminating collagenase digestion with the implications that it entails. A review of literature has concluded that subcutaneous human adipose tissue is an accessible and abundant cell source for clinical applications (Kapur et al. 2015). Numerous studies using both ASC and SVF can be found in the clinical trials.org database in this context.

Umbilical Cord The umbilical cord (UC) connects the fetus to the placenta. The two arteries and a vein in the UC are wrapped in Wharton’s jelly (WJ), a gelatinous connective tissue. The UC has characteristics ideal for procuring stem/progenitor cells as it provides abundant stem cells. Their clinical use would not generate ethical conflicts, they are easily obtainable, display low immunogenicity, and have the potential for use in autologous cell therapy (Moreira et al. 2019). Besides Wharton’s jelly, MSCs are also isolated from umbilical cord blood (UCB).

Umbilical Cord Blood Once considered biological waste, UCB contains different subpopulations of stem cells, a unique feature not shared with peripheral blood. They have been isolated and characterized from fetuses/infants of various ages at 19–40 weeks (Iwatani et al. 2019). Besides being a source of cells for hematopoietic stem cells (HSCs), they are under extensive investigation and characterization as an alternative source of MSCs to the BM-derived MSCs for use in cell-based therapy (Weiss and Troyer 2006). From the ease of availability and simple collection/processing protocols to a lower rejection rate and a higher rate of acceptance, UCB stem cells are considered at par or even superior to the BM-derived stem cells. Given their low immunogenic nature, they have also been used from the allogenic source, which incidentally helps overcome their limited availability from the autologous source. However, the quality and quantity of UCB-derived cells are influenced by various methodological-related parameters, the greatest being the sample volume

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(Vasaghi et al. 2013). Some authors have proposed that UCB-derived MSCs show high morphological and molecular similarities with the BM-derived MSCs, including the lack of hematopoietic surface antigens (Erices et al. 2000). Similar to BM cells, UCB-cells constitute a heterogeneous population of cells. In a recently published study, two distinct subpopulations of MSCs were reported and named as short- and long-living cells based on their growth capacity and colony-forming efficiency (Amati et al. 2017). Besides revealing a differential trilineage differentiation potential, the study’s immunophenotyping results showed intense surface marker expression of CD90, CD105, CD44, CD13, and HLA-DRA while lacking in HSCs-specific markers, that is, CD31, CD34, and CD45. The cells were also negative CD271. There are reports about the presence of UCB-derived MSCs co-expressing neuronal markers, which show spontaneous neuronal differentiation (Divya et al. 2012). A recently published study has used a combinatorial expression pattern of CD105, CD90, and CD73 (Mishra et al. 2020). They observed that UCB-derived MSCs double-positive for CD105 +CD90+ were CD45+CD34+ immediately after isolation but started to reduce CD45 and CD34 markers during in vitro expansion. The authors also reported that umbilical cord tissues were a much rich source of MSCs than the UCB. Molecular profiling for stemness-related markers revealed little difference between MSCs derived from UCB, BM, and adipose tissues (Heo et al. 2016). A direct comparison of UCB-derived MSCs and BM-derived MSCs revealed that the former showed more robust chondrogenic differentiation. Bioinformatic analysis revealed donor-to-donor variations in their inherent genetic expression profile pertaining to the pro-angiogenic gene under hypoxic culture conditions (Kang et al. 2018). The presence or absence of MSCs in UCB has remained controversial for some time. For example, Yu et al. (2004) reported that early fetal blood was rich in MSCs; however, full-term UCB was devoid of these cells (Yu et al. 2004). Alternatively, several authors have proposed that the difficulty of obtaining MSCs from UCB was due to their low propensity, as low as 1–2 clones per 108 mononuclear cells of UCB (Martins et al. 2009; Bieback et al. 2004; Kern et al. 2006). These data seriously argue against the possibility of considering the UCB as a resource for acquiring MSCs. However, several authors have successfully differentiated UCB-MSCs in vitro to osteogenic, chondrogenic, neural, and hepatic lineages (Liu et al. 2011; Tio et al. 2010; Zhang et al. 2011). Although well-studied and well-characterized, up until now, UCB is not unanimously accepted as a source of MSCs for routine clinical applications.

Wharton’s Jelly Thomas Wharton was the first to describe WJ in 1656, a gelatinous substance composed of various isoforms of collagen and proteoglycans with the principal function to protect the arteries and veins from compression and torsion. They provide a bidirectional flow, delivering oxygen and nutrients that contribute to the adequate development of the fetus and moreover eliminating the waste and carbon dioxide. Human WJ-derived MSCs are emerging as an efficient and advantageous

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source of stem cells for experimental and clinical application. Their comparison with MSCs derived from other compartments of the umbilical cord revealed that they are a better choice of cells for clinical application (Subramanian et al. 2015). They exhibit a high degree of self-renewal capacity and multi-lineage differentiation potential, similar to that of MSCs derived from BM (Kern et al. 2006). The ease of availability and noninvasive collection protocols adds up to these advantages. Phenotypically and genetically, they show close resemblance with the embryonic stem cells, but their use and availability are without ethical or moral issues (Marino et al. 2019). Transcriptome profiling of WJ-derived stem cells revealed very low-level expression of pluripotency markers akin to the embryonic stem cells, that is, Nanog, Sox2, Lin28, and POUF1, thus providing a reason for their safety in terms of teratogenicity (Fong et al. 2011). Besides, they also express genes associated with immunomodulation, apoptosis, and chemotaxis. They also release a plethora of growth factors, cytokines, and extracellular vesicles as part of their paracrine activity contributing to their beneficial effects (Puig-Pijuan et al. 2020). There are two popular protocols for the isolation of WJ-derived MSCs, including the one based on the explant technique and the enzymatic digestion method. The enzymatic digestion protocol offers better results because the cell populations obtained are more uniform and homogeneous (Ding et al. 2015). WJ-derived MSCs are attractive cells due to their capacity for proliferation and other characteristics of MSCs like immunomodulatory properties and because the UC is an easy resource to obtain.

Placenta The human placenta plays a fundamental and essential role in fetal development, nutrition. The placenta’s fetal part originates from the blastocyst, whereas the maternal component (decidua) is derived from the endometrium. It is generally believed that it might be a source of primitive cells with immunomodulatory characteristics that render these cells worthwhile for use in regenerative medicine (Evangelista et al. 2008). Besides other types of cells, various studies have demonstrated that the human term placenta is a rich source of MSCs as it is considered one of the structures developed during the earliest stages of embryogenesis (Battula et al. 2008). Considering the complexity of the structure of the placenta, Parolini and coworkers published a paper to show the origin and define a protocol for the isolation of cells from the placenta. One of the main characteristic features of the placenta is its four regions, including the amniotic epithelial, amniotic mesenchymal, chorionic mesenchymal, and chorionic trophoblastic, each one of which respectively offers the following cell populations: amniotic epithelial cells, amniotic mesenchymal stromal cells, chorionic mesenchymal stromal cells, chorionic trophoblastic stromal cells (Parolini et al. 2008). A direct comparison of MSCs derived from these tissues has revealed that MSCs from fetal tissue had higher expansion potential than those derived from the maternal tissue, but they all had different levels of paracrine activity (Wu et al. 2018a). Soncini et al. achieved the isolation of amniotic and chorionic mesenchymal cells. These MSCs were isolated by a mechanical

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separation followed by enzymatic digestion (Soncini et al. 2007). Both show MSCs’ characteristics, such as fibroblastic morphology, adherence to plastic, and the capacity to form colonies. Both cell types, when analyzed by flow cytometry, showed phenotypes similar to BM MSCs. Diaz-Prado reported the isolation of amniotic membrane MSCs from human placenta by two different protocols for comparison and successfully isolated and in vitro expanded their derived cells for further characterization; however, the cell yield was much higher by Soncini’s protocol (Díaz-Prado et al. 2011). These amniotic MSCs are identified by the expression of CD105, CD90, and CD73, plastic adherence ability, and trilineage differentiation and showed excellent osteointegration and bone regeneration potential post engraftment in a rabbit model (Yin et al. 2019). Despite these encouraging data, the discrepancy in their characterization makes it difficult for their progress to regular use in the clinic (Ghamari et al. 2020).

Characterization MSCs Before Culture As discussed earlier, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy in 2006 proposed minimal criteria to define human MSCs that included preferential adherence to a plastic surface; specific surface antigen expression, that is, CD105, CD73, and CD90, among others, and a simultaneous lack of expression of hematopoietic and endothelial cell-specific surface antigens, that is, CD45, CD34, CD14 or CD11b and HLA-DR); and multilineage differentiation potential to adopt phenotypes similar to osteoblasts, adipocytes, and chondrocytes. These criteria define the minimal requirements to ascertain the purity of MSCs preparation in culture. However, the in vivo identification of MSCs in their “natural habitat” before ex vivo culture has not been well established by any such criterion. They are generally accepted to have a fibroblastic or pericytic origin (de Souza et al. 2016; Soundararajan and Kannan 2018). It would be exciting to establish an appropriate optimized protocol of cell selection that allows the employment of MSCs before their in vitro expansion. A standard protocol to define pre-culture identification markers would guarantee higher purity of the cell preparations than that obtained with selection based on plastic adherence. Many investigators direct their efforts to find a marker to ensure their selection. In an exciting review, Lv and co-workers (Lv et al. 2014) have in-depth reviewed several molecules proposed as markers to identify MSCs before culturing (Lv et al. 2014). The authors have primarily focused on four markers, that is, Stro-1, CD271, SSEA-4 (stage embryonic antigen-4), and CD146 for consideration.

STRO-1 STRO-1 is a cell membrane 75 kd endothelial antigen expressed on MSCs membrane (Ning et al. 2011). It is highly expressed on MSCs. It moves out from the endoplasmic reticulum in response to a decrease in intracellular calcium (Lv et al. 2014). In 1991,

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Simmons and Torok-Storb demonstrated Stro-1 enriched F-CFU from human BM with multipotency (Simmons and Torok-Storb 1991). However, this marker’s use as an in vivo MSCs “enricher” did not work with the various tissues tested for its expression. Lv et al. (2014) reviewed that Stro-1 is not universally expressed in all reported types of MSCs. Adipose tissue-derived stem cells highly express STRO-1 in culture when differentiated into endothelial lineage (Ning et al. 2011). Its expression is lost from MSCs when the cells are cultured for a longer time in vitro (Gronthos et al. 2003). Various research groups have attempted to establish a relationship between STRO-1 expressions with the MSCs functionality. STRO-1 expression has been related to the immunosuppressive properties of the MSCs (Francois et al. 2005). STRO-1+ cells could stimulate T-cell proliferation more than STRO-1- MSCs. Similarly, another study has attributed the expression of STRO-1 positivity with the paracrine activity of the cells (Psaltis et al. 2010). Pekozer and colleagues have recently shown that STRO-1 positivity is related to trilineage differentiation potential (except for osteogenesis, which was similar to STRO-1- cells) of the cells besides higher clonogenicity and proliferation (Pekozer et al. 2014). Despite these sporadic data, the exact relationship between STRO-1 expression and MSCs functions has not yet been fully established; its expression is related to the primitive status of MSCs.

CD271 CD271 is one of the most specific and prolific markers for the purification of MSCs from the human BM besides UCB. However, CD217+ MSCs derived from UCB were slow in proliferation compared to the ones derived from the BM (Watson et al. 2013). CD271 is also referred to as the low-affinity nerve growth factor receptor (LNGFR), nerve growth factor receptor (NGFR), or p75NTR (neurotrophin receptor). It has been included as a member of the tumor necrosis factor (TNF) superfamily (Thomson et al. 1988). A review performed by Alvarez Viejo and colleagues concluded that CD271 would not be regarded as a universal marker to identify MSCs before culture in vitro. In the BM or adipose tissue, CD271 could be considered a quite suitable marker to isolate MSCs. However, CD271 is inadequate for MSCs’ isolation from other tissues such as UC or UCB. Moreover, in the placenta, contradictory results have been obtained by different groups (Álvarez-Viejo et al. 2015). MSCs isolated based on CD271 are highly immunosuppressive and possess lymphohematopoietic engraftment promoting properties. Kuci and colleagues have reported a functional heterogeneity between CD271+ MSCs subpopulations compared with the plastic adherent MSCs. They have shown that both MSCs populations showed differential proliferation and differentiation potentials besides allosuppression but following different mechanisms (Kuçi et al. 2013). Within their derivative clones, the cells were monopotent, bipotent, and tripotent, while their immunosuppressive properties were not consistent with their proliferation or differentiation capacity. A recently published study has reported that CD271-selected MSCs were less angiogenic than their counterparts isolated based on preferential

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plastic adherence, although both cell types could populate the scaffolds equally (Kohli et al. 2019).

SSEA-4 SSEA-4 is an early embryonic glycolipid antigen and one of the panels of reliable markers for identification of the undifferentiated human ESCs and cleavage to blastocyst stage embryos (Wright and Andrews 2009). Gang et al. identified the adult BM-MSCs population using SSEA4 (Gang et al. 2007). Conversely, other authors reported no detection of SSEA-4 expressing cells in the unsorted BM (Tormin et al. 2011; Wagner et al. 2005). Therefore, these data suggest that this marker would not be considered an excellent marker to isolate MSCs before culture. A recent study has isolated SSEA-4 expressing cell population from WJ that was also positive for CD90, CD105, CD70, Nanog, and Sox2 (Li et al. 2017). These cells could spontaneously differentiate cells of all three germ layers in vitro. Moreover, they were able to undergo adipogenic and osteogenic differentiation much easier than SSEA4- cells. Maddox and colleagues have reported the presence of stromal cells expressing SSEA-4 in the breast and abdominal adipose tissue and showed the higher potential of osteogenic and adipogenic differentiation potential (Maddox et al. 2012). It is pertinent to mention that breast adipose tissue contained only 0.48% SSEA-4 positive cells sub-population against the 12% propensity of their counterparts in abdominal fat tissue. It is also expressed by some of the cancer cells identifying their malignant character and resistance to chemotherapy (Aloia et al. 2015; Nakamura et al. 2019). Given its association with disease progression in cancers, SSEA-4 expression results in the loosening of the cell-to-cell interaction, loss of epithelial phenotype, and adoption of the mesenchymal phenotype. These cellular and molecular changes are important for the cells to attain migratory capacity, thus contributing to their metastasis (Sivasubramaniyan et al. 2015).

CD146 CD146, also known as Mel-CAM, MUC18, A32 antigen, is a 113 kDa melanoma cell adhesion molecule (CAM) (Wang and Yan 2013). This is primarily expressed at the intercellular junction of endothelial cells. A more recently published study has reported that CD146 is more than merely an adhesion molecule to serve as a receptor for various signaling molecules, thus participating in diverse physiological and pathological processes encompassing angiogenesis to lymphogenesis (Wang et al. 2020c). The soluble form of CD146 derived from endothelial cells is being considered a reliable marker of neuroinflammatory disease (Wang et al. 2020a). Moreover, it mediates FAK activation to help melanoma cells to traverse vessel walls during metastasis in response to VEGF signaling (Jouve et al. 2015). Harkness et al. (2016) published a study showing that CD146 defines a subpopulation of human MSCs capable of bone formation and in vivo trans-endothelial migration. They also

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showed that the CD146 sub-population of the BM cells was ideal for use in clinical protocols of bone tissue regeneration (Harkness et al. 2016). The expression of CD146 was found in human MSCs and other sources of MSCs such as adipose tissue, placenta, or dermis, among others (Lv et al. 2014). CD146 expression on MSCs has been attributed to their better therapeutic potential than the cells lacking in CD146 expression (Wu et al. 2016). It is also considered a surrogate marker for MSCs cultured in vitro to predict their differentiation potential. MSCs in the confluent cultures downregulate CD146 expression with concomitant decrease in differentiation potential. Therefore, a moderate rate of medium addition during MSCs culture may ensure their confluence good enough to maintain their differentiation capacity (Jones et al. 2018).

MSCs in the Clinical Perspective Given their remarkable reparability, immunomodulatory, and anti-inflammatory properties generally attributed to their paracrine behavior and differentiation potential, MSCs have entered into advanced phases of clinical trials for the treatment of various diseases. As discussed earlier, their near ideal characteristics, as discussed in the previous sections, make them best candidates for cell-based therapy. Currently, they are part of numerous clinical trials worldwide that use MSCs to treat various pathologies some of which will be discussed in the following sections of the chapter (Table 1).

MSCs-Based Therapy for Cardiovascular Pathologies Since the pioneering work of Hamano et al. in a group of five patients in 2001 that the BM cell transplantation for myocardial repair is safe (Hamano et al. 2001), they have been extensively studied in both the small and large experimental animal models of myocardial infarction and heart failure for the repair and regeneration of the injured myocardium (Haider et al. 2008a; Kim et al. 2012; Van der Spoel et al. 2015; Cai et al. 2016). There is mounting evidence in the literature that transplantation of MSCs (both native, genetically modified, and physiologically or pharmacologically preconditioned) in the experimentally injured heart results in decreased infarction size, reduced area of fibrosis, attenuated remodeling, and preserved global heart function (Haider et al. 2008a, b, 2010, 2012; Afzal et al. 2010; Lai et al. 2012; Kim et al. 2012; Haider and Aziz 2017). MSCs have also combined with other cell types to enhance their survival and differentiation potential (Hosseini et al. 2018). BM-derive MSCs have also been reprogrammed to achieve pluripotency status and their derived cells have been successfully using for myocardial repair in experimental animal models (Buccini et al. 2012). These therapeutic benefits have been attributed to a multifactorial mechanism wherein cardiogenesis and vasculogenesis due to cardiac differentiation and paracrine activity of the transplanted cells. Based

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Table 1 Summary of the studies involving MSCs from different tissue sources for various disease conditions Disease Acute graft-versus-host disease

MSCs source BMMSC

Inflammatory bowel disease: Perianal Crohn’s disease Crohn’s Disease

ASC

Ulcerative colitis

BMMSC

Knee osteoarthritis

ASC

Knee osteoarthritis

ASC

Knee osteoarthritis

BMMSC

Multiple Sclerosis

BMMSC

Multiple Sclerosis

BMMSC

Rheumatoid arthritis

BMMSC

Rheumatoid arthritis

ASC

Rheumatoid arthritis

UCMSC

UCMSC

Results BM-MSCs was considered second-line treatment for GvHD Results phase III clinical trial, improved results as compared with placebo, after 1 year of follow-up UC-MSCs were effective in the treatment of Crohn’s Disease and produced mild side effects Studied the efficacy and safety of BM-MSCs for patients with ulcerative colitis. Two-year follow-up showed immunomodulatory effects and reduction in inflammation and decreased risk of recurrence Evaluated the efficacy of autologous ASCs therapy on pain, function, and disease modification in knee osteoarthritis. Results showed that the therapy was safe and effective Satisfactory functional improvement and pain relief for patients that received this ASCs treatment Compared the results of MSC with hyaluronic acid, after a year better results were observed with MSCs Observed that transplantation of BM-MSCs in patients with multiple sclerosis is a clinically feasible and relatively safe procedure and induces immediate immunomodulatory effect The administration of the cells was intrathecal and the results in some of the patients were encouraging enough for Phase II Suitable safety profile of autologous MSCs therapy in rheumatoid arthritis patients with promising trend for clinical efficacy Observed that the intravenous dose of ASCs in patients was well-tolerated, recommended it appropriate to carry out one more phase of this clinical trial Authors indicated that the intravenous infusion of was safe

References Le Blanc et al. 2004, 2008 Panés et al. 2018 Zhang et al. 2018 Lazebnik et al. 2010

Freitag et al. 2019

Lee et al. 2019 Kim et al. 2020 Karussis et al. 2010

Harris et al. 2018

Ra et al. 2011

ÁlvaroGracia et al. 2017 Park et al. 2018 (continued)

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Table 1 (continued) Disease Heart failure

MSCs source BMMSC

Heart failure

BMMSC

Heart failure

BMMSC

Heart failure

BMMSC

Heart failure

UCMSC

Heart failure

ASC

Results Intramyocardial injection of autologous MSCs into akinetic non-revascularized segments produced comprehensive regional functional restitution, which in turn improved global LV function Successfully reported the safety and feasibility of allogenic MSCs in patients with dilated cardiomyopathy The intracoronary infusion of human BM-derived MSCs at 1 month was tolerable and safe with modest improvement in LVE Intracoronary BMC therapy improved ventricular performance, quality of life, and survival in patients with heart failure Assessed the safety and efficacy in patients with chronic stable heart failure and reduced LV-ejection fraction ASC treatment was safe but did not improve exercise capacity compared to placebo

References Karantalis et al. 2014

Hare et al. 2017 Lee et al. 2014

Bartolucci et al. 2017

on these data, and encouraged by the favorable data in Phase-I and Phase-II trials (Karantalis et al. 2014; PROMETHEUS trial (Prospective Randomized Study Of Mesenchymal Stem Cell Therapy in Patients Undergoing Cardiac Surgery; Clinical trial identifier: NCT00587990), MSCs-based studies have currently advanced to multiple Phase III clinical trials worldwide, that is, RELIEF trials (Clinical trials identifier: NCT01652209; Randomized, Open labEled, muLticenter Trial for Safety and Efficacy of Intracoronary Adult Human MSCs AMI). Allogenic MSCs have also been assessed to overcome the problem of autologous cells from the aging and diseased donors. A phase I/II pilot clinical study POSEIDON (Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis; Clinical trials Identifier: NCT01087996) was performed to ascertain a dose-range comparison between autologous and allogenic BM-derived MSCs in 31 randomized patients (1:1) between the two cell type treatment with escalating doses (Hare et al. 2012). Other research groups have also reported similar clinical trials to ascertain the use of allogeneic MSCs (Perin et al. 2015; Jansen of Lorkeers et al. 2015). Hare et al. have successfully reported the safety and feasibility of allogenic MSCs in patients with dilated cardiomyopathy (Hare et al. 2017). Some of the other trials using TENDO I/M delivery of MSCs include ESTIMATION trial (Clinical trial identifier: NCT01394432); CEP-41750 (Clinical trial Identifier: NCT02032004; Allogeneic Mesenchymal Precursor Cells) for the Treatment of Chronic Heart Failure; CHART-1 trials (Clinical trial identifier: NCT01768702); and CHART-2 trial (Clinical trial identifier: NCT02317458).

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A randomized, open-label, multicenter, Phase-II/III SEED-MSC (Clinical trial identifier: NCT01392105) pilot trial assessed the safety and efficacy of BM-derived MSCs (Lee et al. 2014). The cells were delivered by I/C infusion at 1 month after successful revascularization of the infarct-related artery in 58 acute myocardial infarction (AMI) patients. Similarly, Dr. Timothy Henry is leading another Phase-II clinical study AMICI to assess the safety of Allogeneic Mesenchymal Precursor Cell Infusion in MyoCardial Infarction (Clinical trial identifier: NCT01781390). The research involves 105 AMI patients and is expected to complete and release its findings very soon. Some other on-going Phase-II/III clinical trials including RELIEF (Clinical trial identifier: NCT0165209), CIRCULATE (Clinical trials identifier: NCT03404063), SEEDMSCs (Clinical trial identifier: NCT01392105; Safety and Efficacy of Intracoronary Adult Human Mesenchymal Stem Cells After Acute Myocardial Infarction), etc. have also opted for I/C delivery of MSCs in patients with AMI. I/C route of cell delivery has also been used for myocardial cell therapy using cells other than BM-derived MSCs, that is, CSCs (CAREMI trial; Clinical trial identifier: NCT02439398), BM-derived CD133+ (COMPARE-AMI; ACTRN12609001045202), and BM-derived AC133+ cells (STAR; Stem cell Transplantation in patients with chronic heARt failure) which is incidentally one of the most extensive clinical trials using I/C route for BM cell delivery. Interesting use of the I/C route has been the delivery of CDCs in children with hypoplastic left heart syndrome (HLHS) after staged surgery (Clinical trial identifier: NCT01273857). More recently, Bartolucci and colleagues assessed the safety and efficacy of the UC-MSCs in 15 patients with chronic stable heart failure and reduced LV-ejection fraction and compared them with placebo treated patients (Bartolucci et al. 2017). They reported that intravenous infusion of 1
106 cells/kg UC-MSCs was safe. Moreover, they also observed significant improvement in LV-function, and quality of life in patients treated with UC-MSCs during 3, 6, and 12 months of follow-up. Other than BM, MSCs-derived from other human tissues have been used to assess their efficacy in the patients which include ASC (Clinical trial Identifier: NCT01449032; MesenchYmal STROMAL CELL Therapy in Patients With Chronic Myocardial Ischemia; MyStromalCell Trial), and UC-MSCs (Clinical trial Identifier: NCT01739777; RIMECARD Trial (Randomized Clinical Trial of Intravenous Infusion UC Mesenchymal Stem Cells on Cardiopathy).

MSCs-Based Therapy for Acute Graft-Versus-Host Disease Graft-versus-host disease (GvHD) is a severe and sometimes life-threatening complication due to interaction between donor-derived immunocompetent T cells and recipient tissue antigens. Acute GvHD reaction severity is graded from I (mild) to IV (very severe) after allogenic HSCs transplantation that maybe even fatal in some cases (Moreno and Cid 2019; Nassereddine et al. 2017). Although GvHD offers diverse therapeutic targets during its progression, the use of MSCs with

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immunomodulatory has been extensively studied as a potential therapeutic option. In 2004, substantial research in The Lancet was published, which provided a leap forward in the treatment of GvHD using BM-MSCs (Le Blanc et al. 2004). The study successfully exploited the immunomodulatory properties of haploidentical BM MSCs in patients with treatment-resistant grade IV acute GvHD. Since the publication of these data, Le Blanc and colleagues besides many other research groups have been part of various clinical trials which have advanced to Phase III trials, and in some countries, MSCs-based intervention has achieved a status of second-line treatment for GvHD (Le Blanc et al. 2008; Martin et al. 2010; Galipeau 2013; Wu et al. 2013; Szabolcs et al. 2010). These data provide evidence that the use of BM-MSCs for the treatment of GvHD is safe and feasible (Cheung et al. 2020). On the contrary, the authors of a recently published systematic review based on 12 studies and 13 on-going clinical trials have concluded that the published literature well supports the safety of MSCs-based treatment for GvHD. Still, it could only provide low-quality evidence that MSCs reduce the risk of chronic GvHD (Fisher et al. 2019). The systematic review results also suggested future studies to optimize the therapeutic intervention’s protocol. The systematic review findings can also be interpreted that the outcome of the studies and clinical trials is influenced by various factors (Wang et al. 2017). For example, it is now generally perceived that not all MSCs’ preparations are equally effective, necessitating the optimization of MSCs isolation and purification protocols, besides optimizing the dose and injection route (Elgaz et al. 2019). The pro-inflammatory immune profile in the gut of the recipient at the time of MSCs treatment is one of the primary determinants of the therapeutic outcome (Gavin et al. 2019).

MSCs-Based Therapy for Crohn’s Disease Inflammatory bowel disease (IBD) includes two chronic inflammatory idiopathies: ulcerative colitis and Crohn’s disease that severely affect the quality of life. Crohn’s disease is characterized by the presence of perianal fistulas, treatment of which remains a challenge for therapy with the contemporary therapeutic options. The merging of cell-based therapies using MSCs has reasonable success thus far in the clinical trials to treat IBD via systemic and local delivery (perianal Crohn’s disease) (Adak et al. 2017). Lazebnik et al. (2010) studied the safety and feasibility of BM-MSCs in patients with ulcerative colitis (Lazebnik et al. 2010). A 2-year follow-up of the patients revealed the significant immunomodulatory potential of MSCs, leading to a reduction in inflammation and a decrease in the risk of recurrence. These data were substantiated by a recently published meta-analysis that supported the use of MSCs for treating ulcerative colitis (Shi et al. 2019). Similarly, a previously published meta-analysis by Dave et al. had suggested a promising role for MSCs in cell-based therapy in IBD patients despite many challenges in their routine clinical use (Dave et al. 2015). Searching Clinicaltrials.org, 62 clinical trials for Crohn’s disease have either been completed or in progress using cell-based therapy, mostly using MSCs (https:// clinicaltrials.gov/ct2/results?cond¼Crohn+Disease&term¼stem+cells&cntry).

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Panés et al. (2018) have proposed the allogeneic ASCs to treat complex perianal fistulas in patients with Crohn’s Disease (Panés et al. 2018). The authors published a double-blind phase III clinical trial reporting encouraging data compared to the placebo-treated patients during the 1-year follow-up. They concluded that ASCs provided a safe and effective option in closing fistulas. Although its etiology is unclear, most researchers believe that Crohn’s disease is associated with autoimmune response. Considering this hypothesis, Zhang et al. proposed UC-MSCs to treat this disease in 82 patients with confirmed Crohn’s disease (Zhang et al. 2018). During a randomized controlled clinical trial, the patients received a weekly dose of 1
106 cells/kg for 4 weeks. During the 12-month follow-up, they observed UC-MSCs were effective in reducing Crohn’s disease index, Harvey-Bradshaw index, and steroid therapy. Only four patients showed mild signs of the ill-effects relevant to the treatment. Although the use of MSCs has progressed to Phase-III clinical trials, the underlying mechanism remains elusive. A recent study in an experimental mice model of dextran sulfate sodium (DSS)-induced colitis has revealed that ASCs induce an innate immune memory response in the MSCs-treated animals (Lopez-Santalla et al. 2020b). Cell therapy-treated animals, which received MSCs during the acute phase after DDS treatment, showed sustained protection against inflammation when re-challenged after 12 weeks. The authors concluded that MSCs treatment incurs long-term benefits as they change the regulatory to inflammatory macrophage ratio in lamina propria of the colon. Future studies are warranted to have a head-to-head comparison of the safety and effectiveness of MSCs from different tissue sources, although their use in individual studies has generated encouraging data.

MSCs-Based Therapy for Multiple Sclerosis Multiple sclerosis is an autoimmune disease involving the appearance of focal inflammatory lesions in the substance white matter of the brain characterized by demyelination of the nerve fibers. Karussis et al. 2010 observed that transplantation of BM-derived MSCs in patients with multiple sclerosis is a clinically feasible and relatively safe procedure and induces immediate immunomodulatory effects (Karussis et al. 2010). MSCs also release biologically active secretome due to their paracrine activity that adds to the therapeutic benefits (Gugliandolo et al. 2020). On the same note, MSCs from tissue sources other than BM have also shown encouraging results (Giacoppo et al. 2017; Riordan et al. 2018). These results have been corroborated among many research groups as reviewed by Lotfy et al. (2020). In 2018, a study was published showing the results from a Phase I clinical trial (ClinicalTrials ID: NCT01933802) that used MSCs-derived neural progenitor cells to treat multiple sclerosis (Harris et al. 2018). The cells’ administration was via intrathecal injection, and the results in some of the patients were encouraging enough to propose the execution of Phase II. The safety and efficacy of autologous MSCs to treat multiple sclerosis were further assessed in a Phase I/II clinical study

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MEsenchymal StEm cells for Multiple Sclerosis (MESEMS) (Uccelli et al. 2019). MESEMS study has been designed to merge partially independent clinical trials, following harmonized protocols and sharing some critical centralized procedures, including data collection and analyses. With this model, where various clinical trials are grouped, the authors suggest that the results will provide patients and the scientific community with data on the safety and efficacy of MSC for multiple sclerosis. Although not assessed against all forms of multiple sclerosis, treatment with MSCs is a future hope for the patients.

MSCs-Based Therapy for Rheumatoid Arthritis and Osteoarthritis Encouraged by the experimental animal data that revealed the safety and effectiveness of cell-based therapy for rheumatoid arthritis, the cell-based therapy approach has progressed to the clinical phase of assessment. A direct comparison of MSCs from various sources, including BM, UC, and human deciduous tooth revealed superior outcomes from UC-derived MSCs (Zhang et al. 2019). A recently published meta-analysis of the preclinical studies has demonstrated consistent therapeutic benefits of cell-based therapy in the experimental animal models (Liu et al. 2019b). Although the mechanism of action and beneficial therapeutic outcome is considered multifactorial, it has been reported that the transplanted MSCs control the memory T-cell response (Noymar et al. 2019). The first pilot clinical study with MSCs therapy autoimmune disorders including rheumatoid arthritis was conducted in 2010 by the Stem Cell Research Centre in Korea. The results were published in almost a decade later (Ra et al. 2011). This study involved the use of ASC. It was considered the first proof-of-concept clinical study that has reported a suitable safety profile of autologous MSCs therapy in 10 patients with various autoimmune disorders, including rheumatoid arthritis patients with promising clinical outcomes’ efficacy. Tested in Balb/c nude mice for tumorigenicity even at larger doses, the cell therapy was safe in the patients. Among several studies published in this line, Álvaro-Gracia et al. evaluated the safety and tolerability of the intravenous administration of allogenic ASC in patients with refractory rheumatoid arthritis as a part of Phase Ib/IIb trials including 53 patients (Álvaro-Gracia et al. 2017). They observed that the intravenous dose of adipose tissue-derived stem cells in these patients was well-tolerated. These results agree with the data published by Park et al. (2018), who reported a phase I, uncontrolled, open clinical trial to treat patients with moderate intensity rheumatoid arthritis using UC- MSCs (Park et al. 2018). The authors noted that the intravenous infusion of UC-derived MSCs showed a similar safety profile to BM-derived MSCs. LopezSantalla et al. have recently published a comprehensive review of literature documenting both active and closed clinical trials that have focused on using MSCs in rheumatoid arthritis (Lopez-Santalla et al. 2020a). The authors have concluded that a toxicity-free and adverse effects-free use of MSCs in rheumatoid arthritis patients during all the reported clinical trials conducted. However, insufficient data on efficacy have been obtained from the completed clinical trials, most

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likely because a large majority of the rheumatoid arthritis patients enrolled in the studies were refractory to conventional rheumatoid arthritis treatments with a long history of the disease. It is pertinent to mention that only a few examples of the clinical trials published until today wherein a regeneration of the tissue is necessary to treat autoimmune disorders (Lopez-Santalla et al. 2020a). Moreover, there are still concerns about the use of cells from the autologous tissue sources. For example, it has been reported that autologous MSCs obtained from patients with rheumatoid arthritis were functionally impaired, as was observed by their failure to inhibit Th17 (Sun et al. 2015). These data are significant in optimizing the protocols for future cell therapy clinical trials to harness maximum therapeutic benefits from MSCs-based treatment. Knee osteoarthritis is one of the most commonly diagnosed forms of arthritis, especially in elderly patients after age 65. The underlying cause is the slow but progressive degeneration of the cartilage that protects the knee joint due to physiological aging. Freitag et al. evaluate the efficacy of ASC-based therapy on pain, function, and disease modification in knee osteoarthritis (Freitag et al. 2019). Upon completing the clinical trial, the authors concluded that the therapy was a safe and effective therapy for knee osteoarthritis. These results were substantiated by Lee et al., who used an intra-articular injection of autologous ASC to treat knee osteoarthritis (Lee et al. 2019). They described satisfactory functional improvement and pain relief for patients that received cell therapy. Given that intraarticular injection of hyaluronic acid is a medical option for knee osteoarthritis, Kim et al. (2020) conducted a comparative study between the MSCs and hyaluronic acid-based treatment (Kim et al. 2020). They observed that the MSCs group showed better results during 1-year post-treatment follow-up than the hyaluronic acid group.

Exosomes-Based Therapeutic Intervention Despite encouraging data emanating from the clinical studies using the cell-based therapy approach, the underlying mechanism of the therapeutic benefits is only partly understood. In addition to undergoing fusion with the host cells and differentiation to adopt morphofunctional phenotype post engraftment to participate in the regeneration process, the paracrine hypothesis has gained wide-spread acceptance amongst the researchers in the field (Haider and Aziz 2017). These proposed mechanisms are correct and supported by substantial data; however, they are not exclusive (Álvarez-Viejo 2020). It is widely accepted that MSCs produce a plethora of bioactive molecules, including cytokines, growth factors, and extracellular vesicles (EVs) containing specific payload (Altanerova et al. 2017). Almost every cell type, including MSCs, releases exosomes as part of intercellular signaling, and given their specific payload, they could serve as a novel cell-free therapeutic tool in the field of regenerative medicine (Haider and Aramini 2020; Nikfarjam et al. 2020). However, there is little headway made in exosomal use in the clinical perspective as is evident from the literature search which did not fetch any records of clinical trials

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at least for the diseases included in our chapter. This limited progress of exosomes from bench to the clinic is attributed to the challenges faced in this regard (Forsberg et al. 2020).

MSCs-Derived Exosome for Therapy “Without Cells” The term EVs includes microvesicles, nanoparticles, vesicles, apoptotic bodies, and exosomes (Pinheiro et al. 2018). The difference between these terms is the size of the vesicles released. Due to the size-heterogeneity between these particles, it was imperative to develop guidelines for their definition. In this regard, The International Society for Extracellular Vesicles proposed the minimum criteria to characterize extracellular vesicles, which requires EVs as the generic term for particles naturally released from the cell that is determined by a lipid bilayer and cannot replicate, i.e., do not contains a functional nucleus (Théry et al. 2018). The size of exosomes is 60–100 nm with a diverse protein composition between cells, but they share some proteins, such as CD9, CD63, or CD81, for use as exosome markers (Yamashita et al. 2018). Similar to exosomes derived from other cell types, MSCs-derived exosomes participate in intercellular communication and carry proteins, mRNA, and microRNA (miRNA) for delivery to the target cells to facilitate intercellular signaling (Mendt et al. 2019). The use of exosomes offers several advantages, such as the aversion of introducing exogenous cells, thus avoidance to bring in mutated or damaged genetic material that could harmfully affect the recipient. They are low in immunogenicity (Elahi et al. 2019), but given their inability to divide or replicate, they are static, thus restricting their therapeutic benefits for time duration until they get eliminated from the biological system post-delivery (Phinney and Pittenger 2017). Currently, there are nearly a hundred clinical trials registered in Clinicaltrial.gov fetched by the term exosomes. On the same note, there are nearly a thousand trials that include MSCs. The novelty of exosome-based “cell-free” therapy, however, still require optimization of GMP grade exosome production protocols (Chen et al. 2019), their standard markers, ideal donor cells, standardization of their payload, indicated dose, route of administration, etc., and sufficient preclinical data to support their routine clinical use.

MSCs-Derived Exosomes and Acute Graft-Versus-Host Disease Given the paracrine hypothesis’s wide-spread acceptance that the therapeutic benefits of MSC-based cell therapy are mediated via paracrine release of immunosuppressive and immunomodulating factors, cell-free therapy using MSCs-derived exosomes is now fast-emerging as a treatment option for refractory GvHD in an increasingly standardized way (Zhou et al. 2020). Several preclinical studies have reported that MSCs-derived exosomes could be as effective as MSCs-based cell therapy. For example, using a mice model of acute GvHD (aGvHD), Fujii et al. have

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shown that systemic infusion of BM MSCs-derived exosomes prolonged the survival of mice with aGvHD, besides alleviating pathologic damage to aGvHD targeted organs via suppression of CD4+ and CD8+ cells (Fujii et al. 2018). The authors attributed the therapeutic benefits of exosomal treatment to the unique miRNA profile of the MSC-derived exosome preparation used for treatment. Similarly, Wang et al. reported that human UC-MSCs derived exosomes could prevent aGvHD in a mouse model after allogeneic HSCs transplantation (Wang et al. 2016). The authors noted that EVs derived from UC-MSC could prevent life-threatening a GvHD by modulating the immune response. Moreover, they could represent a prophylactic method to prevent aGvHD as well. On the other hand, working with a chronic mouse model of GvHD, Lai et al. studied the efficacy and safety of MSCsderived exosomes to treat this disease. They suggested that MSCs-derived exosomes could improve survival and ameliorate the pathologic damage of chronic GvHD (Lai et al. 2018). A recently published meta-analysis of four studies involving the use of MSCs-derived exosomes for the treatment and prevention (two studies each) of GvHD showed that exosomes were an effective treatment tool as well as for prophylactic application (Gupta et al. 2020). The authors concluded that the use of MSCs-derived exosomes successfully enhanced survival and attenuated histologic findings of GvHD in all four studies. Supported by the preclinical data, the cell-free therapy approach has progressed to clinical assessment of MSC-derived exosome to treat the GvHD patients but only very cautiously. There is only one Phase I clinical study registered at ClinicalTrials. gov entitle “Effect of UM-MSCs-derived exosomes on dry eyes in patients with GvHD” (Clinicaltrials.gov Identifier: NTC04213248). The study will enroll 27 patients who will receive UC-MSCs derived exodrops (UM-exo). However, the study results are still awaited. Also, we have only found a letter to the Editor in which their use to treat graft GvHD is described. In one patient, Kordelas et al. used an exosome-enriched fraction processed from collected MSCs supernatants instead of administering the MSCs themselves (Kordelas et al. 2014). The patient was stable for several months of post-exosome application. Although the patient died of pneumonia 7 months after treatment, the authors concluded that BM MSC exosomes could be a potentially new and safe tool to treat therapy-refractory GvHD, and most likely other inflammation-associated diseases. Despite encouraging data, there is slow progress for the use of MSCs-derived exosomes for clinical applications, which may be due to the technical and methodological hindrances involved therein.

MSCs-Derived Exosomes for Inflammatory Bowel Disease The role of MSCs-derived exosomes for the treatment of IBD in general and Crohn’s disease, in particular, has been extensively studied in the experimental animal models (Ocansey et al. 2020). Mao et al. investigated the effects of exosomes derived from UC-MSCs in a model of induced inflammatory bowel disease (Mao et al. 2017). According to their findings, UC-MSCs exosomes could substantially alleviate experimentally induced inflammatory bowel disease in a mice model.

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While elucidating the underlying mechanism, IL-10 expression was elevated while pro-inflammatory TNFa, IL-1b, IL-6, IL-7, and iNOS were significantly downregulated in the colonic tissue and spleen of exosome-treated animals. Besides, ubiquitination played a significant role in UC-MSCs derived-exosome treated experimental animals (Wu et al. 2018b). The exosome-treated animals were able to better recover tissue structural integrity. These results agree with those published by Liu et al., who demonstrated that systemic administration of exosomes from human BM-MSCs substantially mitigated colitis in various models of inflammatory bowel disease (Liu et al. 2019a). Mechanistically speaking, the colonic macrophages were pivotal in alleviating the disease process as was observed by abrogation of the beneficial effects of exosome treatment by macrophage depletion. They also agreed with Yang et al., who investigated the potential alleviating effects of BM-MSCs EVs in the colitis model (Yang et al. 2015). The authors concluded that the beneficial effects of exosomes from the BM-MSCs were due to the downregulation of the pro-inflammatory cytokines, inhibition of NF-κBp65 signal transduction pathways, modulation of antioxidant/oxidant balance, and moderation of the occurrence of apoptosis, as the possible underlying mechanism. Despite finding several papers published in the preclinical phase, there are no clinical trials registered in clinicaltrials.gov, although their diagnostic use has been attempted (Larabi et al. 2020). The possible explanation for the lack of exosome-based clinical data is the unavailability of standardized protocols for isolation, large-scale reproducible exosome preparations to their protocols for optimum clinical use (Harrell et al. 2020).

MSCs-Derived Exosomes for Rheumatoid Arthritis and Knee Osteoarthritis Continuing the success story of MSCs-based cell therapy for rheumatoid arthritis and knee arthritis, Chen et al. investigated the therapeutic effects of MSCs-derived exosomes on joint destruction in rheumatoid arthritis (Chen et al. 2018). After treatment with experimentally induced arthritis in the mice model, the authors showed the safety and feasibility of exosome derived from MSC overexpressing miRNA-150. The authors observed a reduction in joint destruction with concomitant inhibition of synoviocyte hyperplasia and angiogenesis. From the analysis of the results, they conclude exosomes facilitate the direct intracellular transfer of miRNAs between cells. Given the pivotal role of miRNAs and their regulatory network in the pathogenesis of rheumatoid arthritis (Zakeri et al. 2019), many other research groups have adopted a similar approach for their use as biomarkers as well as therapeutic targets (Huang et al. 2019). The recently published study by Zheng et al. substantiates these data (Zheng et al. 2020). They also proposed that exosomes derived from BM-MSCs, and more specifically, BM-MSCs secreted exosomal miR-192-5p could delay the inflammatory response events in rheumatoid arthritis. Meng et al. have used exosomes derived from miRNA-320a-loaded MSCs to regulate fibroblasts-like synoviocytes activity by interacting with CXCl9, which are mechanistically involved in rheumatoid arthritis pathology (Meng and Qiu 2020). Cosenza et al. compared the

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anti-inflammatory and immunosuppressive properties of MSCs-derived exosomes and microparticles in a mice model of collagen-induced rheumatoid arthritis (Cosenza et al. 2018). The authors observed that both exosomes and microparticles were equally effective in suppressing the inflammatory response, but exosomes were more efficient in anti-inflammatory activity in vivo. The role of MSCs-derived exosomes and their mechanism remains an area of intense investigation wherein most researchers have used them for both diagnostic and therapeutic purpose, as summarized by Ni et al. and Mianehsaz et al. in their recently published literature reviews (Ni et al. 2020; Mianehsaz et al. 2019). TofiñoVian et al. investigated chondroprotective action of exosomes from adipose tissuederived MSCs from healthy volunteers in vitro using chondrocytes from arthritic patients (Tofiño-Vian et al. 2018). Treatment with exosomes significantly reduced MMP activity and MMP13 expression besides upregulation of anti-inflammatory IL10 in the treated chondrocytes. Their results support the interest of MSCs-derived EVs to develop new therapeutic approaches in arthritic joint conditions. These in vitro results were reinforced by He and colleagues, who investigated the effect of exosome derived from BM-MSCs on damaged cartilage repair and pain in an experimental animal model of osteoarthritis (He et al. 2020). They used a rat model of osteoarthritis established by injection of sodium iodoacetate. After analyzing these results, the authors concluded that exosomes successfully promoted cartilage repair and extracellular matrix synthesis, as well as relieve knee pain in rats with osteoarthritis. A recently published systematic review analyzing 20 published in vivo and in vitro studies revealed positive findings in reduced inflammation, downregulation of catabolic processes, and increased anabolic studies D’Arrigo et al. 2019). However, for optimal benefits of this approach, it is essential that exosome production protocols and identification of patients who could benefit from this novel approach must be identified. As with most of the diseases discussed in this chapter, rheumatoid arthritis and knee arthritis treatment with exosomes have not yet progressed to the clinical trials.

MSCs-Derived Exosomes for the Heart Exosomes from various stem cells, that is, ESCs, iPSCs and their derivative progenitors and cardiomyocytes (Khan et al. 2015; Arslan et al. 2013; Kervadec et al. 2016; El Harane et al. 2018), cardiosphere-derived progenitors (Xiao et al. 2016), and MSCs-derived exosomes (Zhang et al. 2016), have been studied for their reparability of the injured myocardium. Exosomes derived from MSCs have also been used to precondition other cells to enhance their survival post-engraftment and promote angiogenesis and reduce cardiac fibrosis in the experimentally induced cardiac fibrosis with concomitantly improved cardiac function (Zhang et al. 2016). Lai et al. (2010) used purified exosomes from MSCs to treat a mouse model of ischemia-reperfusion injury (Lai et al. 2010). The authors reported a successful reduction in infarct size. These data also strengthened the paracrine hypothesis according to which the cardioprotective effects of stem cell-based therapy were

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related to paracrine secretions of stem cells that also included exosomes (Haider and Aziz 2017). These conclusions were later supported by Wang et al., who tested EVs secreted by BM-MSC secreted EVs for their pro-angiogenic activity in the infarcted heart (Wang et al. 2017). They concluded that exosome-derived MSCs are sufficient to improve angiogenesis and exerted therapeutic benefits in the experimental myocardial infarction model. Study of the mechanisms involved in exosome-mediated cytoprotection showed AMPK/Akt signaling in the H2C9 cells treated with MSCsderived exosomes after subjecting the cells to oxidative stress. On the same note, treatment with UC-MSCs-derived exosome was protective for cardiomyocytes in the infarcted myocardium by transferring miR-19a, targeting SOX6, activating Akt, and inhibiting JNK3/caspase-3 activation (Huang et al. 2020). The exosomal payload’s role of miRNAs has been eloquently reviewed for their role in myocardial repair Haider and Aramini (2020). Despite extensive studies in the small animal model and encouraging data from large animal models with clinical relevance, there are still no studies registered for their clinical use (Gallet et al. 2017; de Couto et al. 2017).

Multiple Sclerosis Given the immunomodulatory role of MSCs, their exosomes are being assessed extensively in experimental animal models of multiple sclerosis to impede autoimmune diseases’ progression (Baharlooi et al. 2020). Employing an experimental rat model of autoimmune encephalomyelitis, Li and colleagues investigated the effect of exosomes derived from BM-MSCs on microglia polarization and inflammation in the central nervous system (Li et al. 2019). They observed that exosome treatment significantly decreased neural behavioral scores, reduced the infiltration of inflammatory cells into the central nervous system, and reduced demyelination compared to untreated experimental model rats. At molecular levels, there was a significant increase in M2-related and TGF-b IL-10, whereas M1-related TNF-a and IL-12 decreased significantly in exosome treatment. Based on these data, they proposed that therapy using exosomes derived from BM-MSCs could be an interesting alternative for treating multiple sclerosis. On the same note, experiments are underway to exploit exosomes’ ability to cross the blood–brain barrier using aptamer bio-conjugated exosomes to enhance oligodendroglia cell line in vitro and reduce demyelinated lesions in the brain of experimental mice model (Hosseini et al. 2019). Intravenous administration of MSCs-exosomes also helped improve recovery in experimental mice with progressive multiple sclerosis (Laso-García et al. 2018). Despite these encouraging data, no clinical trials have been designed to assess their safety and feasibility in human patients as yet.

Conclusion In conclusion, in this chapter, we have focused only on a few of the diseases treated with MSCs in the clinical studies after extensive characterization in experimental animal models. More recently, the same conditions are being investigated to treat

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with exosomes derived from MSCs as part of the emerging cell-free therapy. Despite encouraging data from the experimental animal studies, little progress has been made using exosomes in the clinics. Most of the clinical trials with exosomes are relevant to cancer, that is, Metastatic pancreatic cancer (NCT03608631), Colon cancer (NCT01294072), Malignant ascites and pleural effusion (NCT01854866), Type 1 diabetes, that is, (NCT02138331) and acute ischemic stroke, that is, (NCT03384433).

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Mesenchymal Stromal Cells for COVID-19 Critical Care Patients A Present Hope Abdelkrim Hmadcha, Tarik Smani, Jose Miguel Sempere-Ortells, Robert Chunhua Zhao, and Bernat Soria

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Mechanism of Infection and Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 The Rationale for the Clinical Use of MSCs for COVID-19 Patients . . . . . . . . . . . . . . . . . . . . . . . . . 178 A. Hmadcha (*) Department of Biotechnology, University of Alicante, Alicante, Spain University of Pablo de Olavide, Sevilla, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain e-mail: [email protected] T. Smani Cardiovascular Pathophysiology, Institute of Biomedicine of Seville (IBiS), University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain Department of Medical Physiology and Biophysics, University of Seville, Seville, Spain Biomedical Research Networking Centers of Cardiovascular Diseases (CIBERCV), Madrid, Spain e-mail: [email protected] J. M. Sempere-Ortells Department of Biotechnology, Immunology Division, University of Alicante, Alicante, Spain e-mail: [email protected] R. C. Zhao Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Cell Biology, School of Life Sciences, Shanghai University, Shanghai, China e-mail: [email protected] B. Soria University of Pablo de Olavide, Sevilla, Spain Department of Physiology, Institute of Bioengineering-Alicante Institute for Health and Biomedical Research (ISABIAL), University Miguel Hernández School of Medicine, Alicante, Spain e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_7

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Progress in MSC-Based Therapy for COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Cell-Based Innovation for COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Side Effects of Mesenchymal Stem Cell-Based Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unmet Challenges of Adoptive MSCs Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Source of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Route of Cell Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dosage Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risks Associated with Stem Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179 181 182 183 183 183 184 184 184 185 185

Abstract

Despite the titanic efforts of health systems worldwide through the implementation of severe public health measures, the number of patients with the current coronavirus disease 2019 (COVID-19) has been dramatically increasing since December 2019. COVID-19 is a real threat that is currently becoming a major concern worldwide. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a virulent infection leading to a high mortality rate. Although, the emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of COVID-19, there remains a need for additional effective vaccines to deal with SARS-CoV-2, a virus characterized by its unpredictable nature, high morbidity, and rapid ability to spread and to meet the global demand and address the potential new viral variants. Still there is a significantly increased demand for the development of new therapeutic alternatives to palliate the ongoing pandemic. Actually, treating critical COVID-19 patients is challenging as no specific treatment options against SARS-CoV-2 are available. The main pathologic features of critical COVID-19 were consistent with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Therefore, regenerative, immunomodulatory, and antiinflammatory properties of mesenchymal stromal cells (MSCs) can reduce the manifestation of cytokine storm and can restore ARDS and ALI, exhibiting an important option to be applied to critical COVID-19 patients. Here we propose MSCs as a potential alternative therapy for COVID-19 patients and discussed specific aspects of this proposed cell therapy. Keywords

ARDS · CAR-T cells · Cell therapy · COVID-19 · Cytokine storm · Immunomodulation · Inflammation · Mesenchymal stromal cells · Organoids · SARS-CoV-2 · Stem cells List of Abbreviations

3D ACE2 ALI Ang-1 ARB

Three-dimensional Angiotensin-converting enzyme 2 Acute lung injuries Angiopoietin-1 Angiotensin receptor blocker

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Mesenchymal Stromal Cells for COVID-19 Critical Care Patients

ARDS AT2s CAR-T CCN1 CFTR CLDN1 COVID-19 CPE Cyr61 DEX ECM EMMPRIN FDA GFP GI Gsis HACE2 HCQ HE HESCs HiPSCs HPSC Hrsace2 Hs-cTnI HSV1 ICU IFN Ifnar1-/IL-1α IL1-β IL-2 Il28r-/Il2rg Isgs KGF KRT18 LPS MAS Mascp6 MERS MODS MPA MSCs NPC

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Acute respiratory distress syndrome Type 2 alveolar epithelial cells Chimeric antigen receptor T cells CCN family number 1 Cystic fibrosis transmembrane conductance regulator Claudin1 Coronavirus disease 2019 Cytopathologic effect Cysteine-rich protein 61 Dexamethasone Extracellular matrix Extracellular matrix metalloproteinase inhibitor (or CD147) Food and Drug Administration Green fluorescent protein Gastrointestinal γ-Secretase inhibitors Human angiotensin-converting enzyme 2 Hydroxychloroquine Heme agglutinin esterase Human embryonic stem cells Human-induced pluripotent stem cells Human pluripotent stem cell Human recombinant soluble ACE2 Highly sensitive troponin-I Herpes simplex virus-1 Intensive care units Interferon C57BL/6 mice with a genetic ablation of their type I interferon receptors Interleukin-alpha Interleukin-beta Interleukin-2 C57BL/6 mice with a genetic ablation of their type III interferon receptors Interleukin-2 receptor gamma chain Interferon-stimulated genes Keratinocyte growth factor Cytokeratin 18 Lipopolysaccharide Macrophage activation syndrome Mouse-adapted strain at passage 6 Middle East respiratory syndrome Multiple organ dysfunction syndromes Mycophenolic acid Mesenchymal stem cells Neural progenitor cells

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NSCs: NSG mouse PAMPs PDGFb PMN QNHC RIG-I RLU RM SARS-cov-2 SFTPC SLC10A2 SOS S-protein TF TMPRSS2 TNFα WHO WT

A. Hmadcha et al.

Neural stem cells NOD-SCID with null mutation in the gene encoding the il2rgl Pathogen-associated molecular patterns Platelet-derived growth factor subunit b Polymorphonuclear Quinacrine dihydrochloride Retinoic acid-inducible gene-I-like Relative luciferase units Regenerative medicine Severe acute respiratory syndrome coronavirus-2 Surfactant protein-C Solute carrier family 10 member 2 Sinusoidal obstructive syndrome Spike protein Tissue factor (or CD142) Transmembrane serine protease 2 Tumor necrosis factor alpha World Health Organization Wild type

Introduction Late in December 2019, an outbreak of atypical pneumonia of unknown etiology was described in Wuhan Province in China. A novel coronavirus named “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” was then identified as the etiologic agent (Gorbalenya et al. 2020; Wu et al. 2010). Later, the disease was designated COrona VIrus Disease 2019 (COVID-19) (World Health Organization 2020). The rapid expansion of COVID-19 cases in number and geographic distribution prompted the World Health Organization (WHO) to declare a global health emergency. Containment of the disease was hindered by the lack of antiviral treatment, lack of vaccines, and asymptomatic carriers. On March 11, 2020, COVID-19 was officially classified by the WHO as a pandemic. The WHO has declared coronavirus disease 2019 (COVID-19) a pandemic due to the rapid increase in infections worldwide. The initial outbreak occurred in Wuhan City of Hubei Province of the People’s Republic of China in December 2019 and has since spread to nearly every country and territory globally. As of April 24, 2021, there have been more than 145 million confirmed cases of COVID-19 in the world, including about 3 million deaths, reported to WHO (WHO 2020). However, despite strict worldwide containment strategies and national closures in many countries, prevalence rates continue to increase with significant mortality. Since COVID-19 affects different people in different ways, most infected people develop mild-to-moderate disease and recover without hospitalization, but a subset of patients progresses to severe illness, with a high mortality rate and limited treatment options. The clinical feature of COVID-19 varies from asymptomatic

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forms to conditions involving multiorgan and systemic manifestations in terms of septic shock and multiple organ dysfunction syndromes (MODS). The primary pathologic features of critical COVID-19 were consistent with acute lung injuries (ALI) and acute respiratory distress syndrome (ARDS). The majority of infected persons is usually asymptomatic or has mild symptoms, and about 15% are affected by ARDS, of which 5% progress to multiple organ dysfunction syndromes or failure. From the point of view of prevention, evasive carriers still in the early stage of infection, and that therefore do not show any clinical manifestation of the disease, are the most infectious and the least tractable. This pathology involves direct attacks by the virus on the cells and secondary attacks on the body after activating the immune system. This means that both the virus and the immune response can cause damage to the body, and common complications or secondary infection can occur. At the cellular level, the spike protein (S-protein) of SARS-CoV-2 interacts with cell receptors to infect target cells. SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2), triggering the endocytosis of virus particles. Consequently, ACE2 receptor would represent a potential therapeutic candidate to study SARS-CoV-2 infection mechanisms. Treating COVID-19 patients is challenging as no specific treatment options against SARS-CoV-2 are available. The current supportive but not curative treatments consist of the use of experimental medication. These include remdesivir, hydroxychloroquine, abidol, lopinavir/ritonavir, plasma from convalescent patients, antibody, and other nonspecific vaccines (Haider and Hyder 2020). Currently, remdesivir appears to be the most promising pharmacological intervention for the treatment of pneumonia caused by COVID-19. The exact pathogenesis of the virus and the dynamics of the disease are not yet fully understood; therefore, the available treatment options are limited. These consist mainly of supportive therapies for symptomatic treatment. Several antiviral drugs (Grein et al. 2020), corticosteroids (Wang et al. 2020b; Al-Rasheed et al. 2021), convalescent plasma (Shen et al. 2020; Verma et al. 2020), and neutralizing monoclonal antibodies (Shanmugaraj et al. 2020) have been tested and have undergone different phases of clinical trials, but none have been approved explicitly for COVID-19. Another alarming fact is the report from some countries of recurrence of infection in recovered as well as vaccinated individuals, which calls into question the efficacy of available treatments (Lan et al. 2020). In the absence of a recommended treatment, observance of general principle of resorting to take preventive measures, including social distancing, hygienic precautions, and use of face masks, remains the preferred strategy (Hyder and Haider 2020). Within this scenario, investigations have been conducted at a dizzying speed to achieve a vaccine. The pioneering manufacturer’s platforms of vaccines (BioNTechPfizer, University of Oxford-AstraZeneca, Moderna, Johnson & Johnson, SanofiGlaxoSmithKline, CanSino Biologics, Inovio, Sinovac, Novavax, Gamaleya Research Institute, CureVac, Clover Biopharmaceuticals, Merck & Co.) have been able to ensure their historic and rapid development. According to WHO, as of February 18, 2021, at least seven different vaccines across three platforms have been rolled out in countries. Vulnerable populations in all countries are the highest priority for vaccination. At the same time, more than 200 additional vaccine

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candidates are in development, of which more than 60 are in clinical development (García-Montero et al. 2021; Yan et al. 2021a). The first mass vaccination program was initiated in early December 2020, and as of February 15, 2021, 175.3 million vaccine doses have been administered (Hasan et al. 2021). At least seven different vaccines (three platforms) have been developed and administered as part of the worldwide vaccination program. WHO issued an Emergency Use Listing (EUL) for the Pfizer COVID-19 vaccine (BNT162b2) on December 31, 2020. On February 15, 2021, WHO issued EUL for two versions of the AstraZeneca/Oxford COVID-19 vaccine, manufactured by the Serum Institute of India and SKBio. On March 12, 2021, WHO issued an EUL for the COVID-19 vaccine Ad26.COV2.S, developed by Janssen (Johnson & Johnson). WHO is on track to EUL other vaccine products through June. Although many biotechnology companies have developed different vaccines and millions of people have been vaccinated to date, the complete process of safety evaluation, manufacturing, and scale-up are still under question, and longer followup is needed (Yan et al. 2021b; Kadkhoda 2021). As such, the development of feasible, safe, and effective therapies is extremely urgent. Therefore, increasing experimental and clinical evidence has given credibility to the claim that advanced therapies research could change the future of COVID-19 and the forthcoming emergence of virulent viruses. Notably, cell-based therapies will impact, not yet foreseen, on the present and future sequels of COVID-19. In this regard, mesenchymal stem cells (MSCs) have long been associated with the repairing and rejuvenating damaged tissues due to their broad pharmacological effects, including anti-inflammation, immunomodulation, anti-apoptosis, angiogenesis, and trans-differentiation to specific cell types. They also secrete a myriad of soluble factors and vesicles altogether involved in restoring tissue homeostasis and functionality. The efficacy of MSCs and their secretory factors has been proven in successfully reducing inflammation, dampening immune responses, and repairing lung damage in various preclinical and clinical models (Hmadcha et al. 2009). Therefore, the potential of MSC-based therapy as an option for severe or critically ill COVID-19 patients is being explored in the current scenario (Leng et al. 2020; Sánchez-Guijo et al. 2020) (Table 1). On the one hand, recent studies focus on regenerative, immunomodulatory, and anti-inflammatory properties of mesenchymal stromal cells (MSCs) to reduce the manifestation of cytokine storm and to restore ARDS and ALI, exhibiting an important option to be applied to critical COVID-19 patients, or on MSCs secretome to treat COVID-19 pneumonia (Tang et al. 2020; Meng et al. 2020; Li et al. 2020b; Liang et al. 2020; Lanzoni et al. 2021). Other research includes the use of hematopoietic stem cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, as well as immune chimeric antigen receptor T cell (CAR-T cell). On the other hand, the understanding of the mechanism of infection and pathogenesis are still limited. In this regard, the use of human pluripotent stem cells, both embryonic stem cells (hESCs) and induced stem cells (hiPSCs), to generate tissuespecific human organoids (lung, intestinal, liver, vascular, heart, and kidney

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Table 1 Registered clinical trials using MSC-based therapy to COVID-19 (NIH-ClinicalTrial. gov) NCT number NCT04444271

Title Mesenchymal Stem Cell Infusion for COVID-19 Infection

Study results No results available

NCT04416139

Mesenchymal Stem Cell for Acute Respiratory Distress Syndrome Due for COVID-19

No results available

Phase 2

NCT04713878

Mesenchymal Stem Cells Therapy in Patients With COVID-19 Pneumonia

No results available

Not applicable

NCT04429763

Safety and Efficacy of Mesenchymal Stem Cells in the Management of Severe COVID19 Pneumonia A Proof of Concept Study for the DNA Repair Driven by the Mesenchymal Stem Cells in Critical COVID-19 Patients NestaCell ® Mesenchymal Stem Cell to Treat Patients With Severe COVID-19 Pneumonia

No results available

Phase 2

No results available

Not applicable

No results available

Phase 2

NCT04611256

Mesenchymal Stem Cells in Patients Diagnosed With COVID-19

No results available

Phase 1

NCT04302519

Novel Coronavirus Induced Severe Pneumonia Treated by Dental Pulp Mesenchymal Stem Cells Use of Mesenchymal Stem Cells in Acute Respiratory Distress Syndrome Caused by COVID19 Efficacy of Infusions of MSC From Wharton Jelly in the SARS-Cov-2 (COVID-19) Related Acute Respiratory Distress Syndrome Clinical Trial of Allogeneic Mesenchymal Cells From Umbilical Cord Tissue in Patients With COVID-19

No results available

Early Phase 1

No results available

Early Phase 1

No results available

Phase 2

No results available

Phase 2

NCT04898088

NCT04315987

NCT04456361

NCT04625738

NCT04366271

Phases Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04444271 https:// ClinicalTrials. gov/show/ NCT04416139 https:// ClinicalTrials. gov/show/ NCT04713878 https:// ClinicalTrials. gov/show/ NCT04429763 https:// ClinicalTrials. gov/show/ NCT04898088 https:// ClinicalTrials. gov/show/ NCT04315987 https:// ClinicalTrials. gov/show/ NCT04611256 https:// ClinicalTrials. gov/show/ NCT04302519 https:// ClinicalTrials. gov/show/ NCT04456361 https:// ClinicalTrials. gov/show/ NCT04625738 https:// ClinicalTrials. gov/show/ NCT04366271 (continued)

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Table 1 (continued) NCT number NCT04366323

NCT04252118

NCT04313322

NCT04909892

NCT04905836

NCT04753476

NCT04336254

NCT04346368

NCT04288102

NCT04629105

Title Clinical Trial to Assess the Safety and Efficacy of Intravenous Administration of Allogeneic Adult Mesenchymal Stem Cells of Expanded Adipose Tissue in Patients With Severe Pneumonia Due to COVID-19 Mesenchymal Stem Cell Treatment for Pneumonia Patients Infected With COVID19 Treatment of COVID-19 Patients Using Wharton’s JellyMesenchymal Stem Cells Study of Allogeneic AdiposeDerived Mesenchymal Stem Cells to Treat Post COVID-19 “Long Haul” Pulmonary Compromise Study of Allogeneic AdiposeDerived Mesenchymal Stem Cells for Treatment of COVID19 Acute Respiratory Distress Treatment of Severe COVID-19 Patients Using Secretome of Hypoxia-Mesenchymal Stem Cells in Indonesia Safety and Efficacy Study of Allogeneic Human Dental Pulp Mesenchymal Stem Cells to Treat Severe COVID-19 Patients Bone Marrow-Derived Mesenchymal Stem Cell Treatment for Severe Patients With Coronavirus Disease 2019 (COVID-19) Treatment With Human Umbilical Cord-derived Mesenchymal Stem Cells for Severe Corona Virus Disease 2019 (COVID-19) Regenerative Medicine for COVID-19 and Flu-Elicited ARDS Using Longeveron Mesenchymal Stem Cells (LMSCs) (RECOVER)

Study results No results available

Phases Phase 1| Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04366323

No results available

Phase 1

No results available

Phase 1

No results available

Phase 2

https:// ClinicalTrials. gov/show/ NCT04252118 https:// ClinicalTrials. gov/show/ NCT04313322 https:// ClinicalTrials. gov/show/ NCT04909892

No results available

Phase 2

No results available

Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 2

https:// ClinicalTrials. gov/show/ NCT04288102

No results available

Phase 1

https:// ClinicalTrials. gov/show/ NCT04629105

https:// ClinicalTrials. gov/show/ NCT04905836 https:// ClinicalTrials. gov/show/ NCT04753476 https:// ClinicalTrials. gov/show/ NCT04336254 https:// ClinicalTrials. gov/show/ NCT04346368

(continued)

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Table 1 (continued) Study results No results available

NCT number NCT04273646

Title Study of Human Umbilical Cord Mesenchymal Stem Cells in the Treatment of Severe COVID-19

NCT04371601

Safety and Effectiveness of Mesenchymal Stem Cells in the Treatment of Pneumonia of Coronavirus Disease 2019 Study to Evaluate the Efficacy and Safety of AstroStem-V in Treatment of COVID-19 Pneumonia Study of Intravenous Administration of Allogeneic Adipose-Derived Mesenchymal Stem Cells for COVID-19Induced Acute Respiratory Distress Expanded Access Protocol on Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicle Infusion Treatment for Patients With COVID-19 Associated ARDS A Randomized, Double-Blind, Placebo-Controlled Clinical Trial to Determine the Safety and Efficacy of Hope Biosciences Allogeneic Mesenchymal Stem Cell Therapy (HB-adMSCs) to Provide Protection Against COVID-19 Clinical Research of Human Mesenchymal Stem Cells in the Treatment of COVID-19 Pneumonia Autologous Adipose-derived Stem Cells (AdMSCs) for COVID-19

No results available

Early Phase 1

No results available

Phase 1| Phase 2

No results available

Phase 2

Administration of Allogenic UC-MSCs as Adjuvant Therapy for Critically-Ill COVID-19 Patients Treatment of Covid-19 Associated Pneumonia With Allogenic Pooled Olfactory Mucosa-derived Mesenchymal Stem Cells

NCT04527224

NCT04728698

NCT04657458

NCT04348435

NCT04339660

NCT04428801

NCT04457609

NCT04382547

Phases Not applicable

No results available

URL https:// ClinicalTrials. gov/show/ NCT04273646 https:// ClinicalTrials. gov/show/ NCT04371601 https:// ClinicalTrials. gov/show/ NCT04527224 https:// ClinicalTrials. gov/show/ NCT04728698

https:// ClinicalTrials. gov/show/ NCT04657458

No results available

Phase 2

https:// ClinicalTrials. gov/show/ NCT04348435

No results available

Phase 1| Phase 2

No results available

Phase 2

No results available

Phase 1

No results available

Phase 1| Phase 2

https:// ClinicalTrials. gov/show/ NCT04339660 https:// ClinicalTrials. gov/show/ NCT04428801 https:// ClinicalTrials. gov/show/ NCT04457609 https:// ClinicalTrials. gov/show/ NCT04382547 (continued)

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Table 1 (continued) NCT number NCT04349631

NCT04366063

NCT04352803

NCT04573270

NCT04490486

NCT04355728

NCT04888949

NCT04461925

NCT04522986

NCT04903327

Title A Clinical Trial to Determine the Safety and Efficacy of Hope Biosciences Autologous Mesenchymal Stem Cell Therapy (HB-adMSCs) to Provide Protection Against COVID-19 Mesenchymal Stem Cell Therapy for SARS-CoV-2related Acute Respiratory Distress Syndrome Adipose Mesenchymal Cells for Abatement of SARS-CoV-2 Respiratory Compromise in COVID-19 Disease Mesenchymal Stem Cells for the Treatment of COVID-19

Study results No results available

Phases Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04349631

No results available

Phase 2| Phase 3

No results available

Phase 1

No results available

Phase 1

Umbilical Cord Tissue (UC) Derived Mesenchymal Stem Cells (MSCs) Versus Placebo to Treat Acute Pulmonary Inflammation Due to COVID-19 Use of UC-MSCs for COVID-19 Patients

No results available

Phase 1

https:// ClinicalTrials. gov/show/ NCT04366063 https:// ClinicalTrials. gov/show/ NCT04352803 https:// ClinicalTrials. gov/show/ NCT04573270 https:// ClinicalTrials. gov/show/ NCT04490486

No results available

Phase 1| Phase 2

A Study of ADR-001 in Patients With Severe Pneumonia Caused by SARS-CoV-2 Infection (COVID-19) Treatment of Coronavirus COVID-19 Pneumonia (Pathogen SARS-CoV-2) With Cryopreserved Allogeneic P_MMSCs and UC-MMSCs An Exploratory Study of ADR-001 in Patients With Severe Pneumonia Caused by SARS-CoV-2 Infection Study of Intravenous COVIMSC for Treatment of COVID19-Induced Acute Respiratory Distress

No results available

Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 1

No results available

Phase 2

https:// ClinicalTrials. gov/show/ NCT04355728 https:// ClinicalTrials. gov/show/ NCT04888949 https:// ClinicalTrials. gov/show/ NCT04461925 https:// ClinicalTrials. gov/show/ NCT04522986 https:// ClinicalTrials. gov/show/ NCT04903327 (continued)

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Table 1 (continued) Study results No results available

NCT number NCT04348461

Title BAttLe Against COVID-19 Using MesenchYmal Stromal Cells

NCT04565665

Cord Blood-Derived Mesenchymal Stem Cells for the Treatment of COVID-19 Related Acute Respiratory Distress Syndrome Therapeutic Study to Evaluate the Safety and Efficacy of DW-MSC in COVID-19 Patients

No results available

Phase 1| Phase 2

No results available

Phase 1

NCT04362189

Efficacy and Safety Study of Allogeneic HB-adMSCs for the Treatment of COVID-19

No results available

Phase 2

NCT04293692

Therapy for Pneumonia Patients Infected by 2019 Novel Coronavirus

No results available

Not applicable

NCT04390152

Safety and Efficacy of Intravenous Wharton’s Jelly Derived Mesenchymal Stem Cells in Acute Respiratory Distress Syndrome Due to COVID 19 Umbilical Cord Lining Stem Cells (ULSC) in Patients With COVID-19 ARDS

No results available

Phase 1| Phase 2

No results available

Phase 1| Phase 2

Study of the Safety of Therapeutic Tx With Immunomodulatory MSC in Adults With COVID-19 Infection Requiring Mechanical Ventilation A Phase II Study in Patients With Moderate to Severe ARDS Due to COVID-19

No results available

Phase 1

No results available

Phase 2

No results available

Phase 2

NCT04535856

NCT04494386

NCT04397796

NCT04780685

NCT04377334

Mesenchymal Stem Cells (MSCs) in InflammationResolution Programs of Coronavirus Disease 2019 (COVID-19) Induced Acute Respiratory Distress Syndrome (ARDS)

Phases Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04348461 https:// ClinicalTrials. gov/show/ NCT04565665 https:// ClinicalTrials. gov/show/ NCT04535856 https:// ClinicalTrials. gov/show/ NCT04362189 https:// ClinicalTrials. gov/show/ NCT04293692 https:// ClinicalTrials. gov/show/ NCT04390152

https:// ClinicalTrials. gov/show/ NCT04494386 https:// ClinicalTrials. gov/show/ NCT04397796

https:// ClinicalTrials. gov/show/ NCT04780685 https:// ClinicalTrials. gov/show/ NCT04377334

(continued)

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A. Hmadcha et al.

Table 1 (continued) Study results No results available

NCT number NCT04452097

Title Use of hUC-MSC Product (BX-U001) for the Treatment of COVID-19 With ARDS

NCT04345601

Mesenchymal Stromal Cells for the Treatment of SARS-CoV-2 Induced Acute Respiratory Failure (COVID-19 Disease) Investigational Treatments for COVID-19 in Tertiary Care Hospital of Pakistan

No results available

Phase 1| Phase 2

No results available

Not applicable

Efficacy and Safety Evaluation of Mesenchymal Stem Cells for the Treatment of Patients With Respiratory Distress Due to COVID-19 The Use of Exosomes for the Treatment of Acute Respiratory Distress Syndrome or Novel Coronavirus Pneumonia Caused by COVID-19 A Pilot Clinical Study on Inhalation of Mesenchymal Stem Cells Exosomes Treating Severe Novel Coronavirus Pneumonia Clinical Use of Stem Cells for the Treatment of Covid-19

No results available

Phase 1| Phase 2

No results available

Phase 1| Phase 2

https:// ClinicalTrials. gov/show/ NCT04798716

No results available

Phase 1

No results available

Phase 1| Phase 2

NCT04467047

Safety and Feasibility of Allogenic MSC in the Treatment of COVID-19

No results available

Phase 1

NCT04798066

Intermediate Size Expanded Access Protocol Evaluating HB-adMSC’s for the Treatment of Post-COVID-19 Syndrome Study of Allogeneic AdiposeDerived Mesenchymal Stem Cells for Non-COVID-19 Acute Respiratory Distress Syndrome ACT-20 in Patients With Severe COVID-19 Pneumonia

No results available

https:// ClinicalTrials. gov/show/ NCT04276987 https:// ClinicalTrials. gov/show/ NCT04392778 https:// ClinicalTrials. gov/show/ NCT04467047 https:// ClinicalTrials. gov/show/ NCT04798066 https:// ClinicalTrials. gov/show/ NCT04909879 https:// ClinicalTrials. gov/show/ NCT04398303

NCT04492501

NCT04390139

NCT04798716

NCT04276987

NCT04392778

NCT04909879

NCT04398303

Phases Phase 1| Phase 2

No results available

Phase 2

No results available

Phase 1| Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04452097 https:// ClinicalTrials. gov/show/ NCT04345601 https:// ClinicalTrials. gov/show/ NCT04492501 https:// ClinicalTrials. gov/show/ NCT04390139

(continued)

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Table 1 (continued) NCT number NCT04361942

NCT03042143

NCT04537351

NCT04437823

NCT04269525

NCT04602442

NCT04447833

NCT04371393

NCT04491240

NCT04333368

NCT04299152

Title Treatment of Severe COVID-19 Pneumonia With Allogeneic Mesenchymal Stromal Cells (COVID_MSV) Repair of Acute Respiratory Distress Syndrome by Stromal Cell Administration (REALIST) (COVID-19) The MEseNchymal coviD-19 Trial: MSCs in Adults With Respiratory Failure Due to COVID-19 or Another Underlying Cause Efficacy of Intravenous Infusions of Stem Cells in the Treatment of COVID-19 Patients

Study results No results available

Phases Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 2

Umbilical Cord (UC)-Derived Mesenchymal Stem Cells (MSCs) Treatment for the 2019novel Coronavirus (nCOV) Pneumonia Safety and Efficiency of Method of Exosome Inhalation in COVID-19 Associated Pneumonia Mesenchymal Stromal Cell Therapy For The Treatment Of Acute Respiratory Distress Syndrome MSCs in COVID-19 ARDS

No results available

Phase 2

No results available

Phase 2

No results available

Phase 1

No results available

Phase 3

Evaluation of Safety and Efficiency of Method of Exosome Inhalation in SARSCoV-2 Associated Pneumonia. Cell Therapy Using Umbilical Cord-derived Mesenchymal Stromal Cells in SARS-CoV-2related ARDS Stem Cell Educator Therapy Treat the Viral Inflammation in COVID-19

Has results

Phase 1| Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04361942 https:// ClinicalTrials. gov/show/ NCT03042143 https:// ClinicalTrials. gov/show/ NCT04537351 https:// ClinicalTrials. gov/show/ NCT04437823 https:// ClinicalTrials. gov/show/ NCT04269525 https:// ClinicalTrials. gov/show/ NCT04602442 https:// ClinicalTrials. gov/show/ NCT04447833 https:// ClinicalTrials. gov/show/ NCT04371393 https:// ClinicalTrials. gov/show/ NCT04491240 https:// ClinicalTrials. gov/show/ NCT04333368 https:// ClinicalTrials. gov/show/ NCT04299152 (continued)

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Table 1 (continued) NCT number NCT04524962

Title Study of Descartes-30 in Acute Respiratory Distress Syndrome

Study results No results available

NCT04541680

Nintedanib for the Treatment of SARS-Cov-2 Induced Pulmonary Fibrosis

No results available

Phase 3

NCT04466098

Multiple Dosing of Mesenchymal Stromal Cells in Patients With ARDS (COVID19) A Study of Cell Therapy in COVID-19 Subjects With Acute Kidney Injury Who Are Receiving Renal Replacement Therapy ASC Therapy for Patients With Severe Respiratory COVID-19

No results available

Phase 2

No results available

Phase 1| Phase 2

No results available

Phase 1| Phase 2

NCT04400032

Cellular Immuno-Therapy for COVID-19 Acute Respiratory Distress Syndrome

No results available

Phase 1| Phase 2

NCT04684602

Mesenchymal Stem Cells for the Treatment of Various Chronic and Acute Conditions

No results available

Phase 1| Phase 2

NCT04445220

NCT04341610

Phases Phase 1| Phase 2

URL https:// ClinicalTrials. gov/show/ NCT04524962 https:// ClinicalTrials. gov/show/ NCT04541680 https:// ClinicalTrials. gov/show/ NCT04466098 https:// ClinicalTrials. gov/show/ NCT04445220 https:// ClinicalTrials. gov/show/ NCT04341610 https:// ClinicalTrials. gov/show/ NCT04400032 https:// ClinicalTrials. gov/show/ NCT04684602

organoids) may provide a next-generation cellular model for investigating viral infection and drug screening. Altogether, the ultimate goal of all these strategies is to achieve a definitive and efficient therapy for COVID-19.

Mechanism of Infection and Immune Response Several types of coronavirus are known to have the potential for human infection; only six are known to cause disease in humans: HCoV-229E, HCoV-OC43, HCoVNL63, HCoV-HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV (Skariyachan et al. 2019; Bonilla-Aldana et al. 2020). The 2019nCoV, a single-stranded RNA virus, is closely related to SARS-CoV that emerged in 2003–2004 and caused an epidemic disease (Racaniello 2016). The virus was provisionally designated 2019-nCoV and later given the official name SARS-CoV2 (Gorbalenya et al. 2020). SARS-CoV-2 was characterized as a beta-coronavirus

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and recognized as the seventh discrete coronavirus species capable of causing human disease (Zhu et al. 2020a). The virion nucleocapsid consists of an RNA genome complexed with a nucleoprotein and is enveloped by a phospholipid bilayer. This bilayer is covered by two types of spike proteins: protein S, which is present in all known coronaviruses and forms peplomers on the surface, which gives it a corona solar appearance, and protein hemagglutinin esterase (HE), which is present in only a few types of coronaviruses. The spike S protein interacts with the host angiotensinconverting enzyme 2 (ACE2) receptor protein, resulting in membrane fusion with subsequent release of the viral genome into the cell, or clathrin-dependent and clathrin-independent endocytosis of the virus (Li et al. 2020a). Moreover, the ACE2 receptor is widely expressed by human cells, particularly in the lungs by type 2 alveolar epithelial cells (AT2s) and capillary epithelial cells, and cells of the heart, kidney, and intestine. In addition, two proteases, transmembrane serine protease 2 (TMPRSS2) and extracellular matrix metalloproteinase inhibitor (EMMPRIN or CD147), have been reported to be essential for virus entry into the host cell (Chen et al. 2020). The damage to the lungs is caused by the virus either directly, by the destruction of AT2s and capillary endothelial cells, which disrupts the renin-angiotensin system, or indirectly, by dampening the immune response (Jin et al. 2020). The precise pathogenesis of this particular virus remains unknown. Most of the information on the cycle of infection and subsequent immune response is primarily derived from the SARS and MERS coronaviruses due to the correlation in the clinical features of patients with COVID-19 with these viral infections (Guan et al. 2020; Huang et al. 2020). Upon infection, the virus is recognized by the innate immune system through pathogen-associated molecular patterns (PAMPs), which in this case is the genomic RNA of the virion. This leads to activation of the NFkB pathway and the IRF3 pathway, which results in the expression of type I interferon (IFN). IFN then activates the JAK/STAT pathway and induces the expression of IFN-stimulated genes (ISGs) that have antiviral activity (Prompetchara et al. 2020). Successful viral clearance and amelioration of the clinical manifestations of the disease depend on this effective immune response. However, the virus can evade IFN- and ISG-mediated killing and often results in a delayed IFN response. This results in the infiltration of hyper-inflammatory neutrophils and macrophages into the lung site, along with pro-inflammatory cytokines, mainly IL-1b, IL-2, IL-6, IL-7, IL-8, IL-17, G-CSF, GM-CSF, CCL3, MCP1, and TNF (Cao 2020). This so-called cytokine storm is a result of the innate response (neutrophils and macrophages). Moreover, hyperactivation of T lymphocytes (especially the Th1 response) is actually responsible for pulmonary dysfunction and abnormalities such as pneumonitis, ARDS, respiratory failure, viral sepsis, and organ failure. Elevated pro-inflammatory cytokines also induce the synthesis of hyaluronan synthase 2, which produces hyaluronan in the lungs, leading to the characteristic opacity or fluid accumulation in the lungs (Shi et al. 2020). In critical cases, the virus can also enter the peripheral blood (viremia) and translocate to other target organs expressing the ACE2 receptor, such as the heart, kidney, and intestines, resulting in multiple organ dysfunctions. Thus, there is a great

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need to discover the specific virulence mechanisms during which cell and tissue injury occurs. As it is not always possible to capture the underlying mechanisms of pathophysiology in humans, several modeling methods have been developed, including 3D-engineered organoids derived from pluripotent stem cells (ESCs and iPSCs), different types of stem cells, and animals (Sun et al. 2020; Yang et al. 2020; Youk et al. 2020; Zheng et al. 2021; Boudewijns et al. 2020).

The Rationale for the Clinical Use of MSCs for COVID-19 Patients MSCs administration tends to unbalance the pro-inflammatory cytokines, immune cells, and tissue damage toward an anti-inflammatory and regenerative microenvironment. MSCs have been widely used in cell-based therapy, from basic research to clinical trials (Acosta et al. 2013; Capilla-González et al. 2018; Soria-Juan et al. 2019). Safety and efficacy have been avidly documented in many clinical trials, especially in both systemic and local immune-mediated inflammatory diseases, such as GvHD, Crohn’s complex fistula, and type 2 diabetes complications (Soria-Juan et al. 2019; García-Gómez et al. 2010; Herreros et al. 2012). MSCs play a positive role mainly in two ways: immunomodulatory effects and anti-inflammatory abilities both linked to regeneration (Pacienza et al. 2017; Lee and Kang 2020). MSCs, when activated, can secrete many types of cytokines by paracrine secretion or make direct interactions with immune cells (Leng et al. 2020). MSCs may act as suppressors of the cytokine storm, specifically through IL-1 blockade. Recent data support that IL-1 receptor antagonist, a naturally occurring antagonist of IL-1α/IL1-β signaling pathways, has been attributed to the immunosuppressive effects of MSCs (Harrell et al. 2020). So, IL-1 blockade seems to activate MSCs toward anti-inflammatory phenotype able of releasing antiinflammatory cytokines, of increasing Treg, and of favoring polarization of M1 (pro-inflammatory) macrophages into M2 (anti-inflammatory), which could contribute to revascularization and regeneration of lung tissue (Varghese et al. 2017). In summary, MSCs tend to unbalance the pro-inflammatory cytokines, immune cells, and tissue damage toward an anti-inflammatory and regenerative microenvironment (Fig. 1). This is why these cells have been proposed for use in pulmonary sepsis and cystic fibrosis. They are safe when used for ARDS (Wilson et al. 2015). The intravenous route is the most appropriate for the current intensive care unit (ICU) setting. Additionally, MSCs of any origin injected intravenously are rapidly located in the pulmonary microcirculation network because the cells are significant than the diameter of the capillaries, based on previous data in preclinical animal models. They can also be captured by local macrophages, which subsequently stimulate MSCs to produce IL-10, indirectly providing a source of immunosuppressive cytokines (Argibay et al. 2017). Altogether, MSCs have been successfully tested in other inflammatory diseases. Preclinical data suggest that they may be beneficial in patients with COVID-19 with severe pulmonary inflammation and oxygen therapy (mechanical ventilation) without excluding their use in earlier stages of the disease.

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Fig. 1 Schematic of the anti-inflammatory effects of mesenchymal stem cells-derived exosomes, secretome, and combinatory treatment on lung inflammation and tissue damage caused by COVID19. Abbreviations: Breg, regulatory B cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; IDO, indoleamine-pyrrole 2,3-dioxygenase; IFNγ, interferon gamma; IL-1RA, interleukin-1 receptor antagonist; IL, interleukin; IP-10, interferon gamma-induced protein 10 (or CXCL10, C-XC motif chemokine ligand 10); M1-macrophages, classically activated macrophages; M2-macrophages, alternatively activated macrophages; MCP, monocyte chemoattractant protein; MIP-1, macrophage inflammatory protein 1; NK, natural killer; PGE2, prostaglandin E2; SARSCoV-2, severe acute respiratory syndrome coronavirus 2; Treg, regulatory T cell; TGF-β, transforming growth factor-beta; Th1, T helper type 1; Th2, T helper type 2; TNFα, tumor necrosis factor-alpha; TSG-6, stimulated gene-6; VEGF, vascular endothelial growth factor

Progress in MSC-Based Therapy for COVID-19 MSCs have been widely used for their capacity to differentiate into diverse cell lineages, migration, and secretion of cellular regulators (secretome), together with their immunosuppressive and immunomodulatory potential. Moreover, their isolation is almost easy and presents no major ethical problems, making them the most suitable stem cells among many others (Larijani et al. 2015). On the one hand, adipose tissue, umbilical cord, bone marrow, and blood are some important sources of MSCs. Although adipose tissue-derived MSCs have been shown to have more exciting results initially, the best source of stem cells still needs to be found (Gentile and Sterodimas 2020; Song et al. 2021). On the other hand, through their impacts on T and B cells, macrophages, and dendritic cells, they help establish a tolerogenic environment leading to an optimal therapeutic condition (Wang et al. 2018; Lee and Song 2018). Consequently, by inhibiting T- and B-cell proliferation and successfully regulating pro-inflammatory cytokines to optimize the microenvironment for intrinsic recovery, MSCs can reduce the cytokine storm. Moreover, MSCs can restrict the infiltration of innate immune system cells, consequently decreasing the secretion of inflammatory cytokines, which may indirectly

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attenuate the cytokine storm (Zhu et al. 2020b; Ellison-Hughes et al. 2020; Song et al. 2021; Jeyaraman et al. 2021). Under COVID conditions, a few days after infusion of MSCs, immune cells related to the cytokine storm are shown to decrease. Increased levels of lymphocytes and regulatory dendritic cells along with decreased levels of CRP, IL-1, IL-6, IL-12, IFN-γ, and TNF are also other results of this type of MSC-based therapy. MSCs can also deliver antimicrobial peptides and anti-inflammatory cytokines (Rajarshi et al. 2020; Wang et al. 2020a; Leng et al. 2020). In addition to these anti-inflammatory characteristics, the secretion of IL-10 and some growth factors, together with their regenerative and repair capacity, renders MSCs a potent therapeutic tool for lung repair and treatment of ARDS (Azmi et al. 2020). Administration of MSCs has also been shown to have beneficial effects in conditions like sepsis and septic shock due to their ability to normalize inflammatory biomarkers, oxygen saturation, and pulmonary improvements. For sepsis condition, umbilical cord Wharton’s jelly-derived MSCs are mentioned as the best source of MSCs due to their effectiveness and acceptability (Laroye et al. 2020). Furthermore, the gene expression profile showed that MSCs were negatively expressed angiotensin-converting enzyme 2 (ACE2-), an essential protein for viral infection along with transmembrane serine protease 2 (TMPRSS2-), which indicated that MSCs are free from COVID-19 infection (Leng et al. 2020; Hernandez et al. 2021). In this regard, the FDA has confirmed the safety and efficacy of MSCs for widespread application for COVID-19 cases (Choudhery and Harris 2020; Kavianpour et al. 2020). Despite the aforementioned benefits on the administration of MSCs, there are still some challenges; MSCs-related characteristics regarding their dosage, route of administration, frequency, and location of MSCs in the damaged sites have posed some limitations. Ethical concerns that remain, along with the lack of standardized protocols in the preparation and isolation processes, are other challenges. Another primary concern about MSCs therapy is their side effect of increased hypercoagulability (Jeyaraman et al. 2021). Therefore, in accordance with the increased risk of thrombosis, cell-free therapies, including MSCs secretome and MSCs extracellular vesicles, appear to be an interesting treatment approach for COVID-19 having no risk of mutagenesis and/or tumorigenicity. Exosomes harbor different types of microRNAs and mRNAs and various protein components and have a lower risk of escort and lower transmission of infection. However, a clearer understanding of the dose, timing, and route of administration of the cells is needed. Their ability for nebulized administration (Hmadcha et al. 2020; Aguilera et al. 2021) and more extended storage periods also make them promising alternative therapeutic approaches (Maron-Gutierrez and Rocco 2020; Kheirkhah et al. 2021). One of the first clinical trials to study the efficacy of intravenous infusion of MSCs in ten patients with confirmed moderate, severe, or critical forms of COVID19 aged 45–75 years was conducted in China (Chinese Clinical Trial Registry – ChiCTR2000029990). Seven patients (one with the critical, four with the severe, and two with the moderate form of the disease) received an intravenous infusion of MSCs; three patients with the severe disease received a placebo. Two days after

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MSCs infusion, all seven patients showed a significant improvement in lung function and a decrease in the expression of disease symptoms. All seven patients who received MSCs recovered and were discharged 10 days after the procedure. Due to the immunosuppressive properties of MSCs, after infusion, they contributed to a significant decrease in the level of pro-inflammatory cytokines and chemokines in serum, which attracted a lower number of mononuclear cells/macrophages to the damaged lungs. At the same time, MSCs promoted the recruitment of many regulatory dendritic cells (DCs) to the site of inflammation. In addition, there was an increase in IL-10 and vascular endothelial growth factor (VEGF) levels in this trial, which may contribute to lung recovery. Among the three patients in the control group (who received a placebo), one died, the second developed ARDS, and the third remained in severe condition (Leng et al. 2020). Since then and over a year, many clinical trials have been launched with different types of MSCs to treat COVID-19 disease.

Other Cell-Based Innovation for COVID-19 Cell therapy is emerging as one of the most promising strategies for regenerating damaged or failed tissues and organs in the healthcare system. In this regard, in addition to the extensive use of MSCs of both autologous and allogeneic origin, there is a wide range of treatments using various cell types (e.g., T cells, NK lymphocytes, and different stem cells). In this context, the adoptive T-cell therapy approach or CAR-T cell therapy as a type of immunotherapy has proven effective against some infections and diseases. In this case, T cells are extracted from the patient’s immune system (autologous source) and sent to the laboratory for genetic modification. The modified cells are then reinfused into the patient (Bonifant et al. 2016; Maus and Levine 2016; Seif et al. 2019). Despite the impressive efficacy of CAR-T cell therapy in treatment, it has several serious side effects, including cytokine release syndrome (CRS) and neurological difficulties. Immediate-onset CRS tends to be a cytokine storm (Chen et al. 2019; Hong et al. 2021). Currently, T-cell therapy has also shown promise in immunosuppressed individuals as a preventive measure against COVID-19. Thus, peripheral blood cells from convalescent subjects who had been at risk for the virus were used (Keller et al. 2020). Regulatory T-cell-related strategies have also been suggested as treatment approaches for disease management based on their ability to inactivate innate/ adaptive immunity through inhibitory molecules (Stephen-Victor et al. 2020). In addition, antigen-specific modified/unmodified T-cell transfer has shown promising results in treating different disorders by reconstituting T-cell subsets (effector/memory cells). In this context, it is mentioned that adoptive T-cell therapy by transferring immune subsets of T cells has therapeutic benefits that may be the same as the characteristics of adult tissue stem cells. However, the required high maintenance of memory T cells and engraftment processes may create some limitations (Busch et al. 2016). In this regard, specific COVID-19-related T cells (within CD45RA memory T cells) have been recognized that can be feasibly received by depleting CD45RA

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from convalescent donors. These cells can provide a cell population for the condition of lymphopenia along with rapid reactions to infection. Memory T cells can respond quickly to infection and provide long-term immune protection to reduce the severity of COVID-19 symptoms. Also, CD45RA memory T cells confer protection from other pathogens encountered by the donors throughout their life (Ferreras et al. 2021). CD45RA memory T cells also provide immunity against probable secondary infections that may be found in COVID-19 hospitalized individuals (Ferreras et al. 2021). HLA-matched cytotoxic T cells isolated from convalescent patients are other promising approaches for treating COVID-19, as are EBV-specific cytotoxic T cells used for EBV+-related lymphomas (Hanley et al. 2020). Another promising candidate for a significant advance has been natural killer (NK) cell therapy. In this case, autologous or allogeneic origins can be used to create pure populations of NK cells. The use of allogeneic NK cells as a platform for CAR engineering has been augmented by the limitations of autologous NK cells (such as a diminished effector role and the requirement for a patient-specific stock) (Veluchamy et al. 2017; Daher and Rezvani 2018). Given that the decrease in NK cell numbers may be related to the severity of COVID-19 infection, some clinical trials used engineered NK cells to help combat COVID-19 (Market et al. 2020; van Eeden et al. 2020). However, the use of NK cells also has many drawbacks that may clinically hinder their efficacy. Their short lifespan (due to the lack of cytokine support), low cell number, and vulnerability to immunosuppressed status could limit their trafficking and function (Nayyar et al. 2019; Liu et al. 2021).

Side Effects of Mesenchymal Stem Cell-Based Therapy Although the safety and efficacy of MSCs infusion have been demonstrated in hundreds of clinical trials, this treatment can lead to potential complications (Acosta et al. 2013; Capilla-González et al. 2018; Soria-Juan et al. 2019), and possibly it is the time for combinatory therapies. Preclinical studies have shown that the lungs act as a filter that retains most of the cells injected intravenously (Zhang et al. 2020). Numerous critically ill COVID-19 patients are in a hypercoagulable procoagulant state. Hence, they are at high risk for disseminated intravascular coagulation, thromboembolism, and thrombotic multiorgan failure, another cause of high lethality of the infection. It remains unclear whether intravenous (IV) infusion is a safe and effective route of MSCs infusion for COVID-19 patients. This information is important as MSC-based products express variable levels of highly procoagulant tissue factor (TF or CD142), which compromises the hemocompatibility and safety profile of the cells. Of potential concern is that intravenous infusions of poorly characterized MSC products with uncontrolled (high) TF (CD142) expression may trigger blood clotting in COVID-19 subjects and other vulnerable patient populations and further promote the risk of thromboembolism. By contrast, well-characterized products with robust manufacturing procedures and optimized clinical delivery modes hold great promise for improving COVID-19 patients by exerting their beneficial immunomodulatory effects, inducing tissue

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repair and organ protection. While the need for MSCs therapy for COVID-19 subjects is evident, integrating innate and adaptive immune compatibility testing into current cell, tissue, and organ transplantation guidelines is critical for safe and effective therapies. Thus, it is essential to use only well-characterized and safe MSCs for even the most urgent and experimental treatments (Moll et al. 2020). Because the COVID-19 patients suffer a prothrombotic state, concomitant use of heparin and defibrotide, a drug used in sinusoidal obstructive syndrome (SOS) after hematopoietic stem cell transplantation, has been proposed. Defibrotide is a mixture of single-stranded oligonucleotide aptamers with multi-target pharmacology limiting endothelial cell activation (Pescador et al. 2013). Given its antithrombotic, antiTNFα, anti-atherosclerotic, etc., the US Food and Drug Administration (FDA) approved its use in SOS. It will be consistent both to block the cytokine storm and prevent pulmonary thromboembolism. With HIV infections, we learned that combinatorial therapies show higher effectiveness in controlling the disease. Case reports, pilot studies, and well-designed clinical trials are needed to fight this pandemic. Now and when it comes back.

Unmet Challenges of Adoptive MSCs Therapy Despite the promising preliminary results, specific challenges demand attention before MSCs can be adopted at a larger scale in treating coronaviruses-induced infections. These challenges include the study design, source of MSCs, route of administration, dosage requirements, and their laboratory preparation and manipulation (Escacena et al. 2015; Soria-Juan et al. 2019; García-Bernal et al. 2021).

Study Design We believe that most of the trials that have been registered utilizing MSCs or their derived products do not have any appropriate control to conclusively determine the efficacy of cellular or cell-free therapy (Table 1). In most of these cases, MSCs are used in combination with adjunct antiviral drugs and supportive therapy. In such cases, it would become almost impossible to determine if the observed clinical improvements are actually attributed to MSCs or not. Therefore, a strategy including an appropriate control should be included in the design of such trials along with a greater sample size to validate the clinical efficacy of stem cells technology fully.

Source of Cells Different tissue sources, whether adult- or fetus-associated tissues, like adipose tissue, umbilical cord, dental pulp, and bone marrow, vary in their capacity to generate MSCs. Therefore, choosing an ideal source for harvesting MSCs and subsequently generating cell-free products is equally important as cell-free therapy

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is fast emerging as a therapeutic option (Haider and Aslam 2018). MSCs tissue sources, like umbilical cord and adipose tissue, are easily accessible without discomfort to the donor and generate a greater amount of MSCs with equivalent differentiation potential from the same amount of tissue in comparison to bone marrow, which incidentally is more invasive.

Route of Cell Administration Another major factor to consider here is their route of administration. MSCs and their products can either be systemically or locally injected. There are only limited studies that have compared the different routes of MSCs administration and have reported different outcomes. Therefore, it is difficult to conclude which route can be considered as the safest and most effective (Antunes et al. 2014; Cardenes et al. 2019). For COVID-19 disease, MSCs can be administered both systemically and locally with equal efficacy as both of these routes would result in their delivery first to the lungs only. However, in the case of cell-free products, like exosomes, delivery directly to the lungs via intranasal or intratracheal route is a more tenable option as systemic delivery often leads to a substantial loss in the amount of these products, mainly due to the activity of circulatory proteases and their distribution to the liver and spleen first, thus calling for booster doses (Gardin et al. 2020; Mahajan and Bhattacharyya 2021).

Dosage Strategies The number of MSCs required per dose and the total number of doses required are quite crucial in determining the treatment outcome. Based on the previous studies, it is estimated that approximately 4  108 MSCs are required for every patient regardless of the clinical indication (Olsen et al. 2018). The trials registered for COVID-19 have reported varied dosages, with an average of 1  106 cells/kg body weight up to 2–5 times to this average dose, but the actual number of MSCs required to produce such doses is not mentioned in any report, which needs to be optimized. Furthermore, while MSCs can be injected directly, the products like exosomes need to be prepared into stable formulations for their delivery to the patients (Gardin et al. 2020; Mahajan and Bhattacharyya 2021).

Risks Associated with Stem Cell Therapy While MSCs and their products have proven beneficial in the current scenario, we should not overlook the risks associated with stem cell therapy. Many stem cells clinics have opened up in recent years, marketing unethical and unauthorized stem cell treatment for various ailments, including COVID-19, by feeding on people’s fears and anxieties (Turner 2020). These less than clinical-grade stem cells and unlicensed stem cell treatments are potentially dangerous to the general public and undermine the efforts at determining stem cells efficacy in clinical trials. Undoubtedly, the use of

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MSCs and their factors has proven to be quite promising in the current scenario but is mainly dependent on the functional quality and integrity of stem cells. Therefore, stringent regulatory control should be maintained for the manufacturing and distribution of these products (see ▶ Chap. 3, “Considerations for Clinical Use of Mesenchymal Stromal Cells” of this book for review on clinical use of MSCs).

Concluding Remarks Considering the prevalence of COVID-19 and its complications, such as cytokine storm, which is followed by ARDS and death of patients, finding a way to treat and improve patients is of great importance. As previously mentioned, currently available vaccines are not a cure for COVID-19; there is no specific therapy for this virus, and supportive therapies as well as nonspecific antiviral drugs are mainly used for this purpose. Cell-based therapy is a modern method to treat various diseases. Recently, a number of studies have been conducted to treat COVID-19 using stem cells, suggesting the application of MSCs or immune cells such as T, CAR-T, or NK cells. Accordingly, the safety and immunomodulatory role of MSCs in ARDS has been approved. The MSCs can secrete factors that improve the pulmonary microenvironment, inhibit immune system over-activation, promote tissue repair, rejuvenate alveolar epithelial cells, inhibit counteract pulmonary fibrosis, or improve function in lung tissue damaged by SARS-CoV-2 infection. Nonetheless, many issues related to the application of MSCs, such as the ideal dose and optimal timing of administration, need further study. Of note, in several animal models of human disease, the use of MSC-secreting exosomes has been claimed to mimic the beneficial effects of MSCs in antiviral therapy against the influenza virus by reducing virus replication in the lungs and virus-induced release of pro-inflammatory cytokines, which highlights the great potential of cell-freebased therapies. In addition, considering the impossibility of studying the detailed mechanism of pathogenicity and the sequence of suggested drugs or vaccine candidates in human beings, these significant steps toward cell-based therapies in the SARS-CoV-2 field of study should be continued. Ongoing experimental studies and randomized trials will play an essential role in elucidating the therapeutic potential of MSCs, leading to a better understanding of how MSCs interact with SARS-CoV-2infected lung tissue. Although current progress on COVID-19 vaccinations is promising, the world population will have to continue to adapt to the “new normal” and practice social distancing and hygienic measures, at least until an effective cure is available to the general public.

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Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease Mikhail Konoplyannikov, Oleg Knyazev, Peter Timashev, and Vladimir Baklaushev

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IBD Therapy with MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of MSCs’ Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs-Derived Exosomes for Experimental IBD Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M. Konoplyannikov (*) Federal Research Clinical Center of Specialized Medical Care and Medical Technologies of the FMBA of Russia, Moscow, Russia Department of Advanced Biomaterials, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia e-mail: [email protected] O. Knyazev Department of Inflammatory Bowel Diseases, Moscow Clinical Scientific Center Named After A. S. Loginov, Moscow, Russia Department of Inflammatory Bowel Diseases, State Scientific Centre of Coloproctology Named After A. N. Ryzhih, Moscow, Russia P. Timashev Department of Advanced Biomaterials, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia Laboratory of Clinical Smart Nanotechnologies, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia Department of Polymers and Composites, N. N. Semenov Institute of Chemical Physics, Moscow, Russia Chemistry Department, Lomonosov Moscow State University, Moscow, Russia V. Baklaushev Federal Research Clinical Center of Specialized Medical Care and Medical Technologies of the FMBA of Russia, Moscow, Russia © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_8

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Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Completed Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proceeding Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Inflammatory bowel disease (IBD) belongs to the group of diseases characterized by idiopathic inflammation of the gastrointestinal organs. Two basic IBD types are distinguished: ulcerative colitis and Crohn’s disease. The IBD symptoms including vomiting and diarrhea, abdominal pain, rectal hemorrhage, and anemia have a significant negative impact on the general patient’s state of health. More than four million people in the USA and Europe suffer from IBD, while the general incidence of this disease in the developed countries exceeds 0.5% of the population. Besides, IBD is associated with a significant risk of colitis-associated malignancy. In the last decades, considerable progress has been achieved in the IBD therapy due to application of drugs suppressing the local gastrointestinal tract inflammation, such as antibodies to TNF-α (infliximab and adalimumab), corticosteroids, salicylates, etc. At the same time, this strategy, unfortunately, does not result in the repair of the damaged tissues, primarily ulcers of the colon, in many IBD patients. To achieve the mucosa healing and stable remission in IBD patients, novel approaches are required, cell therapy, actively used since the beginning of 2000s, being one of them. In our book chapter, we discuss the advantages and problems of application of mesenchymal stem cells (MSCs) which are most actively used in the cell therapy of IBD. The results of the most important preclinical and clinical studies are covered. Keywords

Clinical trials · Inflammatory bowel disease · Ulcerative colitis · Crohn’s disease · Cell therapy · Mesenchymal stem cells · Regenerative medicine Abbreviations

5-АSA APC ASC AZA BM CCL-2 CD CDAI CI DC DSS GCS

5-Aminosalicylic acid Antigen-presenting cells Adipose tissue–derived stem cells Azathioprine Bone marrow С-С-chemokine ligand 2 Crohn’s disease Crohn’ Disease Activity Index Confidence interval Dendritic cells Dextran sodium sulfate Glucocorticosteroids

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GI HBI HGF HLA-G hUC I/A I/V IBD IBDQ ICG IDO IFN IL iNOS LPS MHC miR MMP MSCs NK-cells PDAI PGE2 PSC RR SMA SVF TGF-β TNBS TNF UC

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Gastrointestinal Harvey-Bradshaw Index Hepatocyte growth factor Human leukocyte antigen-G Human umbilical cord Intra-arterial Intravenous Inflammatory bowel disease Inflammatory Bowel Disease Questionnaire Lindocyanine green Indoleamine-2,3-dioxygenase Interferon Interleukin Inducible nitric oxide synthase Lipopolysaccharide Major histocompatibility complex MicroRNA Matrix metalloproteinases Mesenchymal stem cells Natural killer cells Perianal Disease Activity Index Prostaglandin E2 Primary sclerosing cholangitis Relative risk Smooth muscle actin Stromal vascular fraction β-transforming growth factor Trinitrobenzenesulfonic acid Tumor necrosis factor Ulcerative colitis

Introduction Inflammatory bowel disease (IBD) is a group of chronic inflammatory conditions of the gastrointestinal (GI) tract characterized by the augmented immune response of the mucosa. Crohn’s disease (CD) and ulcerative colitis (UC) are the two basic types of IBD. Long-lasting IBD results in GI tract damage. CD may affect any part of the GI tract from the mouth to the anus. The terminal part of the small intestine (ileum) is most frequently affected near the place where it joins the large intestine. CD may manifest itself in the form of “patches,” involving some parts of the GI tract and leaving the other parts intact. The inflammation in CD may spread through the whole colon wall thickness (Sairenji et al. 2017). In UC, only the colon and rectum are affected. The inflammation appears only in the innermost layer of the colon mucosa. It usually starts in the rectum and lower parts of the colon, but may also spread

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continuously and affect the entire large intestine. IBD shares some symptoms such as persistent diarrhea, abdominal pain, rectal bleeding/bloody stool, weight loss, and fatigue. In some cases, it is difficult to determine whether a patient has CD or UC. Such cases are classified as indeterminate colitis (Guindi and Riddell 2004). The exact cause of IBD is unknown, but there is an assumption that it results from a defective immune system. The immune system of an IBD patient wrongly reacts to the environmental triggers that cause the GI tract inflammation. Such a wrong reaction of the immune system arises, supposedly, in people with a corresponding family history who inherited genes determining the susceptibility to IBD (Khor et al. 2011). More than four million people in the USA and Europe suffer from IBD, while the general incidence of this disease in the developed countries exceeds 0.5% of the population. Seventy thousand new IBD cases are diagnosed yearly in the USA only, and in general, the yearly financial burden of IBD in the USA exceeds 31 billion dollars (CCFA 2014; GBD 2020). The majority of patients receive the diagnosis of IBD at the age of less than 35 years. In particular, 80,000 children suffer from IBD in the USA. These lifelong chronic conditions essentially affect the quality of life and medical expenses of patients. Besides, IBD patients are susceptible to the risk of developing of other serious diseases such as colon cancer, thrombosis, and primary sclerosing cholangitis (PSC). In some cases, surgical removal of the damaged GI parts is required for the therapy of severe IBD forms. However, due to the achieved success of the drug therapy of IBD, it has been generally used in the last decades, with five basic types of drugs (CCFA 2014). Aminosalicylates such as sulfasalazine, balsalazide, mesalamine, and olsalazine administered per os or rectally reduce the colon wall inflammation and are applied primarily for the UC treatment. At the same time, they are less efficient in the CD treatment. Corticosteroids, such as prednisone, prednisolone, and budesonide, keep the immune system under control. Therefore, they are efficient in the short-term management of exacerbations. But unfortunately, their side effects include infections, weight gain, sleep disorders, etc. Immunomodulators, such as azathioprine, 6-mercaptopurine, and methotrexate, influence the immune system activity; they are toxic and usually used to sustain the remission in those patients who do not respond to other drugs, or respond to steroids only. Antibiotics, such as ciprofloxacin and metronidazole, are of moderate use in treating CD patients with the affected colon or perianal region. In particular, antibiotics are administered in the case of infections, e.g., abscesses. TNF inhibitors include adalimumab, certolizumab pegol, golimumab, and infliximab. These drugs have a pronounced anti-inflammatory effect and are used in the therapy of patients suffering from severe forms of IBD in the absence of a satisfactory and sound effect from the standard treatment. However, the application of these drugs, regretfully, is not always efficient, as well. In particular, the long-term infliximab administration has shown that up to one-third of patients do not respond

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to the anti-cytokine therapy, either due to primary resistance to the drug or the development of secondary resistance (Magro and Portela 2010). Besides, severe complications may occasionally emerge, including bacterial, viral, and fungal infections, increased risk of lymphoma, colorectal cancer, and other oncological diseases.

IBD Therapy with MSCs According to estimates, application of contemporary methods of IBD therapy leads to a 20–30% rate of remission, with a maximum of 50% when using a combinatorial therapy approach (Ocansey et al. 2020). Furthermore, cell therapy has shown to be very effective and extremely promising in treating IBD (Cassinotti et al. 2012; Irhimeh and Cooney 2016; Lopez-Santalla et al. 2020). Therefore, the use of mesenchymal stem cells (MSCs) is of particular interest regarding this approach.

MSCs Properties MSCs are multipotent stromal cells which may be derived from the bone marrow, adipose tissue, dental pulp, skeletal muscle, etc. (Lei et al. 2006; Tolar et al. 2010; Williams et al. 1999; Zuk et al. 2001; Gronthos et al. 2011). MSCs express molecules of the major histocompatibility complex (MHC) class I at a low level and do not express molecules of MHC class II, hence they may be used in allogeneic transplantation (Prockop 2009; Haider et al. 2011). They constitute a heterogeneous population of cells and are characterized by the expression of specific surface markers including CD73, CD90, and CD105 markers, while lacking the expression of CD14, CD11b, CD79 and Cd19, CD34 and CD45 hemopoietic stem cell–specific markers, as well as CD31 endothelial markers (Lv et al. 2014; Haider 2018). Besides surface marker expression, they show specific adherence to the plastic surface and possess trilineage differentiation potential to adopt adipocyte, osteoblast, and chondrocyte phenotypes (Caplan and Correa 2011; Wang et al. 2018). This criterion of characterization has been set forth by the International Society of Cell Therapy (ISCT) which has significantly helped in harmonizing the nomenclature and biological characterization of the cell preparations being used in the experimental and clinical studies. Their autologous availability and robust nature, therefore, can be genetically manipulated to delivery genes of interest to the target organ for angiomyogenic repair as well as to enhance their therapeutic potential (Jiang et al. 2006; Haider et al. 2008) and reprogramming in to pluripotency (Buccini et al. 2012). They have also been combined with other stem cell types for combinatorial cell therapy approach (Hosseini et al. 2018).

Mechanisms of MSCs’ Action The first and the primary mechanism of MSCs’ action is their transdifferentiation into morphofunctionally competent cell types and achieve the phenotype of interest,

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which allows the replacement of damaged cells and contribute to the repair and restoration of damaged tissues (cartilage, bones, etc.). The second mechanism of MSCs’ action is associated with the ability of MSCs to move to the sites of damage and inflammation, and secrete cytokines and growth factors, and lipid vesicles rich in bioactive cargo of proteins, lipids, and RNA as part of their paracrine activity to reduce inflammation and restore the damaged tissues (Caplan and Correa 2011; Caplan 2016; Bernardo and Fibbe 2013) (Fig. 1). Besides the abovementioned two primary mechanisms, MSCs also have immunosuppressive and anti-inflammatory effects via the suppression of proliferation and differentiation of T cells (CD4+ and CD8+ lymphocytes), reducing the activity of NK cells and activating Т regulatory cells. In addition, MSCs reduce the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNFα, and IFN-γ) and boost the secretion of anti-inflammatory IL-4 and IL-10 (Spaggiari et al. 2008; Ghannam et al. 2010). More recently, MSCs surface markers including PDL1, Gal-9, CXCR4 etc., have been implicated as part of the immunosuppressive activity of MSCs (Siyu et al. 2020). Concomitantly, proangiogenic activity of MSCs induces neovascularization regionally at the site of engraftment to restore regional blood flow (Maacha et al. 2020), while apoptosis and oxidative stress are inhibited (Terai and Tsuchiya 2017). Put together, the mechanism of MSCs’ therapeutic benefits is multifactorial as summarized in Fig. 1.

Fig. 1 General MSCs’ effects grouped by the two fundamental mechanisms: (1) direct transdifferentiation of the transplanted MSCs (into cells of adipose, bone, cartilage, and muscle tissues) to replace damaged cells and (2) induction of cytokines secreted by MSCs as a part of their paracrine activity into the inflammatory medium, affecting the recipient’s immune system. Abbreviations: (IL-6: Interleukin-6; PGE2: Prostaglandin E2; TGF-β: β-transforming growth factor; IDO: Indoleamine-2,3-dioxygenase; CCL-2: С-С-chemokine ligand 2; IL-10: Interleukin
10; HGF: Hepatocyte growth factor; MMP: Matrix metalloproteinases; HLA-G: Human leukocyte antigen-G)

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Preclinical Studies Yabana et al. demonstrated that in rats with dextran sodium sulfate (DSS)-induced colitis, MSCs, administered intravenously to the animals, migrated to the lamina propria of the damaged colon, where they activated the expression of alpha-smooth muscle actin (α-SMA), which facilitated the restoration of the epithelium (Yabana et al. 2009). It was also shown that MSCs participated in sustaining the epithelial barrier function by the repeated assembly of claudins, apical proteins of tight junctions. The most critical role in the IBD pathogenesis is evidently played by enhanced proliferation and defective apoptosis of immune cells, which is likely related to the imbalance of Bcl-2 and Bax, essential proteins controlling apoptosis (Dias et al. 2014). Akiyama et al. showed that systemic infusion of bone marrow–derived MSCs (BM-MSCs) induced apoptosis of T cells via the Fas-ligand (FasL)-dependent pathway and could improve the disease course in experimental murine DSS-induced colitis (Akiyama et al. 2012). However, FasL/MSCs did not induce apoptosis of recipients’ T -cells and could not positively influence the colitis course. It was shown that Fas-regulated secretion of MCP-1 protein by BM-MSCs recruited T cells for FasL-mediated apoptosis. Apoptosis of T cells, in turn, leads to the induction of macrophages producing a high level of TGFβ. This results in an increased number of T-regulatory cells and, finally, in the immune tolerance of the organism. IBD is also associated with the imbalance in subpopulations of T cells. As a result, the pro-inflammatory cytokines level grows: in CD – due to differentiation of Th1 and Th17 cells, and in UC – due to differentiation of Th2 cells. In contrast, the level of T-regulatory (Treg) cells is depressed in the peripheral blood of IBD patients (Sisakhtnezhad et al. 2017). Among Treg cells, the crucial role in the immune system suppression and sustaining the tolerance belongs to CD4+CD25+FoxP3+ cells (Akiyama et al. 2012). Chen et al. demonstrated that intravenous MSCs administration significantly reduced the clinical severity of murine UC (weight loss, diarrhea, and inflammation) induced by trinitrobenzene sulfonic acid (TNBS) and enhanced the survival of animals (Chen et al. 2013). It was shown that MSCs reached the damaged colon and facilitated the proliferation of intestinal epithelial cells and differentiation of intestinal stem cells (determined by detecting Lgr5+-cells). Furthermore, it was mediated by suppressing both Th1 and Th17 cell–induced autoimmune and inflammatory reactions (IL-2, TNF-α, IFN-γ, T-bet; IL-6, IL-17, RORγt), as well as by enhanced activity of Th2 cells (IL-4, IL-10, and GATA-3). Besides, it was shown that MSCs induced activated CD4+CD25+Foxp3+ Т-regulatory cells (TGF-β, IL-10, Foxp3). Macrophages, dendritic cells, and B cells, known as antigen-presenting cells (APC), are also involved in the IBD pathogenesis due to their specialization in presenting an antigen to T cells and the subsequent generation of the T cell response. Macrophages play a critical role in sustaining normal intestinal homeostasis, but they

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may also participate in the IBD pathogenesis (Han et al. 2021). In a healthy colon, resident macrophages exhibit an M2 phenotype, while inflammatory M1 macrophages dominate in the inflamed intestinal mucosa. In this regard, changing the balance of macrophage population to the M2 phenotype is being adopted as a novel approach in IBD therapy (Ahluwalia et al. 2018). Numerous preclinical studies have shown that MSCs can induce immunomodulating macrophages and macrophages mediate their therapeutic efficiency in experimental UC with an M2-like phenotype (Hidalgo-Garcia et al. 2018). Jo et al. cocultured immature dendritic cells and lipopolysaccharide (LPS)-treated mature dendritic cells with MSCs for 48 h, and then analyzed the profiles of surface markers and cytokines and the regulatory role of those DC for primary splenocytes (Jo et al. 2018). Besides, the therapeutic effects of MSCs and DC cocultured with MSCs were compared for UC-affected mice. The authors demonstrated that following the coculture of MSCs with immature dendritic cells (MSCs-DC) or LPS-treated mature dendritic cells (LPS þ MSCs-DC), the expression of CD11c, CD80, CD86, IL-6, TNF-α, and IFN-γ was significantly decreased. In contrast, the expression of CD11b, IL-10, and TGF-β was elevated. Besides, MSCs-DC and LPS þ MSCs-DC induced CD4, CD25, and Foxp3 in primary mice-derived splenocytes. In mice with DSS-induced UC, MSCs and MSCs-DC increased the length of the colon, body weight, and survival, and caused a histological improvement. Moreover, in the MSCs and MSCs-DC groups, the expression of IL-6, TNF-α, and IFN-γ in the colon tissues was also inhibited, while the expression of IL-10, TGF-β, and Foxp3 was elevated. These data assumed that MSCs stimulate differentiation of dendritic cells into regulatory dendritic cells leading to improved chronic colitis therapy. It was also shown that administration of MSCs could suppress activation and proliferation of B cells secreting IgG and, oppositely, stimulate the formation of CD5 + regulatory B cells (Bregs) producing IL-10. Besides, it was shown that MSCs could depress the proliferation of NK cells secreting pro-inflammatory cytokines (Liu et al. 2020).

MSCs-Derived Exosomes for Experimental IBD Therapy MSCs-derived exosomes – extracellular vesicles obtained from MSCs – contain a large number of essential factors (Haider and Aramini 2020). In intercellular communication, exosomes are identified as efficient carriers for nucleic acids, functional proteins, lipids, mRNA, and microRNA (Samoylova et al. 2017). Thus, MSCsderived exosomes, similar to MSCs themselves, have a potent physiological action affecting the damaged tissue repair (Zhao et al. 2019; Haider and Aramini 2020). At the same time, exosomes are more stable than MSCs and in principle are nonimmunogenic. It was demonstrated earlier by several research groups that exosomes secreted by MSCs had a pronounced regenerative effect in the therapy of many diseases causing tissue damage, including IBD (Mianehsaz et al. 2019; Mendt et al. 2019; Mao et al. 2017). For instance, Mao et al. showed that exosomes released from human

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umbilical cord–derived MSCs (hUC-MSCs) positively influenced the treatment of DSS-induced colitis and studied the primary mechanism of their effect (Mao et al. 2017). Similarly, exosomes labeled with indocyanine green (ICG) reached the colon tissue of IBD-affected mice 12 h after the injection. The IL-10 gene expression was increased, while the expression of TNF-α, IL-1β, IL-6, iNOS, and IL-7 genes was decreased in the colon and spleen tissues of mice treated with MSCs-derived exosomes. Besides, macrophages infiltration in the colon tissues was significantly reduced. It was also shown that coculturing in vitro with exosomes suppressed the expression of iNOS and IL-7 in macrophages isolated from the peritoneal cavity of normal mice. In addition, the researchers found that IL-7 expression in the colon tissue was higher for colitis patients than healthy participants of the control group. In general, the data obtained have demonstrated a potent effect of hUC-MSCs-derived exosomes on the relief of DSS-induced experimental IBD. The observed effects may be mechanistically mediated via the modulation of IL-7 expression in macrophages at molecular levels. In a study by Yang et al., exosomes derived from MSCs preconditioned with IFN-γ were transplanted in an experimental mice model of DSS-induced colitis that essentially improved the index of activity and histological assessment of colitis, as well as reduced the fraction of Th17 cells and augmented the fraction of Treg cells (Yang et al. 2020). Molecular studies revealed that the administration of exosomes markedly inhibited the expression of Stat3 and p-Stat3, suppressing differentiation of Th17 cells. Interestingly, treatment with exosomes derived from MSCs preconditioned with IFN-γ showed the highest inhibition. Furthermore, the preliminary treatment with IFN-γ increased the level of miR-125a and miR-125b in MSCsderived exosomes, which directly targeted Stat3, suppressing differentiation of Th17 cells. Moreover, concomitant infusion of miR-125a and miR-125b also demonstrated a therapeutic effect in colitis, accompanied by a simultaneous decrease in the Th17 cell fraction. In general, this study demonstrated that the IFN-γ treatment enhanced the efficiency of MSCs-derived exosomes in the relief of colitis, owing to increasing the level of miR-125a and miR-125b, which are bound to 30 -UTR of Stat3, to suppress differentiation of Th17 cells.

Clinical Studies Completed Clinical Studies Due to their therapeutic properties, MSCs (obtained mainly from the bone marrow and adipose tissue) have been actively used in numerous clinical trials on IBD therapy, with both local injections of cells and intravenous (systemic) infusions (Table 1).

Local MSCs Injections Local administration of MSCs is used primarily for the therapy of fistulizing (extraluminal) form of CD (Ko et al. 2021). For example, Panes et al. have conducted a

Disease Perianal CD

Perianal CD

Perianal CD

Perianal CD

No 1.

2.

3.

4.

24

36

18 (45 total)

Patients 212

Allogeneic ASC

Autologous ASC

Allogeneic ASC, autologous ASC, and stromal vascular fraction (SVF)

Cells Allogeneic ASC

20–40  106/local injection

3–6  107/local injection

40–42  106 ASC, 6.5–15  106 SVF/local injection

Cell dose/delivery 120  106 (n ¼ 107) vs placebo (n ¼ 105)/local injection

Table 1 Results of major completed clinical trials on the MSCs therapy of IBD

At week 24, 69.2% of the patients showed reduction in the number of draining fistulas, 56.3% of the patients achieved complete closure of the treated fistula, and 30% of the cases presented complete closure of all existing fistula tracts

Key findings At week 52, a significantly larger part of patients receiving ASC reached the combined remission (56.3%, as compared to the control group with 38.6%) and the clinical remission (59.2% vs. 41.6% of the control group). In 1 year, no serious adverse events were observed Healing achieved: 40% of CD patients who received SVF, 66.6% of CD patients who received auto-ASCs, and 55.5% of CD patients who received allo-ASCs At 24 months, complete healing was observed in 27 out of 36 patients (75.0%). No adverse events related to ASC administration were observed

University of Ulsan College of Medicine and Asan Medical Center, Seoul, Republic of Korea; NCT01011244 and NCT01314079 (Cho 2015) Virgen del Rocio University Hospital, Seville, Spain (de la Portilla 2013)

University Hospital Fundación Jiménez Díaz, Madrid, Spain (Herreros 2019)

Study location Forty-nine hospitals in Europe and Israel; NCT01541579 (ADMIRECD) (Panes 2018)

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Perianal CD

UC

5.

6.

70

21

Allogeneic hUC-MSCs

Allogeneic BM-MSCs

Group I (n ¼ 34): I/V injection of 0.5  106 cells/kg, followed by intra-arterial injection of 1.5  107 MSCs 1 week later. Group II (n ¼ 36): placebo (normal saline)

1  107 (n ¼ 5, group 1), 3  107 (n ¼ 5, group 2), or 9  107 (n ¼ 5, group 3) MSCs, or placebo (n ¼ 6)/local injection Thirteen out of fifteen patients (87%) treated with BM-MSCs were available for a long-term follow-up (4 years). No adverse events were associated with local injection of any dose of MSCs. In group 2 [n ¼ 4], all fistulas were closed 4 years after BM-MSCs therapy. In group 1 [n ¼ 4] 63%, and in group 3 [n ¼ 5] 43% of the fistulas were closed, respectively. Pelvic MRI showed significantly smaller fistula tracts after 4 years One month after therapy, 30/36 patients in group I showed a good response, and diffuse and deep ulcer formation and severe inflammatory mucosa were improved markedly. During the follow-up, the median Mayo score and histology score in group I were decreased while IBDQ scores were significantly improved compared with before treatment and group II (P < 0.05). Compared with

Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease (continued)

Qingdao University, Qingdao, Shandong, China; NCT01221428 (Hu 2016)

Leiden University Medical Center, Leiden, The Netherlands (Barnhoorn 2020)

7 203

Disease

Luminal CD

UC and luminal CD

No

7.

8.

90 (69 UC and 21 CD)

82

Patients

Table 1 (continued)

Allogeneic BM-MSCs

Allogeneic hUC-MSCs

Cells

150–200  106 (n ¼ 39 with UC and n ¼ 11 with CD) vs control group (n ¼ 30 with UC and n ¼ 10 with CD)/I/V infusion

Group I (n ¼ 41): 1  106 cells/kg once a week, four times in total. Group II (n ¼ 41): placebo (normal saline)/I/V infusion

Cell dose/delivery group II, there were no evident adverse reactions after MSCs infusion in any of the patients in group I, and no chronic side effects or lingering effects appeared during the follow-up period Twelve months after treatment, the CDAI, HBI, and corticosteroid dosage had decreased by 62.5  23.2, 3.4  1.2, and 4.2  0.84 mg/day, respectively, in the UC-MSCs group and by 23.6  12.4, 1.2  0.58, and 1.2  0.35 mg/day, respectively, in the control group ( p < 0.01, p < 0.05, and p < 0.05 for UC-MSCs vs. control, respectively). Four patients developed a fever after cell infusion. No serious adverse events were observed In MSCs group: statistically significant decrease in the indices of the clinical and morphological activities of an inflammatory process; clinical remission occurred in

Key findings

Moscow Clinical Research Center, Moscow, Russia (Lazebnik 2010)

Shaanxi Provincial People’s Hospital, Xi’an, China (Zhang 2018)

Study location

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Luminal CD

Perianal CD

9.

10.

22

34

Allogeneic BM-MSCs

Allogeneic BM-MSCs

Group 1 (15 patients): 2  106 MSCs/kg (at months 0, 1, and 6), with AZA 2–2.5 mg/kg. Group 2 (19 patients): 2  106 MSCs/kg (at months 0, 1, 6)/ IV infusion 2  106 MSCs/kg I/V infusion and 10–20  106 local injection According to the study results, in 12 weeks the cure of simple fistulas was noted in 8 patients (66.6%) of group I, in 6 patients (60%) of group II, and in 1 patient (7.14%) of group III. In 6 months, the simple fistulas were still healed in 8 patients (66.6%) of group I, in 6 patients (60%) of group II, and in 1 patient (7.14%) of group III. In 12 months, the healing was sustained in 7 patients (58.3%) of group I, in 6 patients (60%) of group II, and in 2 patients (14.3%) of group III. In 24 months, the closure of fistulas was sustained in 5 patients (41.6%) of group I, in 4 patients (40%) of group II, and in 0 patients (0%) in group

40 patients (80%); discontinuing corticosteroids in 34 of the 50 patients (68%) Clinical remission (CDAI 2 cm, confirmed by magnetic resonance imaging; MRI) and clinical remission (absence of draining fistulas). Earlier, the same researchers reported a primary endpoint of a study at week 24 (combined remission in 51.5% patients receiving Сх601, compared to 35.6% in the control group, the difference being 15.8%; 97.5% confidence interval 0.5–31.2; P ¼ 0.021) (Panés et al. 2016). At week 52, a significantly larger section of the patients receiving Сх601 reached the combined remission (56.3%, as compared to the control group with 38.6%, 17.7%difference; 95% confidence interval 4.2–31.2; P ¼ 0.010) and the clinical remission (59.2% vs. 41.6% of the control group with the 17.6% difference; 95% confidence interval 4.1–31.1; P ¼ 0.013). The safety was sustained for 52 weeks; side effects were observed in 76.7% of group Сх601 patients and 72.5% of the control group patients. The researchers concluded that according to the results of the phase 3 study of CD patients with treatment-resistant perianal fistulas, the researchers have concluded that Cx601 is safe and efficient for closures of external fistulas, compared to placebo, in one year of the study. Based on the ADMIRE CD Study results, Darvadstrocel (Alofisel), a medication based on MSCs derived from the adipose tissue, has been developed (Scott 2018). This is the first MSCs-based cell preparation approved in the EU to treat complicated perianal fistulas in adult patients with nonactive/moderately active luminal CD when fistulas do not respond to one or more standard therapies. Herreros et al. have published the data of a clinical study that assessed 45 patients with 52 surgically resistant anal fistulas of various etiology (of them 18 patients with perianal fistulas caused by CD) (Herreros et al. 2019). The patients’ response to MSCs therapy of different types was monitored, with cells, including allogeneic MSCs from the adipose tissue (ASCs), autologous ASCs, and a stromal vascular fraction (SVF), which were believed to contain ASCs with a minimal addition of adipocytes and erythrocytes. In 40 out of 42 cases of perianal fistulas (95.2%), either healing or improvement was shown in 6.6 weeks on average (in the observation time of 2–36 weeks). The cure occurred in 22 out of 42 cases (52.4%). Most of the patients were cured in 5.8 months on average (in the observation time of 0.5–24 months). The disease course in the 42 patients was assessed depending upon the applied cell preparations.

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The degree of cure reached 13/23 (56.5%) for SVF, 3/9 (33.3%) for autologous ASCs, and 6/10 (60%) for allogeneic ASCs. The administered cell dose was also analyzed, with the average value of 43.9 million (in the range of 3–210 million cells) for the cases of cure. If to focus on perianal fistulas caused by CD, 18/18 patients (100%) demonstrated healing or improvement/partial response, beginning from 5.3 weeks on average (in the observation time of 2–12 weeks). The cure occurred in 10/18 (55.5%) cases. Most of the patients were cured in 6.5 months (in the observation time of 0.5–24 months). The disease course in those 18 patients was also assessed depending on the cell preparations used. The degree of cure reached 40% for SVF, 66.6% for autologous ASCs, and 55.5% for allogeneic ASCs. The mean administered dose in the cure cases was 43.9 million (in the range of 3–210 million cells). In all the cases of CD-associated perianal fistulas, the surgical technique was applied: the curettage, closing of the fistula tract, and injection of cells (Herreros et al. 2019). The phase 2 clinical trial on the application of autologous ASCs for CD-associated perianal fistulas with a high rate of recurrence has shown their safety and therapeutic potential with a stable response for 2 years (Cho et al. 2015). In this phase 2 study, 41 patients initially participated. In 24 months, the complete cure was observed in 27 out of 36 patients (75.0%) (the data from 5 patients were absent in 24 months). No ASCs-based treatment-related adverse effects were observed. Moreover, the complete closure of the fistula was stable after the initial treatment. These results also testified that autologous ASCs were efficient in the treatment of CD-associated fistulas. De la Portilla et al. have conducted an open-label, single-arm clinical trial which included 24 CD patients with perianal fistulas from six hospitals in Spain (de la Portilla et al. 2013). Twenty million ASCs were administered locally in one draining fistula tract. At week 12, if the fistula had not completely closed, 40 million more ASCs were administered. The patients were monitored up to week 24 after the first treatment. During 6 months of follow-up, no serious adverse events were observed, attesting the treatment as sufficiently safe. At week 24, the number of fistulas was reduced in 69.2% of patients, the complete closure of treated fistulas was observed in 56.3% of the patients, and in 30% of the cases all the fistulas were completely closed. The criteria used to grade the extent of closure were the following: absence of draining and complete re-epithelization, and the MRI-confirmed absence of accumulations. The MRI Score of Severity showed a noticeable reduction at week 24. Thus, the applied ASCs-based therapy appeared safe and fairly efficient for CD-associated perianal fistulas. A double-blind dose-finding study on the allogeneic BM-MSCs treatment of refractory perianal fistulizing CD was conducted at Leiden University Medical Center in 2012–2014 (Barnhoorn et al. 2020). The study involved 21 patients; three regimes of local MSCs administrations were applied: cohort 1 – five patients, 1  107 cells, cohort 2 – five patients, 3  107 cells, and cohort 3 – five patients, 9  107 cells. The patients were assessed for 4 years, with the registration of

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clinical events, monitoring the fistula closure, and measuring the level of anti-HLA antibodies, pelvic MRI, and rectoscopy. The long-term follow-up was performed in 13 patients (four from cohort 1, four from cohort 2, and five from cohort 3). No serious side effects of the therapy were observed. In two patients, malignancies were observed; however, these were reported as unrelated to the cell-based therapy. During 4 years of follow-up, the closure of fistulas was observed in all the cohort 2 patients, in 63% of cohort 1 patients, and in 43% of cohort 3 patients. No anti-HLA antibodies were detected in 24 weeks and 4 years of posttreatment follow-up. The fistula tracts became notably smaller, according to the MRI data. This study demonstrated that local application of BM-MSCs was safe and efficient for fistula closures. A promising variation of the MSCs treatment for perianal fistulas is the use of a bioabsorbable matrix as a carrier for the cells. A Gore BioA Fistula Plug based on a bioabsorbable material was earlier tried in a multicenter study of high anal fistulas, including those in CD patients (Ommer et al. 2012). The study showed a rather high efficiency of such plugs in the treatment of fistulas; in particular, two out of four study participants with CD had complete healing in 6 months. Another development of the plug technique was its combined use with MSCs. A six-months-long study at Mayo Clinic (ClinicalTrials.gov Identifier: NCT01915927), including 12 patients, was dedicated to the treatment of fistulas with autologous ASCs deposited onto a Gore BioA Fistula Plug (Dietz et al. 2017). ASCs were harvested from the patients for autologous transplantation, and 6 weeks later the fistula plug loaded with autologous ASCs (named MSCs-MATRIX) was placed during a surgical intervention. Before the surgical procedure, ASCs were thawed and cultured on a Gore Bio-A Fistula Plug for 3–6 days in a polypropylene bioreactor. Each plug contained about 20  106 cells. The primary study objective was to establish the safety and efficacy of autologous MSCs-MATRIX in the treatment of recurrent anal fistulas. The criteria for the secondary endpoint of the study were both clinical and radiographical. The former included: (1) partial response, when the drainage and symptoms reported by a patient were notably reduced, and (2) complete healing, when the drainage was not seen either without any action or with a mild pressure in 6 months after the treatment. The latter criterion included the narrowing and shortening of the fistula tract, as well as the absence of an abscess, as visualized by MRI (T2-weighted hyperintense fistula tract on a T2-weighted fast spin echo image). Quantitatively, the MRI results were presented in percent difference from the baseline and using the Van Assche perianal fistula severity score. The applied MSCs-MATRIX plug for a fistula did not cause any serious effects during the 6 months of observation. Ten of the twelve patients (83%) in 6 months had clinical and radiographic signs of the complete healing. Thus, the bioabsorbable plugs containing MSCs proved themselves safe and efficient for chronic perianal fistulas. A recently published systematic review and meta-analysis by Cao et al. have estimated the efficiency of stem cells (MSCs derived from the bone marrow and adipose tissue) in the treatment of CD-associated fistulas of any form (Cao et al. 2021).

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In total, a total of 29 clinical studies involving 1252 patients were included in the review and analyzed. It was shown that the group of patients with CD-associated fistulas, to whom stem cells were transplanted, demonstrated a higher degree of fistula healing as compared to the placebo-treatment group (61.75% vs. 40.46%, or 2.21, 95% CI 1.19–4.11, P < 0.05). The group of patients who received stem cells in the dose of 3  107 cells/mL had a 71.0% acceleration of fistula healing vs. the groups of stem cell treatment with other doses (RR 1.3, 95% CI 0.76–2.22). The percentage of cured patients with perianal and trans-sphincteric fistulas was higher than patients with rectovaginal fistulas (77.95% vs. 76.41%). It is of interest that Crohn’s Disease Activity Index (CDAI) and Perianal Disease Activity Index (PDAI) temporarily increased 1 month after stem cell–based therapy; however, they returned to the initial level 3 months after the treatment. Moreover, the incidence of side effects related to the treatment was significantly lower in the MSCs-treated group than in the placebotreatment group (RR 0.58, 95% CI 0.30–1.14). The conducted study has shown that the application of stem cells, especially ASC, is a promising approach in the treatment of CD-associated fistulas, based on its higher efficiency and lower incidence of adverse events.

Intravenous MSCs Administration Systemic (intravenous) administration of MSCs is used mainly in the therapy of luminal (inflammatory) forms of IBD (Ko et al. 2021). In a randomized placebo-controlled clinical trial (ClinicalTrials.gov: NCT01221428), Hu et al. studied the safety and efficiency of hUC-MSCs in treating moderate and severe UC (Hu et al. 2016). Thirty-four UC patients were included in group I and received an MSCs infusion in addition to the basic treatment, while 36 patients in group II received saline in addition to the basic treatment. One-month post-treatment, the incidence of diffuse and deep ulcers and severe inflammatory processes in the mucosa was essentially reduced in 30 patients of group I. During the following observation, the average score of the Mayo scale and the histological score were decreased in group I, while the IBDQ score was significantly improved as compared to before the treatment and group II (P < 0.05). Furthermore, in comparison with group II, no apparent adverse reactions were observed after MSCs infusion in group I patients. Again, no chronic or long-lasting side effects were observed during the entire observation period. Thus the authors demonstrated that MSCs infusion was a safe and efficient strategy to treat UC. Zhang et al. studied the safety and efficiency of hUC-MSCs to treat CD (Zhang et al. 2018). Eighty-two patients with diagnosed CD who had received the supporting steroid therapy for more than 6 months were included in the study. Forty-one patients were randomly assigned for administering four peripheral intravenous infusions of 1  106 hUC-MSCs/kg, one infusion per week. The patients were observed in the dynamics for up to 12 months. CDAI, Harvey-Bradshaw Index (HBI), and the dosage of corticosteroids were evaluated. As a result of the treatment, CDAI, HBI, and the dosage of corticosteroids decreased by 62.5  23.2, 3.4  1.2, and 4.2  0.84 mg/day, respectively, in the hUC-MSCs group, and by 23.6  12.4, 1.2  0.58, and 1.2  0.35 mg/day, respectively, as compared to the control group

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(р < 0.01, p < 0.05, and p < 0.05 for the hUC-MSCs group vs. the control, respectively). Four patients developed fever after the cell-based infusion. No serious adverse events were observed. The researchers concluded that hUC-MSCs were efficient in CD treatment, though occasionally may cause mild side effects. In one of our studies, 22 patients with exacerbation of moderate and severe UC were treated with allogeneic BM-MSCs (Knyazev et al. 2016). The patients were divided into two groups. Patients of group I (n ¼ 12), in addition to the standard antiinflammatory therapy, received MSCs according to the following protocol: 0 (first infusion), week 1, and week 26, followed by every 6 months for the subsequent years of observation. Patients of group 2 (n ¼ 10) received the standard antiinflammatory therapy with 5-aminosalicylic acid (5-АSA) and glucocorticosteroids (GCS). Of group I patients, 58.3% had a severe UC exacerbation, and 41.7% had moderate UC exacerbation; in group II, the severe and moderate UC patients constituted 60% and 40%, respectively. Total colitis was established in 33.3% of group I patients and in 40% of group II patients; left-sided colitis was observed in 66.7% and 60% patients, respectively. The efficiency criterion for the therapy was a no-relapse course of the disease for 12 months. The UC clinical activity was estimated by the Rahmilevich score, endoscopic activity – by the Mayo score. The control over the dynamics of clinical, laboratory, and endoscopic indices was performed in 2, 6, and 12 months, then yearly for 3 years. During the first year of observations, in group I, a UC relapse occurred in two patients (16.7%), in group II – in three patients (30%). The relative risk (RR) was 0.3 (95% CI 0.08–1.36; p ¼ 0.2; χ2 ¼ 1.47). The Rahmilevich Clinical Activity Index was 3.33  0.54 points in group I, 4.4  1.13 points in group 2 (р ¼ 0.81), the Mayo score was 3.1  0.85 and 3.9  1.06, respectively (р ¼ 0.66). In 2 years of observation, the risk of a UC relapse in group I was three times lower than that in group 2 (р ¼ 0.03). The average duration of remission in group I was 22 months, in group II – 17 months ( p ¼ 0.049). In 3 years of observation, the duration of remission was 22 and 20 months, respectively ( p ¼ 0.66). The Rahmilevich Clinical Activity Index was 4.75  1.13 points in group I, 8.1  1.1 points in group II (р ¼ 0.001). In conclusion, the study reliably demonstrated that MSCs infusions enhanced the efficiency of antiinflammatory therapy in patients with the acute UC. In another study (Lazebnik et al. 2010), we used intravenous administration of allogeneic BM-MSCs to treat 39 UC patients and 11 CD patients (with the control groups of 30 UC patients and 10 CD patients). A statistically significant decrease in the indices of the clinical and morphological activities of the inflammatory process was noted after the MSCs transplantation in 39 patients with UC and in 11 patients with CD as compared to the control groups. A clinico-morphological remission occurred in 40 patients (80%). In addition, the use of MSCs made it possible to discontinue GCS in 34 out of 50 patients (68%) with the hormone-dependent and hormone-resistant forms of UC and CD and reduced the dose of prednisolone to 5 mg/day in 7 patients, with administering 5-ASA only. Our later study estimated the efficiency of BM-MSCs therapy of CD patients receiving azathioprine (AZA) (Knyazev 2018a). The study included 34 patients with the inflammatory (luminal) CD form. Group I (n ¼ 15) received an anti-inflammatory

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therapy with the use of MSCs culture in combination with AZA. Group II (n ¼ 19) received MSCs without AZA. The severity of attack was estimated in CDAI points. The blood serum was studied, including immunoglobulins (IgA, IgG, IgM), interleukins (IL) 1β, 4, and 10, tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), transforming growth factor-1β (TGF-1β), С-reactive protein (CRP), thrombocytes, and erythrocyte sedimentation rate (ESR) in 2, 6, and 12 months from the MSCs therapy beginning. The initial mean CDAI was 337.6  17.1 points in group I and 332.7  11.0 points in group II ( p ¼ 0.3). In both groups, a significant decrease in CDAI was noted in two and 6 months from the therapy beginning: in 2 months – to 118.9  12.4 in group I and to 120.3  14.1 in group II ( p ¼ 0.7), in 6 months – to 110.3  11.1 in group I and to 114.3  11.8 in group II ( p ¼ 0.8). In 12 months, the CDAI was 99.9  10.8 in group I and 100.6  12.1 in group II ( p ¼ 0.8); in 24 months – 133.2  28.3 in group I and 120.8  15.5 in group II ( p ¼ 0.2); in 36 months – 139.9  23.4 and 141.7  20.8 ( p ¼ 0.9) in group I and II, respectively. The IgA, IgG, and IgM levels were significantly lower in the group of patients with a more extended history of the disease and prolonged use of AZA. After the MSCs infusions, in both groups, we observed a tendency to the increase in the pro- and antiinflammatory cytokines, with a significantly lower level of pro-inflammatory cytokines (INF-γ, TNF-α, and IL-1β) in group I. The latter indicates potentiation of the immunosuppressive effect of MSCs and AZA, which provides a more pronounced anti-inflammatory effect. Moreover, it has been demonstrated that MSCs transplantation stimulates elevation of the initially reduced concentration of immunoglobulins and cytokines in the blood serum and restoring their balance with the setting-in of the clinical remission. Interesting results were obtained when comparing the effects of combined (local and systemic) administration of BM-MSCs, anti-cytokine therapy (infliximab, IFL) and antibiotic, and immunosuppressive therapy on the healing of CD-associated simple perianal fistulas (Knyazev 2018b). The first group of CD patients aged from 19–58 years (Ме – 29; n ¼ 12) received MSCs systemically according to a scheme and locally. The second group aged from 20–68 years (Ме – 36, n ¼ 10) received IFL according to a scheme. The third group aged from 20–62 years (Ме – 28, n ¼ 14) received antibiotics and immunosuppressants. According to the study results, in 12 weeks the cure of simple fistulas was noted in eight patients (66.6%) of group I, in six patients (60%) of group II, and in one patient (7.14%) of group III. In 6 months, the simple fistulas were still healed in eight patients (66.6%) of group I, in six patients (60%) of group II, and in one patient (7.14%) of group III. In 12 months, the healing was sustained in seven patients (58.3%) of group I, in six patients (60%) of group II, and in two patients (14.3%) of group III. During 24 months follow-up, the closure of fistulas was sustained in five patients (41.6%) of group I, in four patients (40%) of group II, and no patient (0%) in group III. In conclusion, it was demonstrated that combined cell and anti-cytokine therapy of CD with perianal lesions reliably provided more frequent, sustained, and prolonged closure of simple fistulas, as compared to antibiotic and immunosuppressive therapy, and reduction of the relapse incidence as well (Fig. 2).

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Fig. 2 Colonoscopy of a 38-year-old female patient with CD, before and after MSCs-based cell therapy [50]. (a) The internal opening of the fistulous tract in the lower part of the rectum ampulla before the treatment; (b) Twelve weeks post-treatment, healed fistula

In their recent publication, Ko et al. have provided an extensive analysis of the safety and efficiency of MSCs-based cell therapy of IBD involving on 24 studies, in 17 of which MSCs were administered locally while in the remaining 7 studies MSCs were administered systemically (Ko et al. 2021). The authors concluded that local MSCs injection-based protocol for fistulizing (extra-luminal) CD form demonstrated long-term efficiency, with the good safety level. However, regarding the efficiency of systemic MSCs infusion, the evidence was ambiguous, in the authors’ opinion. They noted the marked methodological heterogeneity of the studies (first of all, due to different MSCs sources), along with the absence of facts confirming that MSCs reach the colon after an intravenous injection, and found that the safety profile was not always clearly demonstrated. At the same time, in our studies mentioned above, unequivocal pieces of evidence have been obtained for the efficiency of systemic allogeneic MSCs infusions in the IBD therapy (Lazebnik et al. 2010; Knyazev et al. 2016; Knyazev et al. 2018a, b). In a larger and a more extensive study with a 5-year follow-up, we compared the safety profile of BM-MSCs and a standard treatment using 5-ASA, GCS, and immunosuppressive agents (Knyazev et al. 2015). The study included 103 IBD patients (56 UC patients and 47 CD patients) who received the MSCs therapy and 208 patients receiving the standard anti-inflammatory therapy (but not anti-TNF therapy). All the participants were similar in their demographic characteristics and disease features. No differences were found in the development of acute posttransfusion toxicity, infectious complications, exacerbation of chronic inflammatory diseases, serious infectious complications, malignancy, and death, with the exception of fever in some patients treated with MSCs. Thus, cell-based therapy was considered safe for the clinical practice.

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Proceeding Clinical Studies Currently (by March 2021), 14 proceeding clinical trials involving MSCs-based cell therapy for the IBD treatment have been registered at Clinicaltrials.gov (Table 2). Included in these clinical studies are autologous MSCs-based cell therapy (two studies) and allogeneic MSCs-based cell therapy (12 studies). BM-MSCs are used in seven studies, MSCs derived from the adipose tissue will be used in five studies, one study will use MSCs derived from the umbilical cord blood, while one study will use Wharton’s jelly–derived cells. Ten clinical trials are dedicated to the treatment of CD, while the other four trials will focus UC treatment. Local MSCs administration protocol will be used in 12 studies while systemic administration will be used in the other two studies. The mentioned above Mayo Clinic study of MSCs-impregnated plugs for perianal fistulas (Dietz et al. 2017) has a very promising development with young patients (Pediatric MSCs-AFP Sub-Study for Crohn’s Fistula, NCT03449069). A single dose of 20 million autologous MSCs is suggested to use in five pediatric patients aged from 12–17 with CD-associated perianal fistulas. The treatment will begin with a standard therapy of infection drainage and placement of a draining seton. In 6 weeks after the placement of a draining seton, it will be removed and replaced with a fistula plug (MSCs-coated Gore Bio-A Fistula Plug). The follow-up period will be 24 months, with the treatment safety and the fistula response being monitored. Table 2 The ongoing clinical trials on the MSCs therapy of IBD (according to clinicaltrials.gov by March, 2021) No. 1.

2.

3.

4.

Title, ClinicalTrials.gov ID Use of Mesenchymal Stem Cells in Inflammatory Bowel Disease; NCT03299413 Angiographic Delivery of AD-MSCs for Ulcerative Colitis; NCT04312113 Adipose Mesenchymal Stem Cells (AMSCs) for Treatment of Ulcerative Colitis (AMSCs_UC); NCT03609905 Study of Mesenchymal Stem Cells for the Treatment of Medically Refractory Ulcerative Colitis (UC); NCT04543994

Disease UC

Cell type and source Allogeneic MSCs, Wharton’s jelly

Delivery Intravenous

Location Cell Therapy Center, Amman, Jordan

UC

Autologous ASC

Intraarterial

Mayo Clinic in Rochester, Minnesota, USA

UC

Allogeneic ASC

Local

Liaocheng City People’s Hospital, Liaocheng, Shandong, China

UC

RemestemcelL (Allogeneic BM-MSCs)

Local

Cleveland Clinic, Cleveland, Ohio, USA

(continued)

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Table 2 (continued) No. 5.

6.

7.

8.

9.

10.

11.

12.

Title, ClinicalTrials.gov ID Mesenchymal Stem Cells for the Treatment of Perianal Fistulizing Crohn’s Disease; NCT04519671 Study of Mesenchymal Stem Cells for the Treatment of Ileal Pouch Fistula’s in Participants with Crohn’s Disease (IPAAF); NCT04519684 Mesenchymal Stem Cells for the Treatment of Rectovaginal Fistulas in Participants with Crohn’s Disease; NCT04519697 Mesenchymal Stem Cells for the Treatment of Pouch Fistulas in Crohn’s Disease; NCT04073472 Study of Mesenchymal Stem Cells for the Treatment of Medically Refractory Crohn’s Colitis; NCT04548583 A Follow-Up Study to Evaluate the Safety of ALLO-ASC-CD in ALLO-ASC-CD-101 Clinical Trial; NCT03183661 MSCs Intratissular Injection in Crohn’s Disease Patients; NCT03901235 A Study to Evaluate the Safety of ALLOASC-CD for

Disease CD

Cell type and source Allogeneic BM-MSCs

Delivery Local

Location Cleveland Clinic, Cleveland, Ohio, USA

CD

Allogeneic BM-MSCs

Local

Cleveland Clinic, Cleveland, Ohio, USA

CD

Allogeneic BM-MSCs

Local

Cleveland Clinic, Cleveland, Ohio, USA

CD

Allogeneic BM-MSCs

Local

Cleveland Clinic, Cleveland, Ohio, USA

CD

Allogeneic BM-MSCs

Local

Cleveland Clinic, Cleveland, Ohio, USA

CD

Allogeneic ASC

Local

Anterogen Co., Ltd., Seoul, Republic of Korea

CD

Allogeneic BM-MSCs

Local

CHU de Liège, Liège, Belgium

CD

Allogeneic ASC

Local

Yonsei University College of (continued)

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Table 2 (continued) No.

13.

14.

Title, ClinicalTrials.gov ID Treatment of Crohn’s Disease; NCT02580617 Long-Term Safety and Efficacy of FURESTEM-CD Inj. in Patients with Moderately Active Crohn’s Disease (CD); NCT02926300

Pediatric MSCs-AFP Sub-Study for Crohn’s Fistula; NCT03449069

Disease

Cell type and source

Delivery

Location Medicine, Seoul, Korea, Republic of

CD

Allogeneic MSCs, UC

Local

CD

MSCs-AFP (Patch coated with ASC)

Local

Inje University Haeundae Paik Hospital, Busan, Korea, Republic of Yeungnam University Medical Center, Daegu, Korea, Republic of Seoul National Universty Bundang Hospital, Seongnam-si, Korea, Republic of (and 4 more. . .) Mayo Clinic in Rochester, Rochester, Minnesota, USA

Abbreviations: CD Crohn’s disease, UC ulcerative colitis, ASC adipose tissue–derived stem cells, MSCs mesenchymal stem cells, BM-MSCs bone marrow–derived mesenchymal stem cells, hUC human umbilical cord, MSCs-AFP mesenchymal stem cell–coated anal fistula plug

Conclusion The numerous open and randomized clinical studies on MSCs in the IBD therapy have unequivocally shown the safety of this approach and its potential efficiency, including the traditional treatment-resistant cases. The therapeutic action of MSCs originates from the potent immunomodulating effect resulting in the reduction of the autoimmune inflammation and stimulation of reparative processes in the intestinal mucosa. In turn, it prolongs the duration of remission, decreases the risk of relapses, and the frequency of hospital admissions. Based on the conducted clinical trials, a first medication based on allogeneic MSCs derived from the adipose tissue, Darvadstrocel (Alofisel, Takeda), has been approved in the EU for the therapy of complicated perianal fistulas in patients with luminal CD. A promising approach in the treatment of fistulizing CD is the use of biomaterials as carriers for MSCs (fistula plugs coated with MSCs). Firstly, the donor cell survival is higher on a biomaterial. Secondly, the application of autologous MSCs enhances the therapeutic effect of fistula plugs. However, presently there is no single established optimal protocol for MSCs transplantation in IBD therapy. Additional studies are warranted on the optimal MSCs source, dosage, delivery method, and optimal treatment frequency. Despite the achievement of positive results, further preclinical and clinical studies are

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required to enhance the efficiency of both local and systemic MSCs transplantation. Along with BM-MSCs and ASC, the use of MSCs from the placenta appears promising. With the techniques enhancing the efficacy of MSCs production, such as 3D culturing and application of large-volume bioreactors, it may essentially lower the price of MSCs production and make this unique therapy available for a wide circle of patients.

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in patients with Crohn’s disease. Gastroenterology 154(5):1334–1342.e4. https://doi.org/10. 1053/j.gastro.2017.12.020 Prockop DJ (2009) Repair of tissues by adult stem/progenitor cells (MSCss): controversies, myths, and changing paradigms. Mol Ther 17(6):939–946. https://doi.org/10.1038/mt.2009.62 Sairenji T, Collins KL, Evans DV (2017) An update on inflammatory bowel disease. Prim Care 44(4):673–692. https://doi.org/10.1016/j.pop.2017.07.010 Samoylova EM, Kalsin VA, Bespalova VA, Devichensky VM, Baklaushev VP (2017) Exosomes: from biology to clinics. Genes Cells XII(4):7–19. https://doi.org/10.23868/201707024 Scott LJ (2018) Darvadstrocel: a review in treatment-refractory complex perianal fistulas in Crohn’s disease. BioDrugs 32(6):627–634. https://doi.org/10.1007/s40259-018-0311-4 Sisakhtnezhad S, Alimoradi E, Akrami H (2017) External factors influencing mesenchymal stem cell fate in vitro. Eur J Cell Biol 96(1):13–33. https://doi.org/10.1016/j.ejcb.2016.11.003 Siyu L, Fei L, You Z, Baeku J, Qiang S, Shu G (2020) Immunosuppressive property of mscs mediated by cell surface receptors. Front Immunol 11:1076. https://doi.org/10.3389/fimmu. 2020.01076 Spaggiari GM, Capobianco A, Abdelrazik H, Becchetti F, Mingari MC, Moretta L (2008) Mesenchymal stem cells inhibit natural killer–cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood 111(3):1327–1333. https:// doi.org/10.1182/blood-2007-02-074997 Terai S, Tsuchiya A (2017) Status of and candidates for cell therapy in liver cirrhosis: overcoming the “point of no return” in advanced liver cirrhosis. J Gastroenterol 52(2):129–140. https://doi. org/10.1007/s00535-016-1258-1 Tolar J, Le Blanc K, Keating A, Blazar BR (2010) Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells 28(8):1446–1455. https://doi.org/10.1002/stem.459 Wang S, Miao Z, Yang Q, Wang Y, Zhang J (2018) The dynamic roles of mesenchymal stem cells in colon cancer. Can J Gastroenterol Hepatol 7628763:8 pp. https://doi.org/10.1155/2018/7628763 Williams JT, Southerland SS, Souza J, Calcutt AF, Cartledge RG (1999) Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am Surg 65(1):22–26 Yabana T, Arimura Y, Tanaka H, Goto A, Hosokawa M, Nagaishi K, Yamashita K et al (2009) Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity. J Pathol 218(3):350–359. https://doi.org/10.1002/path.2535 Yang R, Huang H, Cui S, Zhou Y, Zhang T, Zhou Y (2020) IFN-γ promoted exosomes from mesenchymal stem cells to attenuate colitis via miR-125a and miR-125b. Cell Death Dis 11(7): 603. https://doi.org/10.1038/s41419-020-02788-0 Zhang J, Lv S, Liu X, Song B, Shi L (2018) Umbilical cord mesenchymal stem cell treatment for Crohn’s disease: a randomized controlled clinical trial. Gut Liver 12(1):73–78. https://doi.org/ 10.5009/gnl17035 Zhao T, Sun F, Liu J, Ding T, She J, Mao F, Xu W et al (2019) Emerging role of mesenchymal stem cell-derived exosomes in regenerative medicine. Curr Stem Cell Res Ther 14(6):482–494. https://doi.org/10.2174/1574888X14666190228103230 Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2):211–228. https://doi.org/10.1089/107632701300062859

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Extracellular Vesicles-Based Cell-Free Therapy for Liver Regeneration Mustapha Najimi and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver and Heart Connected and Non-connected Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Status on Cell-Based Therapy for Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Animal Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-Free Therapy Approaches for the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostic and Therapeutic Role of EVs in Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The substantial worldwide burden of liver diseases and related complications is in line with the regular developments of innovative therapeutic strategies that could alleviate the number of patients requiring liver transplantation, the gold standard of care approved so far. Cell transplantation has brought new perspectives to treat those patients while keeping their own livers. The concept was simple as the transplanted cells were used to promote parenchymal regeneration and/or repairing. Isolated hepatocytes were initially applied and demonstrate the proof of concept of this approach at the clinical level. Stem cells, second-generation advanced therapy medicinal products, have provided many technological and logistical solutions to improve the wide clinical use of cell therapy. Mesenchymal stem cells were extensively developed to this end and show a significant ability to migrate in the recipient diseased liver, to differentiate in situ, and to exhibit M. Najimi (*) Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCLouvain), Brussels, Belgium e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_9

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interesting immunomodulatory, immunosuppressive, and anti-fibrotic features. Most of those paracrine effects were mediated by potent bioactive molecules secreted by those stem cells. Extracellular vesicles represent a significant part of this secretome and display several interesting characteristics that support their development for liver-cell-free therapy. This chapter summarizes and discusses the significant advances related to cell-based and cell-free therapies currently achieved for the treatment of liver diseases. It also addresses the current challenges that extracellular vesicles-based therapy is dealing with before a future clinical use. Keywords

Cargo · Exosomes · Extracellular vesicles · Hepatic · Liver diseases · Noncoding RNAs · Secretome · Stem cells Abbreviations

ACLF ESCs EVs iPSC HSCs

Acute on chronic liver failure Embryonic stem cells Extracellular vesicles Induced pluripotent stem cells Hepatic stellate cells

Introduction The heart and the liver, together with the lungs, constitute the central trio of organs that critically manage the primary physiological activities of the human body and also control the activities of other organ systems. Any defects impacting one or the other organ can consequently be fatal. Due to its contractile function, the heart distributes oxygenated and nutrient-rich blood to all the body tissues and organs besides preserving body temperature (Yilmaz et al. 2013). Since antiquity, much interest has been focused on heartrelated research. It has been described as a hot and dry organ and a center of intelligence and vitality in the human body. The structural and functional development of the heart is critical from birth to weaning as its increased activity has to be perfectly aligned with growing locomotor activity and thermoregulation (Rakusan et al. 1984; MacLellan and Schneider 2000). Such proliferative growth will completely stop after the end of this critical period (Rumyantsev 1977; Brodsky 1990; MacLellan and Schneider 2000). On the other hand, the liver is the largest gland in the human body and concomitantly manages a broad range of high-level physiological and complex functions such as detoxification, protein synthesis, and biochemical production, and any alteration of which significantly impacts the physiological functioning of other body organs (Gebhardt and Matz-Soja 2014). Galen A. has considered the liver the primary organ of the human body because it is the source of the veins and the

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principal instrument of sanguification (Temkin and Straus 1946). Contrary to the heart, the critical period for the liver is from weaning to maturation (Wheatley 1972; Alison 1986; Brodsky 1990) primarily due to dietary changes (Wheatley 1972; Brodsky 1990; Vinogradov et al. 2001). For many years, an established intimate interaction between both vital organs has been extensively reported as in the theoretical system of traditional Chinese and modern-day medicines (Zhang and Fang 2021). Avicenna also pointed out the functional interaction between the two vital organs in his famous book The Canon of Medicine. The blood circulation between the liver and heart is primarily supporting such essential interorgan communication. Indeed, the hepatic artery and portal vein together allow 25% of cardiac output to reach the liver (Silvestre et al. 2014), while hepatic veins and the inferior vena cava are involved in the venous drainage toward the liver (Silvestre et al. 2014). Furthermore, such an interaction has also been described earlier during development (El-Hadi et al. 2020). The determination of the hepatic lineage is dependent on the pre-cardiac mesoderm. Indeed, it is wellaccepted that the emerging ventral endoderm should be spatially close with the pre-cardiac splanchnic mesoderm to follow a hepatic fate (Fukuda 1979; FukudaTaira 1981). The invagination of the foregut will lead the ventral wall of the endoderm abutting the developing heart. The septum transversum, which gives rise to the epicardium of the heart and the diaphragm, also plays a significant role in modulating the differentiation of hepatocytes that constitute metabolically highly active cell population of the liver. The mesenchyme compartment of the septum transversum collaborates with the developing heart to modulate the hepatic lineage specification in a paracrine manner (Rossi et al. 2001).

Liver and Heart Connected and Non-connected Defects Structural and/or functional defects of one or the other organ will seriously impact health by negatively altering the quality of life and decreasing its expectancy. Hence, scientists and clinicians have shown a great interest in consistently developing novel and improving the existing therapeutic strategies dedicated to treating any defects of organs, the heart and liver. Irrespective of the etiology of the disease, perturbation of the functionality of the cells remains the primary cause of various pathologies. Thus, replacing, repairing, and/or reactivating the functionally perturbed cells remains the primary focus of all the innovative therapies currently being developed to address the root cause of the problem and restore the normal function of these organs. Heart diseases are also associated with partial or complete disruption of blood flow to the heart muscle (coronary heart diseases) and altered contractile function, which will also negatively impact the blood flow to other organs like the brain (cerebrovascular diseases) (Ren et al. 2021). Cardiovascular diseases are the leading cause of morbidity and death worldwide, affecting nearly 17.9 million people as reported in 2019. Indeed, acute events, including heart attack and stroke, may be induced due to perturbed blood flow toward the brain, or the heart, due to the

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deposition of fatty plaques in the vessels, blood clots, or bleeding. Deaths related to heart attack and stroke represent 85% of cardiovascular diseases (WHO). Congenital or acquired structural abnormalities of the heart, including the valves or the heart muscle (rheumatic heart disease), as well as inherited disorders, are the other reported defects (Dimmeler 2011). Therefore, damaged or weakened organ will lead to heart failure that may be triggered by a heart attack or abnormally increased high blood pressure. Cardiovascular diseases may be fatal for which no cure is available; however, they mostly remain preventable. Depending on the type and the severity of the illness, rigorous lifestyle changes, early start of proper medication according to the published guidelines, and surgery may ensure these patients have improved quality of life and live longer, with reduced hospitalization occurrences. Akin to cardiovascular diseases, pathologies of the liver may lead to significantly altered liver functions. Such compromised liver function may happen at any age in life and may be caused by several internal and/or extraneous factors. Liver defects may be genetically inherited or acquired after viral infection, excessive alcohol consumption, and obesity. Inborn errors of liver metabolism happen as early as the neonatal stage and cause more than 300 different human diseases (Najimi, 2016). Although presenting features are specific for each genetic alteration, clinical disease manifestations include lethargy, vomiting, seizure, etc. Early diagnosis and appropriate treatment should limit critical extrahepatic impairments, including those occurring at the brain level. Therapeutic solutions applied so far include pharmacological, dietary, and surgical intervention (Najimi 2016). When conventional treatments fail to alleviate the disease symptoms, orthotropic liver transplantation (OLT) remains the gold standard and clinically most validated therapeutic approach to treat liver diseases (Wallot et al. 2002). However, donor shortage limits its widespread use worldwide (Kamath et al. 2001; Struecker et al. 2014; Najimi 2021). Notably, there is mounting evidence in the literature that acute and chronic heart diseases might directly lead to reversible or irreversible deterioration of liver functions or vice versa (Poelzl and Auer 2015). This is well-supported by the existence of heart-liver and liver-heart axes primarily mediated by organokines specifically synthesized and secreted by the endocrine heart (16 cardiokines) and liver (Chiba et al. 2018; Jensen-Cody and Potthoff 2020; Meex and Watt 2017; Stephan and Haring 2013; Cannone et al. 2019). This means that the heart can modulate the liver metabolic functions, while heart diseases significantly affect the liver, an observation made since the nineteenth century (Komatsu et al. 2019). For example, heat failure may induce hypoxic hepatitis, and atrial fibrillation (arrhythmia) obviously induces hyper-coagulation. Heart failure, a general consequence of the cardiac pump dysfunction at the systolic (reduced contractility) or diastolic (altered relaxation and consequent ventricular filling) levels, is associated with severe hepatic congestion, which induces acute hepatocellular necrosis and raises liver enzyme blood concentrations, and direct or indirect serum bilirubin elevation. The diseases of the liver also affect the functional status of the heart, including the role of nonalcoholic fatty liver disease in promoting the development of cardiovascular diseases (i.e., coronary artery disease, structural myocardial

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alterations, and cardiac arrhythmias). Still, the pathophysiological mechanisms involved therein are not yet deciphered, although insulin resistance, visceral adiposity, subclinical generalized inflammation, dyslipidemia, and oxidative stress are potentially related pathways (Tana et al. 2019). End-stage liver diseases are also implicated in developing cirrhotic cardiomyopathies. In an animal model of liver cirrhosis, an abnormally increased expression of collagen isoforms was reported in the ventricular myocardial tissue that led to a raised cardiac stiffness and diastolic dysfunction (Glenn et al. 2011). Hence, sustained hemodynamic variations are behind both bridging fibrosis and cardiac cirrhosis. The consequent dysregulation of hepatic functions will lead to the impairment of the metabolism of cardiovascular drugs and potential, which can lead to an undesirable toxicity. The functional liver-heart connection was also reported in genetic diseases. The best example is Alagille syndrome, a genetic disorder caused by abnormal bile ducts, in which subsequent perturbed bile flow induces significant scarring that prevents the liver from eliminating bloodstream wastes. Alagille syndrome is interestingly linked to an impaired blood flow from the heart to the lungs (pulmonic stenosis) (Tretter and McElhinney 2018). The heart-liver interorgan connection is supported by the nature of the major risk factors of the heart disease (i.e., alcohol intake, unhealthy diet, etc.), whose processing and metabolism are managed primarily by the liver. Inversely, perturbation in the liver functionality (like raised blood lipids, obesity) will influence the quality of heart activity. Therefore, broadening our knowledge on both heart and liver physiopathology is mandatory for the design and dosage of preventive and therapeutic strategies dedicated to one or the other organ.

Status on Cell-Based Therapy for Liver Diseases Although OLT remains the gold standard therapeutic option for treating liver diseases, the significantly increased donor shortage and the consequently enhanced mortality due to long waiting time limit its application worldwide (Kamath et al. 2001; Struecker et al. 2014; Najimi 2021). The more extended living grafts are unfortunately associated with posttransplantation complications and morbidity, including long-term exposure to high levels of immunosuppression regimens, deficits, or delays in development (Moreno and Berenguer 2006). This has prompted the development of innovative strategies that may restore liver function, especially for indications where liver transplantation is not the ultimate treatment, or at least support the patient while waiting for a graft to be transplanted. From the early experiment involving hepatocyte transplantation in a rat model of Crigler-Najjar syndrome by Groth et al. (1977), cell-based therapy has progressed a long way to involve stem cell and progenitor cell engraftment for liver diseases treatment (Sun et al. 2014). The cells are generally infused as a cell suspension via the vascular system without compromising or alteration of the structural integrity of the diseased liver (Najimi et al. 2016; Forbes et al. 2015). The portal vein system remains the optimal injection site from which the suspended cell will be delivered

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(Regmi et al. 2019). The aim is to allow transplanted cells to reach the liver parenchyma, where the transplanted cells can function after engraftment as well as in a paracrine manner (Iansante et al. 2018). Cell-based therapy preserves the recipient’s liver, and it may be repeatedly applied with no significant complications (Wang et al. 2019). The proof of concept of cell-based therapy for treating liver diseases has been demonstrated by using hepatocytes isolated from cadaveric donors (Ibars et al. 2016). Ibars and colleagues at Hepatic Cell Therapy Unit, Valencia, have shared their experience of working with five adults and four children with inborn metabolic diseases and have reported hepatocyte transplantation as a safe and viable option to generate metabolically functioning hepatocyte post engraftment. The transplanted hepatocytes were shown to display an ability to migrate from the injection site, replace the dead cells in the recipient hepatic parenchyma, and function in situ leading to vital restoration of deficient metabolic defects. Those data have also been confirmed by using stem/progenitor cells of intrahepatic and extrahepatic origins that can be expanded and differentiated into hepatocyte-like cells in vitro before transplantation. Some of the important parameters that significantly impact the efficacy and prognosis after cell-based liver therapy include the quality of the source organ from which the cells will be isolated, the quality and yield of cell suspension postisolation and expansion, or post-cryopreservation and thawing, as well as the nature and severity of the targeted disease, etc. (Zhu et al. 2020). Understanding of these impacting factors will not only streamline, optimize, and standardize the protocols, but it will have practical value in terms of the fast-emerging concept of individualized treatment of the patients.

Experimental Animal Studies Cell-based therapy dedicated to treating liver diseases has been firstly assessed on surgical animal models in which liver regeneration can be physiologically induced like after partial and total hepatectomy (Alwahsh et al. 2018; Forbes et al. 2015). Animal models of inherited metabolic diseases with single specific liver enzymatic defects have also been widely used. Mostly genetically modified, those animal models exhibit significant liver damage and hepatocyte structural and functional perturbations. These conditions provide a high advantage for transplanted healthy cells to survive, engraft, and proliferate in situ. The best example is the mouse model overexpressing the urokinase-type plasminogen activator gene within the liver as albumin promoter drove its expression. This experimental mouse model displays a severe hepatic injury, while transplanted hepatocytes are able to effectively reconstitute the whole liver mass (Sandgren et al. 1991). Crossed with the (SCID ¼ Severe Combined Immunodeficient) mice , those genetically modified animal models did allow the deep study of human cells behavior after transplantation like in fumarylacetoacetate hydrolase knockout Fah/, Rag2/Il2rg/ mice (Ohshita and Tateno 2017; He et al. 2010).

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Animal models in which liver diseases were pharmacologically induced and mimicked did allow the evaluation of cell-based therapy efficacy in acquired defect settings, i.e., nonalcoholic steatohepatitis, fibrosis/cirrhosis, etc. (Al-wahsh et al. 2018), but also under acute liver failure conditions like by using acetaminophen (APAP). Although animal models have demonstrated the potential of transplanted healthy cells to integrate into recipient’s livers and to correct hepatic defects and even animal improved rate of survival, the higher number of combinations involving animal models and types of transplanted cells is not yet supporting the establishment of a standardized application of cell-based therapy and its translation to the clinic. Accordingly, preclinical models deeply recapitulating human diseases at the cellular and molecular levels are still lacking although trials using large transgenic or genetically deficient animals, i.e., macaques and pigs, have been reported. On the same note, despite the use of stem cells from different tissue sources, there is an obvious lack of consensus among the stem cell researchers about the ideal cell choice (Gounder et al. 2017). From among the different cell types, mesenchymal stem cells have been extensively studied for cell-based therapy of liver diseases and have progressed to the clinical phase studies more than any other cell type (de Miguel et al. 2019).

Clinical Studies Encouraging preclinical data of liver cell therapy have supported the exploration of its usefulness at the clinical level, primarily when no other therapeutic option can be applied. This also has been supported by the reduced invasiveness of cell delivery intervention, the repeatability of cell infusions, and safety posttransplantation (Najimi et al. 2016). After successfully recovering good quality and significantly high yield of clinically approved human liver cell suspensions, hepatocyte transplantation, under the proof of concept and first in man configurations, has been applied in several centers worldwide, and data from patients with different etiologies have been reported. Indeed, the durability of the effect posttransplantation as shown on several patients with inborn errors of liver metabolism was quite variable and dependent on several factors including the yield of cells infused, the type of the analyses performed, as well as the severity of the disease. The lack of appropriate clinical trials reporting deep investigations on safety and efficacy (only very few were reported so far) makes it very difficult to formulate any conclusions on the definitive clinical use of isolated hepatocytes. Furthermore, although hepatocyte transplantation did show its ability to be used at least as a bridge to transplantation, still limitations are hampering the rapid clinical development of such therapeutic option, including the significant scarcity of good-quality raw material and the inability to long-term preserve the isolated cells due to their poor ability to survive and proliferate in vitro as well as post-cryopreservation/thawing. Recent advances in studying stem/progenitor cells have highlighted their potential in providing several solutions to the limitations encountered when isolated hepatocytes were used, such as self-renewal, plasticity, and paracrine potency. Stem/progenitor cells have been looked for and isolated from different tissues and organs at any age including the liver itself. Although with exciting preclinical results

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like those reported on embryonic stem cells (ESCs) (Woo et al. 2012; Asahina et al. 2006) and FIRST MENTIONED HERE human-induced pluripotent stem cells (iPSCs) (Corbett and Duncan 2019; Sekine et al. 2020; Takeishi et al. 2020), only MSCs are considered one of the most well-studied and extensively characterized cell types currently applied for clinical evaluation on liver diseases (Luan et al. 2021; Zhang et al. 2020). Their use in the humans has also been supported by the excellent safety profile and tolerance reported in the translational experimental studies and the ongoing trials. Fifty-nine clinical studies are registered so far in which both allogeneic and autologous MSCs isolated from different tissues were used to target various acute and chronic liver defects. A recently published pooled analysis of 39 published studies involving MSCs for various liver defects has reported that compared with the conventional treatment, MSC therapy significantly improves liver function in terms of the model of end-stage liver disease score; albumin, alanine aminotransferase, and total bilirubin levels; and prothrombin time, up to 6 months after administration (Zhao et al. 2018). Interestingly, subgroup analysis showed that single injection via hepatic artery of MSCs was more effective than peripheral intravenous injections. Moreover, bone marrow-derived MSCs were more effective than umbilical cordderived MSCs. Recent technological advances did allow knowing and learning more on the optimal conditions for banking, large-scale production and cryopreservation, stability of MSCs, and treatment methodology/approach, which would ultimately lead to provide the best-quality cell suspension to the patient. Liver cells of mesenchymal phenotype and stem/progenitor profile have also been described in both mice and humans. While directly isolated from mouse livers, those cells were obtained after primary culture of human liver parenchymal cell suspensions and displayed variable levels of plasticity (Herrera et al. 2006; Najimi et al. 2007; El-Kehdy et al. 2016). Those cells isolated from the adult human liver are currently developed under industrial settings (GMP large-scale expansion) to be clinically tested as advanced therapy medicinal products to treat liver diseases. For instance, HepaStem ® has been successfully used in patients with urea cycle defects and Crigler-Najjar syndrome in a phase I/II clinical study in Europe aiming at evaluating its safety and preliminary efficacy at 6 and 12 months post-infusion. In parallel to their safety profile, transplantation of those cells was associated with de novo urea synthesis in most urea cycle diseased patients. It also decreased bilirubin level only in some of the Crigler-Najjar patients (Smets et al. 2019). Both metabolic effects were reported at 6 months post-HepaStem ® transplantation. Advances in understanding the behavior of MSCs in targeting liver diseases have also highlighted their potent paracrine features. Indeed, this has been revealed by both in vitro analyses in which the secretome of those cells was shown to contain several bioactive molecules and in vivo, in which many described positive effects were not associated with the engraftment of transplanted cells in the recipients’ livers. HepaStem has been recently applied on cirrhotic patients with acute on chronic liver failure (ACLF) or with acute decompensation at risk of developing ACLF and has been shown to significantly improve the altered liver functions in parallel to systemic inflammation and survival rate of the transplanted patients (Nevens et al. 2021).

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Cell-Free Therapy Approaches for the Liver Thanks to the information recovered from advanced clinical trials, it becomes more consistent that the beneficial clinical effects of transplanted cells were more related to their paracrine potential and aligned with their reported low engraftment (Haider and Aziz 2017). Other lessons learned from those clinical trials are the cell expansion culture timing and costs, the high procoagulant activity of the transplanted cells, their entrapment in the lung tissue when peripherally infused which may reduce their potency, and the cytogenetic abnormalities that may happen during large-scale production and/or long-term culture in vitro that may render the cell tumorigenic post engraftment (Prockop et al. 2010; Nikitina et al. 2018). On the same note, maintenance of the quality of the cell preparation and avoidance of batch-to-batch variations remains one of the major hurdles to achieve optimal prognosis (Haider, 2018). Similarly, survival of the donor cells post engraftment significantly reduces the feasibility, although various strategies, such as transient immunosuppression and preconditioning of donor cells, have been adopted successfully (Haider et al. 2004, Xiao et al. 2004, Haider and Ashraf 2012). The fast-emerging strategy of cell-free therapy using both soluble and particulate components of stem cell paracrine secretions has given encouraging data which is comparable with the cell-based therapy (Haider and Aslam 2018). Extracellular vesicles (EVs), one of the insoluble components of cellular paracrine secretion, have been considered to be a significant component of the MSCs’ secretome that could be delivered precisely and that could have influential paracrine and endocrine contribution toward the intercellular communications (Raposo and Stahl 2019; Devaraj et al. 2021). Significant technological advancements have led to the isolation and purification of these nano-sized entities from the soluble factors of the cell secretome, and they have been extensively characterized for yield and contents – under different physiopathological conditions (Borgovan et al. 2019). EVs have also been proposed to discriminate between stem cell populations depending upon their tissue of origin, which will help in categorizing and characterizing them to reduce batch-to-batch variation in their preparation (Hur et al. 2020). Accordingly, their potential use as a therapeutic alternative to cell transplantation may bring interesting solutions for easy and widespread clinical use. Indeed, infusion of EVs could be safer as many cell-related posttransplantation reactions, like procoagulant activity, thrombogenic effect, ectopic cell migration, and differentiation, might not be considered. The simple recovery and purification of the cell supernatant containing EVs will make the production process much cheaper while following the same production and storage paths as the small molecules. Furthermore, the structural aspects of the EVs are essential to protect the cargo contents for a smooth “physiological” transfer to the target cells after fusion with the cell membrane (Haider and Aramini 2020). This latter feature is quite interesting as it will allow the manipulation of their internal and/or membranous contents for targeted tissue and material delivery. The diverse parenchymal and non-parenchymal cell composition of the liver should be perfectly aligned with a high level of coordinated intercellular

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communication to ensure the complex vital functions that such organ manages (Devaraj et al. 2021). EVs secreted by liver cells may be critical tools for the establishment of a fine-tuned crosstalk between neighboring and distant cells at the physiological as well as pathological levels. The content of EVs has been reported to be involved in the alteration of acute and chronic immune-inflammatory responses associated with several liver diseases, including those chemically or virally induced. Such alteration is due to a perturbed communication between the various liver cell types and between the liver and other organs (Babuta and Szabo 2021). As shown in the normal liver, quiescent hepatic stellate cells (HSCs) can modulate parenchymal regeneration via cell migration and immune responses and facilitate tissue remodeling after damage. Therefore, the EVs of each liver cell type may adapt the content and yield of their cargo depending on the liver defect. Many consider the circulating pools of such entities as noninvasive diagnosis biomarkers and exploit them as potential targets for developing innovative treatments. An increasing number of preclinical studies dealing with evaluating the therapeutic potential of EVs in liver diseases are noticed. Diverse strategies, using both native and modified EVs, have been investigated to target many aspects of liver defects, including inflammation (inhibition of infiltrating cells or potentiation of the intrahepatic immune response), tissue regeneration (inhibiting the injury and/or promoting hepatocyte proliferation), viral intrahepatic infection, fibrosis/cirrhosis, and liver cancer (Driscoll et al. 2021). Transplantation of EVs is associated with an improvement of liver defects. It has confirmed the multiple modes of action of these entities, including inhibition of inflammation, hepatocyte apoptosis, autophagy and HSCs activation, the fundamental cell event behind the initiation, and sustaining of liver fibrosis (Lee et al. 2021). In acute liver failure or injury, the therapeutic effects of EVs have been studied posttransplantation in different appropriate animal models. MSCs derived from bone marrow, umbilical cord, and adipose tissue were mainly used and did show significant mitigation of inflammation, autophagy, and apoptosis in parallel to stimulation of hepatocyte proliferation and activation of the adaptive immune system (Jin et al. 2018; Jiao et al. 2019; Haga et al. 2017; Yao et al. 2019). Such modifications have significantly attenuated the levels of circulating inflammatory markers and the necrotic parenchymal areas and accordingly restored the altered liver functions. Those data have been associated with the presence of noncoding RNAs, like H19, miR-17, and miR-455-3p. In the ischemia/reperfusion animal model, EVs from MSCs fuse with the membranes of the hepatocytes and stimulate their proliferation, which further significantly decreases transaminases levels and histopathological scores (Du et al. 2017). Transplantation of exosomes and their migration to the liver are followed by a significant inhibition of the ALF induced by D-GalN/LPS treatment in mice due to an inhibition of hepatic mononuclear cells and cell apoptosis (Chen et al. 2017). In addition, anti-fibrotic effects of EVs have been reported posttransplantation in the widely used CCl4-treated animal model as demonstrated by the inhibition of epithelial-to-mesenchymal transition, intrahepatic inflammation, and hepatocyte apoptosis (Li et al. 2013; Devaraj et al. 2021).

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iPSCs have also been used to generate MSCs as they display higher survival and proliferation than their adult stem cell counterparts (La Greca et al. 2018). When transplanted in ALF animal models, EVs from those cells display the same therapeutic effects as restoration of normal transaminases levels, thanks to significant inhibition of hepatocellular necrosis and sinusoidal congestion (Povero et al., 2019; Chen et al., 2017). EVs from the iPSCs-derived MSCs effectively protect hepatocytes and stimulate their proliferation via the sphingosine-1-phosphate pathway (Du et al. 2017). In chronic liver disease settings, EVs from MSCs of adult (bone marrow, umbilical cord, adipose tissue, and amniotic fluid) or embryonic origin (ESC, iPSCs) can improve fibrosis as experimentally shown both in vitro and in vivo (Povero et al. 2019; Li et al. 2013; Rong et al. 2019; Mardpour et al. 2018). Several features have improved following EVs transplantation, including the expression of ECM-specific disorganization markers, of activated HSCs and infiltrated immune cells, and hepatocyte survival. So far, EVs-derived MSCs from the bone marrow, adipose tissue, and liver were studied under Hepatocellular carcinoma (HCC) settings both in vitro and in vivo (Weng et al. 2021; Bruno et al. 2013; Ko et al. 2015; Webber et al. 2015; Fonsato et al. 2018). The data reported the potential of those EVs to decrease the expression of HCC markers and inflammatory cytokines as well as the number of altered parenchymal cells (both by inducing apoptosis and decreasing proliferation) which leads to an improvement of liver functions. Furthermore, an increase in the level and activity of circulating NKT cells and their recruitment to the diseased liver have been reported (Lou et al. 2015; Ko et al. 2015). Both coding and noncoding RNAs are implicated in those reported effects (Fonsato et al., 2018). This has led to assess the efficiency of EVs for which the cargo was modified to improve their migration (membrane engineering) as well as the targeted delivery (content engineering) of specific molecules related to the tissue and disease configurations (Tan et al. 2013; Lai et al. 2013; Ishiguro et al. 2020; Psaraki et al. 2021). Advances that may arise from studies using naïve EVs should help in addressing the molecules/pathways that would improve the therapeutic value of these entities. Accordingly, additional information is mandatory to know more on the appropriate EV doses to be applied; their quality, safety, potency; and durability of the effects posttransplantation, with the vision to implement their use at the clinical level.

Diagnostic and Therapeutic Role of EVs in Liver Diseases Intercellular communication is not only crucial for maintaining liver homeostasis but is actively involved in initiating the disease and sustaining its progression. Under pathological conditions, the yield, the size, and the content of EVs are modulated depending on the disease severity (structural intra- and extra-organ alterations) and chronicity. Therefore, the circulating EVs will reach the target cells to deliver their encapsulated content after membrane fusion and/or to modulate the activity of specific signaling pathways consequently to membranous

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protein-protein interactions. The detection of EVs in blood or other fluid samples and their longer half-life support their development as a very attractive diagnostic tool that could deliver important cellular and molecular information to the diseased organ (Newman et al. 2020). Although circulating noncoding RNAs like miRNAs are reported to be concentrated in EVs, the diagnostic potential of EVs in liver diseases is still not well demonstrated due to the very limited data available so far. Ongoing investigations should potentially lead to improve (i) the selection and purity of hepatic EVs derived from the normal and diseased livers mainly at the cellular level and (ii) the discovery of altered biomarkers specific to each hepatic defect and in fine to address a highly sensitive diagnosis of those different and complex liver defects. While the simplest view of one EV type-one disease is difficultly supported, one could expect several layers of complex combination as for instance, of EVs and/or specific contents. Therefore, extensive investigations are mandatory to address several still raised questions before EVs can be applied at the clinical level, as for instance, the determination of (i) the best source of EVs to be applied for each of liver disease type, (ii) the optimal production process, and (iii) the efficient dose of EVs (fresh, cryopreserved, single and/or repeated injection, etc.) to be infused for each specific liver disease indication. Multi-omics analyses may certainly help in compiling and exploiting information related to their biogenesis, cargo, and function (Chitoiu et al. 2020).

Conclusion and Future Perspectives Although OLT remains the gold standard therapeutic option to treat liver diseases, many patients do not have access to it. Furthermore, the waiting time for a graft often increases, while long-term posttransplantation follow-up highlights significant liver graft hepatitis and fibrosis posttransplantation as reported in the pediatric population (Kelly et al. 2016). Liver cell and stem cell transplantation has been developed as an alternative to OLT or a bridge to transplantation. Several successes and achievements of cell-based therapy have been reported both at the preclinical and clinical levels. Cell-based therapy was initially proposed to provide healthy and highly functional cells that will participate in repairing and regenerating the recipient’s diseased liver. Advances in manipulating the trialed stem cells and evaluating their effects posttransplantation have instead revealed their potent paracrine effects in mitigating liver inflammation, fibrosis, and cancer. Thus, cell-free therapy is positioned as an alternative therapeutic approach able to overcome many limitations reported with cell-based therapy. The effects observed with EVs for the MSCs field are equal to those reported with MSC-based therapies. Still, extensive knowledge and investigations are mandatory before this approach can be fully considered at the clinical level. Indeed, more efforts are needed to understand the EVs-based communication between same and different cells under normal and pathological conditions knowing the complex cell composition of the liver. This means that we have to learn more about the identity of the bioactive molecules involved as well as on their respective

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mode(s) of action (direct and/or indirect) both at the cellular and molecular levels, knowing the heterogeneity of cells and factors causing liver defects. Once addressed, the logistical aspect is another milestone to achieve, in terms of cell material used for large-scale production, considering the heterogeneous aspect of MSC cultures, standardized characterization and quantification, and potency evaluation. At the in vivo level, much information is mandatory to determine the optimal formulation (fresh and/or cryopreserved), route of administration, and injection dose that should be aligned with an optimal safety profile of the transplanted patients.

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Current State of Stem Cell Therapy for Heart Diseases Yong Sheng Tan, Qi Hao Looi, Nadiah Sulaiman, Min Hwei Ng, and Daniel Law Jia Xian

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aging of the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional Therapies for Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Action of Stem Cells in Restoring the Heart Function . . . . . . . . . . . . . . . . . . . . . . . . Clinical Evidence of Cell-Based Intervention in Ameliorating Heart Disease . . . . . . . . . . . . . . . . Acute Myocardial Infraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Refractory Angina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations of Stem Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combination with Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Heart disease is very common among older adults and is one of the main causes of death worldwide. With age, the functionality of the heart will decrease following the changes in the cardiomyocytes and cardiac tissue. Generally, the number of Y. S. Tan Cardiothoracic Department, Serdang Hospital, Kajang, Selangor, Malaysia Q. H. Looi Future Cytohealth Sdn Bhd, Kuala Lumpur, Malaysia N. Sulaiman · M. H. Ng · D. Law Jia Xian (*) Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Jalan Yaacob Latif, Kuala Lumpur, Malaysia e-mail: [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_10

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cardiomyocytes will decrease, the number of senescent cells will increase, the cardiac tissue will become thicker, and the contractility will diminish. Besides, heart diseases such as myocardial infarction and heart failure also will reduce the functionality of the heart. Currently, heart disease is normally treated with medication and surgery. In severe conditions, the patient will be recommended to opt for a heart transplant. However, medication and surgery cannot reverse the pathological changes in the heart, and it is very difficult to find a suitable heart for transplantation. Stem cell therapy offers a glimpse of hope to these patients as the cells can stimulate the proliferation of cardiac progenitor cells and cardiomyocytes as well as secrete the paracrine factors which modulate the tissue environment to promote regeneration. Even though stem cells, e.g., mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs), have been shown to differentiate into cardiomyocytes in vitro, however, there is a lack of evidence to prove that the transplanted cells can reconstitute the myocardium in vivo. The number of clinical trials using stem cells to treat heart disease is still very limited. Results from these trials suggested that stem cell therapy is safe and provides certain benefits to the patients. Nonetheless, there is still a long way to go for the researchers to identify the ideal cell source and therapy protocol to achieve a greater therapeutic effect. Keywords

Cardiomyocyte · Heart diseases · Heart failure · Infarction · Myocardial · Stem cell · Therapy List of Abbreviations

ACE AF AMI Ang-1 ARB ASCs bFGF BM BMMC BMSCs CAD CCS CPCs CSCs CT DNA eNOS EPCs ESC HGF

Angiotensin-converting enzyme Atrial fibrillation Acute myocardial infarction Angiopoietin-1 Angiotensin receptor blocker Adipose-derived stromal cells Basic fibroblast growth factor Bone marrow Bone marrow mononuclear cell Bone marrow-derived mesenchymal stem cells Coronary artery disease Canadian Cardiovascular Society Cardiac progenitor cells Cardiac stem cells Computed tomography Deoxyribonucleic acid Human endothelial nitric oxide synthase Endothelial progenitor cells Embryonic stem cell Hepatocyte growth factor

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HLHS I/C I/V IDO IFN-γ IL iPSC ISCT LVAD LVDd LVEDV LVEF LVESV MCP-1 MI MLHFQ MNCs MSC NO NYHA PCL PDGF PGE2 PGF PLLA PSI RA RCTs ROS SAQ SPECT T/E TGF-β TNF-α UC-MSCs VEGF WMSI

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Hypoplastic left heart syndrome Intracoronary Intravenous Indoleamine-2,3-dioxygenase Interferon-gamma Interleukin Induced pluripotent stem cell International Society for Cell & Gene Therapy Left ventricular assist device Left ventricular end-diastolic internal diameter Left ventricular end-diastolic volume Left ventricular ejection fraction Left ventricular end-systolic volume Monocyte chemotactic protein-1 Myocardial infarction Minnesota Living with Heart Failure Questionnaire Mononuclear cells Mesenchymal stem cell Nitric oxide New York Heart Association Polycaprolactone Platelet-derived growth factor Prostaglandin E2 Placental growth factor Poly-L-lactic acid Perfusion score index Refractory angina Randomized controlled trials Reactive oxygen species Seattle Angina Questionnaire Single-photon emission computed tomography Transendocardial Transforming growth factor-beta Tumor necrosis factor-alpha Umbilical cord-derived mesenchymal stem cells Vascular endothelial growth factor Left ventricular-wall motion score index

Introduction A lively heart is vital to keep the body healthy and to maintain the body’s homeostasis. The heart, together with the vascular system, is responsible for delivering blood throughout the body. Nowadays, the world population is growing older rapidly as people live longer due to better healthcare. Aging leads to progressive

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changes in heart structure and deterioration of heart function. As a result, aging is the dominant risk factor for the heart disease. The incidence and prevalence of heart disease increase with age, and heart disease is one of the leading causes of death worldwide (Yazdanyar and Newman 2009). Proper management of the aging heart and prompt treatment of heart disease are vital to extend the healthy lifespan of older adults and patients with heart disease, respectively. Unfortunately, there is no definitive aging heart therapy. On the same note, pharmacological and surgical interventions can achieve limited results in mending the ailing heart as these therapies primarily help to control the signs and symptoms, but fail to reverse the pathological changes and structural damage. The long waiting list is hindering the patients with advanced heart disease from getting a heart transplant. Despite a significant reduction in cardiovascular mortality over the last decades, especially in the developed countries, cardiovascular disease remains the leading cause of death in many parts of the world (Mc Namara et al. 2019). Thus, stem cell therapists have focused on the developing novel cell-based treatment strategy for a wide range of heart diseases. Mesenchymal stem cells (MSCs) and mononuclear cells isolated from the bone marrow and cord blood are more commonly used to treat heart disease. In fewer studies, cardiac stem cells (CSCs), embryonic stem cell (ESC)-derived cardiac progenitor cells, and skeletal myoblasts also have been trialed to treat heart disease. Stem cells modulate cardiac regeneration primarily through paracrine signaling (Gallina et al. 2015). The stem cell secretome consists of two major components, i.e., soluble proteins and extracellular vesicles (Maacha et al. 2020). However, there is inadequate evidence showing that the transplanted cells differentiate into cardiomyocytes to ameliorate the ailing heart, as indicated by the poor survival of transplanted cells (Abdelwahid et al. 2016). Furthermore, the wound environment with intense inflammation, hypoxic, and nutrient deprivation is hostile to the transplanted cells which are not prepared for such condition. In this book chapter, we have discussed the effects of aging on the heart, the pathophysiology of heart diseases, and the contemporary treatment strategies with emphasis on the clinical evidence of using stem cell therapy to repair the damaged heart.

Aging of the Heart Aging leads to progressive decline in body physiological function, eventually causing various diseases and health complications. For example, aging significantly affects the health of the heart, causing more inferior cardiac function and contributing to the development of heart failure and atrial fibrillation (AF) (Strait and Lakatta 2012; Steenman et al. 2017). Characteristics of the aging heart include alteration in left ventricular diastolic function, left ventricular hypertrophy, reduction of left ventricular systolic reserve, reduction of myocardial contractility, decrease in

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maximum heart rate, and decrease in maximum left ventricular ejection fraction (LVEF) (Steenman and Lande 2017; Christou and Seals 2008; Chiao and Rabinovitch 2015). Reduction of left ventricular diastolic filling rate is the first physiological modification observed at the early stage of aging. The reduction of left ventricular diastolic filling rate is compensated by increasing the atrial contraction in order to sustain the stroke volume and to maintain the LVEF (Fleg and Strait 2012). An increase in atrial contraction is associated with atrial hypertrophy and dilation. In addition, the adrenergic signaling will change to reduce the maximum heart rate to permit a longer filling time (Strait and Lakatta 2012). However, left ventricular contractility and response to β-adrenergic receptor activation reduced with age (Lakatta and Levy 2003). Hence, the myocardial mass will increase to compensate for the reduction in cardiac output. Ventricular hypertrophy is the result of an increase in the size and number of cardiomyocytes. Ventricular hypertrophy is reversible when caused by physiological changes such as in healthy athletes and pregnant women or irreversible when the modification is induced by pathology such as heart disease (Marketou et al. 2016). Although ventricular hypertrophy may provide temporary improvement in cardiac output, however, it will lead to deterioration of cardiac function, i.e., defective ventricular relaxation and filling and even heart failure, in the long term (Tardiff 2006). The heart undergoes complex changes at the cellular and molecular level during aging. With age, there will be alteration in cellular composition resulting in a reduction of cardiomyocyte population as more cells undergo apoptosis, necrosis, and a decrease in CSC reservoir (Daniele et al. 2004; Chiong et al. 2011). These are due to the aging cardiomyocytes that are more susceptible to stress, including oxidative stress. Therefore, an increase in reactive oxygen species (ROS) production due to aging often results in stimulation of cardiomyocyte death. During cardiomyocyte necrosis, various toxic cellular components that can affect the survival of neighboring cardiomyocytes are released (North and Sinclair 2012). The remaining cardiomyocytes will become hypertrophic to compensate for the reduction in cell number. In addition, there will be changes in the collagen composition with a shift toward type I collagen and increased tissue fibrosis. Fibrotic tissue is linked with poorer myocardial contractility. The accumulation of amyloid protein in the aging myocardium is also related to reducing myocardial contractility (Steenman and Lande 2017). Cardiac aging is also associated with mitochondrial dysfunction. Tocchi et al. have mentioned that aging mitochondria have inferior functionality, higher production of ROS, higher mitochondrial DNA mutation, higher respiratory chain dysfunction, dysregulation of mitochondrial fission and fusion, as well as suppressed mitophagy (Tocchi et al. 2015). All these lead to the accumulation of dysfunctional mitochondria. Furthermore, dysregulation in calcium signaling, neurohormonal signaling, and nutrient and growth signaling are also related to the impaired cardiac function (Steenman and Lande 2017; Chiao and Rabinovitch 2015). Figure 1 shows the changes in the aging heart at the cellular and tissue level.

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Fig. 1 Changes in aging heart at the cellular and tissue level. With age, the cardiomyocytes will undergo senescence, and the number of cardiomyocytes will reduce as the number of apoptotic and necrotic cells increases. The heart tissue also will become hypertrophic, ischemic, and fibrotic. These pathophysiological changes to the heart tissue are closely related to the changes in aging vascular system whereby the lumen is narrowed by the atherosclerotic plague, leading to poorer perfusion to the heart tissue

Heart Disease Heart disease is prevalent and is one of the leading causes of death worldwide. There are many types of heart disease, e.g., myocardial infarction (MI) and heart failure, which affect a different part of the organ. MI, commonly known as heart attack, is caused by a sudden disruption in blood supply to the myocardium. The lack of oxygen leads to irreversible damage to the cardiomyocytes. At the cellular and tissue levels, prolonged ischemia causes cardiomyocyte apoptosis and necrosis, inflammation, loss of cellular glycogen, mitochondrial dysfunction, myofibril relaxation, and sarcolemmal disruption. Most of the affected cardiomyocyte cell death took place within the first 24 h of injury. Tissue inflammation following the injury will lead to the second wave of cardiomyocyte cell death. The human heart has inadequate regenerative capacity. Thus, the infarcted tissue will be replaced with fibrous scar tissue. Replacement of functional myocardium with nonfunctional fibrotic scar alters the cardiac contraction and impulse conduction and subsequently increases the prevalence of diastolic and systolic dysfunction, arrhythmia, and heart failure (Thygesen et al. 2018; Frangogiannis 2015). Heart failure is a clinical syndrome characterized by the inability of the heart to pump sufficient blood to meet the body’s metabolic demand. Many factors, including MI, can cause heart failure. Other etiologies include cardiomyopathies, valvular disease, hypertension, diabetes mellitus, myocarditis, infections, systemic toxins,

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Fig. 2 Pathophysiology of aged and diseased heart. Aging and heart disease will lead to volume overload, pressure overload, loss of myocardium, and reduced contractility which in turn cause left ventricular dysfunction. Left ventricular dysfunction will reduce cardiac output and increase end-systolic and end-diastolic volume which eventually resulted in hypoperfusion and pulmonary congestion, respectively

and cardiotoxic drugs. Generally, the initial insult induced a destructive cycle that causes metabolic and morphological changes in the remaining cardiomyocytes. These cellular changes progressively lead to structural remodeling of the ventricle. Although remodeling initially occurs as a compensation mechanism or adaptive response to sustain cardiac performance, however, over time, the response becomes counterproductive and leads to ultrastructure abnormality, e.g., including hypertrophy and fibrosis, finally causing heart failure. In addition, with time, diastolic dysfunction and subsequent systolic dysfunction resulted in an enlarged, dilated, and low contracting ventricle (Kemp and Conte 2012; Johnson 2014). Figure 2 shows the pathophysiology of an aged and diseased heart.

Conventional Therapies for Heart Disease Nowadays, treatment for heart disease depends on the type, etiology, symptoms, and severity of the disease. The treatment of ischemic heart disease starts from lifestyle modifications such as regular exercise, smoking cessation, and lipid control. Antiplatelet and antianginal medications are routinely used in those with established diagnosis via coronary angiogram or CT imaging of the heart. Ultimately such a

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domain might require percutaneous coronary intervention (stenting) or coronary artery bypass surgery to tackle the blocked native coronary vessels (Dababneh and Goldstein 2020). Structural heart disease and valvular heart disease deliberately warranted surgical intervention. The progressive valve defect can lead to ventricular dilatation and subsequently dilated cardiomyopathy because of the lengthening and destruction of muscle fibers (Frank-Starling law) (Epstein and Davis 2003). Therefore, cardiothoracic surgeon referral is paramount to address the underlying valve pathology, and valve repair or replacement is mandatory to delineate the structural defect. Another subgroup of patients commonly seen is those with heart failure due to varying pathology. These patients require medications, e.g., angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta-blockers, diuretics, aldosterone antagonists, vasodilators, digoxin, I(f) inhibitor, and angiotensin receptor-neprilysin inhibitor, to control the disease progression and achieve symptom control (Shah et al. 2017). Surgery might have limited benefits for these patients. However, advancements in technologies, such as left ventricular assist devices (LVAD), provide certain advantages in controlling heart failure (Hunt and Ross 2002; Birati and Jessup 2015). Eventually, a heart transplant is the only chance for them. However, there is a critical shortage of donor’s hearts worldwide to meet the current demand for heart transplantation.

Mechanisms of Action of Stem Cells in Restoring the Heart Function Stem cells are multipotent cells that reside in embryos and adult tissues. Stem cells are categorized either by their potency (e.g., totipotent, pluripotent, multipotent, or unipotent) or their source of origin (e.g., embryo, bone marrow, adipose tissue, Wharton’s jelly, etc.) (Los et al. 2018). The most widely studied stem cells are MSCs that could be isolated from many adult tissues, e.g., bone marrow, adipose tissue, peripheral blood, skin, and heart. Researchers considered MSCs as the most attractive stem cells because it is easily obtainable from many adult tissues and can be expanded with ease in the laboratory to fetch many cells (Ding et al. 2011; Haider 2018). According to the characterization guideline proposed by the International Society for Cell & Gene Therapy (ISCT), MSCs are cells that (i) are plastic adherent in vitro; (ii) are more than 95% positive of CD105, CD73, and CD90, while less than 2% positive for surface antigen markers CD34, CD45, CD79/CD19, CD14/CD11b, and HLA-DR; and (iii) have in vitro tri-lineage, i.e., chondrogenic, osteogenic, and adipogenic differentiation capability (Horwitz et al. 2005). Stem cells have been reported to restore heart function via several mechanisms. Generally, the transplanted cells will stimulate proliferation of native cells in the heart and secrete paracrine factors which favor heart repair and regeneration (Fig. 3). Additionally, MSCs can differentiate into cardiomyocytes to repopulate the injured heart tissue (Hafez et al. 2016). However, based on the results from in vivo study, researchers have found that the benefits of MSCs-based therapy do not rely on its

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Fig. 3 Functions of stem cell therapy. Transplanted cells secrete paracrine factors and extracellular vesicles that stimulate the proliferation of cardiac progenitor cells and cardiomyocytes and modulate the tissue environment to enhance the survival and function of preexisting cardiomyocytes

ability to repopulate the infarcted heart but more on its secretion of paracrine factors, which possess the immunomodulatory ability and support tissue regeneration. This is evidenced by the poor MSC retention in the infarcted area (Luger et al. 2017). Despite the poor cell homing in the infarcted area, delivery of MSCs resulted in improved left ventricular function. This improvement has been attributed to the antiinflammatory effect of MSCs, whereby delivery of MSCs reduces the number of NK cells and neutrophils after MI, subsequently improving left ventricular function (Luger et al. 2017). The body reacts to injury by producing pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin12 (IL-12), interleukin-17 (IL-17), and interferon-gamma (IFN-γ) (Chaplin 2010). These factors are produced by both innate and adaptive immune cells and attract the migration of MSCs to the injury site. Thus, regardless of the origin, whether exogenously introduced or endogenously recruited, MSCs will be activated by these pro-inflammatory cytokines and polarized to the immunosuppressive phenotype. Upon activation, MSCs will modulate the innate and adaptive immune response by influencing the function of macrophages, natural killer cells, B cells, T cells, mast cells, neutrophils, and dendritic cells (Liau et al. 2020b; Wang et al. 2014).

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MSCs mediate immunomodulation through paracrine secretion and cell-to-cell contact (Liau et al. 2020a). MSCs have been reported to secrete a myriad of anti-inflammatory factors, including galectin-1, interleukin-10 (IL-10), interleukin-13 (IL-13), prostaglandin E2 (PGE2), nitric oxide (NO), transforming growth factor-beta (TGF-β), and indoleamine-2,3dioxygenase (IDO) (Lim et al. 2018; Kyurkchiev 2014; Haider and Aziz 2017). Even though inflammation is indispensable in wound healing, an overwhelming inflammation upon heart injury is detrimental for the remaining cardiomyocytes. Thus, stem cell therapy, particularly MSCs, can act as a potent modulator of inflammation response to promote heart regeneration. Immunomodulatory role of MSCs is apparent in restoring heart function during heart disease progression. The paracrine effects of MSCs are not limited to the immune-related cells but including the resident cells, i.e., injured cardiomyocytes and cardiac progenitor cells (CPCs). Exosomes secreted by MSCs contain miR-221 and miR-19a that are involved in apoptosis suppression and activation of PI3K-Akt signaling pathway to promote cardiomyocyte survival and growth (Ward et al. 2018; Yu et al. 2015). The paracrine effects of MSCs are studied via conditioned media exposure in vitro or injection in vivo. Conditioned media are spent media or used media collected from cultured cells. In vitro study showed increased migration and proliferation of CPCs when cultured with MSCs-derived conditioned media. MSCs-derived conditioned media also exert protective effect against serum starvation and hypoxia-induced apoptosis (Nakanishi et al. 2008). On the other hand, in vivo study found that MSCs-derived conditioned media injected in a porcine MI model significantly reduced tissue infarct size and improved systolic function (Timmers et al. 2011). These were achieved by abrogation of TGF-β signaling and apoptosis resulting from phospho-SMAD2 suppression and activation of caspase 3 by the conditioned media (Timmers et al. 2008). MSCs also improve the myocardial angiogenesis after injury. In a preclinical study using experimental rat model of MI, transplanted MSCs differentiated into endothelial cells and improved the myocardium angiogenesis (Siamak et al. 2003). Nonetheless, most of the studies found that MSCs promote angiogenesis mainly through paracrine signaling (Teng et al. 2015; Cai et al. 2009). MSCs secrete several proangiogenic factors, such as IL-6, TGF-β, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1), placental growth factor (PGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1) (Maacha et al. 2020; Tao et al. 2016). Moreover, MSCsderived exosomes are also rich in miRNAs, e.g., miR-210, miR-199-5p, miR-423-5p, miR-939, and miR-21-3p, that are proangiogenic (Baruah and Wary 2020).

Clinical Evidence of Cell-Based Intervention in Ameliorating Heart Disease Several clinical trials have published their findings, while many more trials are underway and have been registered in the ClinicalTrials.gov database (Table 1) (Rajab et al. 2019). Worth mentioning is the BAMI trial that will recruit 3000 MI

II I I II I

I

II I/II

I/II I/II I

II/III 1/II II/III III

NCT01758406 NCT02387723 NCT01883076 NCT01829750 NCT03406884

NCT03431480

NCT03092284 NCT03145402

NCT03227198 NCT03180450 NCT02057900

NCT03763136 NCT01693042 NCT00644410 NCT00526253 NCT02032004

HF Chronic post-MI HF Congestive HF Congestive HF post-MI Chronic HF due to systolic dysfunction

HF HF HF

HF HF

HLHS

5 81 59 170 566

5 60 10

81 5

12

50 10 30 34 32

5

Autologous BM-MNCs Allogeneic UC-derived MSCs ESCs-derived CD15+ IsI-1+ progenitors Allogeneic iPSCs-derived CMs Autologous BM-MNCs Autologous BMSCs Autologous SkMs Allogeneic BM-MPCs

Autologous CSCs Allogeneic AT-MSCs Autologous UC-blood MNCs Autologous CPCs Autologous c-kit+ cells derived from the right atrial tissue Autologous placenta cord blood MNCs Allogeneic AT-stem cells Autologous BM-MNCs

Allogeneic BM-MNCs

(continued)

I/M transplantation I/C infusion (cells collected from 100–150 ml of BM) I/M transplantation Intravenous infusion Fibrin patch transplanted at the epicardium I/M transplantation (100  106 cells) 1 or 2 I/C infusions I/M transplantation I/M transplantation T/E transplantation

I/C infusion I/M transplantation (100  106 cells) I/M transplantation (3 106 cells per kg) I/C infusion (0.3  106 cells per kg) 6–10 I/M injections (up to 12,500 cells/ kg) I/C infusion

I/M transplantation

II/III

NCT01759212

Cell delivery 10 I/M injections (20  106 cells)

Phase I/II

ClinicalTrials. gov identifier NCT00869024 Cell source Autologous BM-MNCs

Table 1 List of clinical trials using stem cells to treat cardiac diseases listed in ClinicalTrials.gov database Patients number 25

Current State of Stem Cell Therapy for Heart Diseases

Disease End-stage HF patients undergoing LVAD implantation End-stage HF patients undergoing LVAD implantation Ischemic HF Ischemic HF HLHS HLHS HLHS

9 249

I

II

I II

III

II

II/III I/II I II/III III II

NCT04236479

NCT02501811

NCT00313339 NCT00384982

NCT02672267

NCT01291329

NCT01392105 NCT02439398 NCT00313339 NCT01187654 NCT01569178 NCT00936819

MI MI MI MI MI MI

MI

MI

MI MI

Ischemic cardiomyopathy

Disease Nonischemic HF Chronic ischemic heart disease patients with laser revascularization surgery Congenital heart disease

80 55 31 80 350 47

160

50

31 116

144

36

Patients number 23 10

Allogeneic ischemia-tolerant BMSCs Allogeneic Wharton’s jellyMSCs Autologous BMSCs Allogeneic CSCs Autologous BM-CD34+ cells BM-AC 133+ or MNCs Autologous BM-MNCs Autologous EPCs or EPCs overexpressing eNOS

Autologous BMSCs or c-kit+ cells or both Autologous BM-CD34+ cells BM-MNCs

Allogeneic BMSCs

Cell source Allogeneic BMSCs Autologous BM-CD133+ cells

I/C infusion (1  106 cells/kg) I/C infusion (35  106 cells) I/C infusion I/C infusion I/C infusion I/C infusion (20  106 cells)

I/C infusion

Cardiopulmonary bypass (1  106, 10  106, 20  106, 40  106, 80  106 cells/kg) 15 T/E injections (150  106 MSCs, 5  106 c-kit+ cells) I/C infusion I/C infusion or I/C infusion+ I/M transplantation I/V infusion

Cell delivery I/V infusion (1.5  106 cells /kg) Injection into laser channels during trans-myocardial revascularization

Abbreviations: BM, bone marrow; BMSCs, bone marrow-derived mesenchymal stem cells; EPCs, endothelial progenitor cells; HLHS, hypoplastic left heart syndrome; I/C, intracoronary; I/V, intravenous; LVAD, left ventricular assist device; MI, myocardial infarction; MNCs, mononuclear cells; MSCs, mesenchymal stem cells; eNOS, human endothelial nitric oxide synthase; T/E, transendocardial

Phase II I

ClinicalTrials. gov identifier NCT02467387 NCT03043742

Table 1 (continued)

250 Y. S. Tan et al.

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Fig. 4 Stem cell therapy for heart diseases. Mesenchymal stem cells, cardiac stem cells, skeletal myoblasts, embryonic stem cell-derived cardiac progenitor cells, and bone marrow mononuclear cells have been used to treat heart disease. The cells can be administered directly through the intravenous, intracoronary, and intramyocardial routes or assembled as cell sheet and cardiac patch to be transplanted at the epicardium

patients (Mathur et al. 2017). The patients will be divided equally into the stem cell treatment group, which would receive an intracoronary infusion of autologous bone marrow mononuclear cells (BMMCs), and a control group patient who would be given only the standard therapy. The trial aims to determine if BMMC treatment can reduce all-cause mortality in acute MI (AMI) patients with an LVEF of 45% after successful reperfusion. Another interesting study is the ENACT-AMI trial that applied autologous endothelial progenitor cells overexpressing human endothelial nitric oxide synthase (eNOS) to treat MI patients (Taljaard et al. 2010). This is the first trial combining cell and gene therapy to treat heart disease. Multiple types of stem cells, e.g., MSCs and mononuclear cells isolated from different tissue sources, cardiac stem cells, skeletal myoblasts, and ESCs-derived CPCs, have been tested clinically to treat heart disease (Haider 2006). The cells were administered via different route, most commonly via direct transplantation to the myocardium or through the intracoronary infusion. Furthermore, the cells also can be injected intravenously and transplanted as a cell sheet or cardiac patch (Fig. 4) (Guo et al. 2020).

Acute Myocardial Infraction Bone marrow cells in different forms have been tested in multiple randomized clinical trials to treat AMI. Most of the studies used the heterogeneous cell population, i.e., BMMCs, while some applied specific cell population such as bone marrow-derived mesenchymal stem cells (BMSCs), CD133+ bone marrow progenitor cells, and CD34+ CXCR4+ bone marrow cells.

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Strauer et al. reported the intracoronary infusion of autologous BMMCs in ten AMI patients. They found that cell therapy is safe and effective in reducing the size of the infarcted region and improving cardiac function (Strauer et al. 2002). In another study that also administered autologous BMMCs via intracoronary route, the authors found no changes in left ventricular end-diastolic volume (LVEDV), significant improvement in left ventricular end-systolic volume (LVESV) and LVEF, and better regional contractility in the patients who received cell therapy compared to the patients who received standard therapy (Francisco et al. 2004). Lipiec et al. infused autologous BMMCs via intracoronary route to 26 AMI patients and found no significant changes in LVEF, LVEDV, LVESV, and left ventricular-wall motion score index (WMSI) compared to the 13 patients in the control group after 6 months (Lipiec et al. 2009). However, the infarct area WMSI, perfusion defect extent, left ventricular perfusion score index (PSI), and infarct area PSI improved significantly in the stem cell group. The FINCELL trial divided 80 AMI patients equally to the treatment group that received intracoronary BMMC injection and the placebo group that received media without cells (Huikuri et al. 2008). After 6 months, the treatment group showed more considerable improvement in LVEF compared to the placebo group. Ge et al. infused autologous BMMCs into the infarct-related coronary artery of ten AMI patients and found that LVEF increased while left ventricular end-diastolic internal diameter (LVDd) remained unchanged and myocardial perfusion defect scores decreased. This was compared with ten AMI patients who received bone marrow supernatant and showed no changes in LVEF and myocardial perfusion defect scores as well as larger LVDd (Ge et al. 2006). Cao et al. delivered autologous BMMCs via intracoronary route to 41 AMI patients as compared to normal saline solution without cells to 45 AMI patients in the control group (Cao et al. 2009). After 6 months, the LVEF improved significantly in the stem cell treatment group compared to the control treatment group. The improvement persisted for up to 4 years of follow-up. The HEBE trial recruited 200 AMI patients and randomly assigned them to receive either an intracoronary infusion of BMMCs, peripheral blood mononuclear cells, or standard therapy (Hirsch et al. 2010). The researchers observed no significant difference in the percentage of dysfunctional left ventricular segments that improved after treatment. Similarly, there was no observed difference between the cell treatment groups in terms of improvement in LVEF and changes in left ventricle mass, volume, and infarct size at 4 months after treatment as compared with the control group. The ASTANI trial treated 50 AMI patients with an intracoronary infusion of autologous BMMCs and found that stem cell therapy significantly increases the exercise time, peak heart rate, and percentage of heart rate reserves compared to the control group (50 patients) 6 months after the treatment (Lunde et al. 2007). However, no significant differences were detected for the changes in LVEF, LVEDV, WMSI, and infarct size between the two groups at 6 and 12 months (Lunde et al. 2006, Lunde et al. 2008). The REGENT trial involved intracoronary infusion of BMMCs and CD34+ CXCR4+ bone marrow cells to 80 AMI patients each (Tendera et al. 2009). At 6 months, the LVEF increased by 3% in both stem cell therapy

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groups but remained unchanged in the 40 control group patients. However, the observed improvement was statistically insignificant. Also, no significant changes were observed in LVESV and LVEDV. Nonetheless, the authors reported that stem cell therapy gave better results in patients with extremely poor LVEF. In a study examining the effect of BMMC dosage, 66 MI patients were divided equally into 3 treatment groups, high-dose group (100  106 cells), low-dose group (10  106), and a control group (without cell transplantation) (Meluzín et al.2006). All the three groups had intravenous administration of their respective treatment. The results showed that the high-dose group demonstrated significantly higher improvement in the peak systolic velocity of longitudinal contraction of the infarction wall compared to the low-dose group, and only the high-dose group showed considerable enhancement in LVEF compared to the control group after 3 months of follow-up. The significant improvement in LVEF persisted for up to 12 months (Meluzín et al. 2008). In a study that compared the effectiveness of intracoronary and intravenous administration of BMMCs, the authors reported no significant differences in the changes in echocardiographic parameters, i.e., LVEF, LVEDV, LVESV, and WMSI, at 6 months between the intravenous group, intracoronary group, and control group (Nogueira et al. 2009). Many have raised the question regarding the optimal timing for stem cell therapy in post-MI patients. Thus, the TIME trial was designed to examine the safety and efficacy of intracoronary infusion of 150  106 autologous BMMCs at day 3 or 7 post-MI on 120 patients (Traverse et al. 2012). After 6 months, it was found that the timing of cell infusion does not affect the improvement in global and regional left ventricular function, whereby no significant differences were observed between the stem cell-based treatment groups. Furthermore, no significant differences were detected between the stem cell groups and the placebo group at 6 months and 2 years (Traverse et al. 2012, Traverse et al. 2018). The LateTIME trial used a similar treatment protocol; however, the cells were administered 2–3 weeks post-MI (Traverse et al. 2010). Similarly, results showed no differences in global and regional left ventricular function between the groups after 6 months (Traverse et al. 2011). The SWISS-AMI trial randomized 200 AMI patients in ratio 1:1:1 to receive early (5–7 days after AMI) administration of BMMCs, late (3–4 weeks after AMI) administration of BMMCs, and standard therapy (control group). At 4-month follow-up, there was no real difference in left ventricular function improvement between the two treatment groups and the control group (Sürder et al. 2013). Similarly, no improvement was observed during 12-month follow-up (Sürder et al. 2016). Thus, the timing of cell transplantation might be less important when using bone marrow cells to treat AMI. The BONAMI trial recruited 101 AMI patients divided into the stem cell therapy group (52 patients) who received an intracoronary infusion of autologous bone marrow cells (98.3  8.7  106 cells) and control treatment group (49 patients) who received the sham infusion without cells (Roncalli et al. 2010). The authors reported that cell therapy significantly improved myocardial viability after 3 months. However, no differences were detected for the LVEF, global WMSI, and infarct size. The REGENERATE-AMI trial investigated intracoronary infusion of autologous

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bone marrow cells to 55 AMI patients. Another 45 AMI patients in the placebo treatment group received normal saline without cells (Choudry et al. 2016). The results showed that the stem cell therapy group had greater improvement in LVEF and significantly higher myocardial salvage index than the placebo treatment group. In the BOOST trial that treated 30 AMI patients with an intracoronary infusion of autologous bone marrow cells, the researchers found that cell therapy is safe and helped to improve the diastolic function and LVEF compared to the control patients (30 patients) (Schaefer et al. 2006, Wollert et al. 2004). However, the follow-up study found that the significant improvement in LVEF diminished after 18 months (Meyer et al. 2006, Meyer et al. 2009). Nonetheless, the improvement in LVEF was maintained in the subgroup of patients with more transmural infarcts. In the multiple-arm BOOST-2 trials, the researchers found that intracoronary infusion of high dose and low dose of autologous bone marrow cells, γ-irradiated or not, did not significantly improve the LVEF (Wollert et al. 2017). The LEUVINE-AMI trial applied autologous bone marrow-derived stem cells in 33 AMI patients, and another 34 patients in the control group received the placebo (Janssens et al. 2006). Stem cell-based treatment significantly reduced the myocardial infarct size and improved the regional systolic function compared to the placebo treatment group after 4 months. However, no significant differences were detected in LVEF, LVEDV, and LVESV. Grajek et al. intravenously infused bone marrow stem cells to 31 AMI patients. The authors found that it did not result in any significant improvement in LVEF, LVEDV, and LVESV compared to the 14 patients in the control group during 1-year follow-up (Grajek et al. 2009). In the multicenter REPAIR-AMI trial, 101 patients received bone marrowderived progenitor cell infusion into the infarct-related coronary artery, and another 103 patients received placebo treatment (Schächinger et al. 2006). The progenitor cell-based therapy significantly improved LVEF, LVESV, and regional contractility compared to the placebo treatment at 4 months. Furthermore, at 2 years, the progenitor cell-based therapy group demonstrated a lower incidence of recurring MI, rehospitalization for heart failure, and death as well as higher improvement in LVEF compared to the placebo-treated group (Assmus et al. 2010). In the TOPCARE-AMI trial, 59 patients with AMI received an intracoronary infusion of circulating progenitor cells or bone marrow-derived progenitor cells and are followed of up to 5 years (Leistner et al. 2011; Schächinger et al. 2004). The results showed the long-term safety of intracoronary delivery of autologous circulating progenitor cells and bone marrow-derived progenitor cells in AMI patients. In addition, the patients also showed a sustained improvement in LVEF for 5 years without any significant difference between the two treatment groups. Chen et al. examined the potential of using autologous BMSCs to treat AMI by intracoronary administrating the cells to 34 AMI patients with another 35 AMI patients in the control group receiving only the saline (Chen et al. 2004). Patients who received the stem cell-based therapy demonstrated significant improvement in LVEF, wall movement velocity over the infarcted region, LVEDV, and LVESV after 3 months compared to those in the control group.

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Bartunek et al. reported that intracoronary infusion of CD133+ bone marrow progenitor cells in 19 AMI patients significantly improved the LVEF, left ventricular regional chordae shortening, and reduction of the perfusion defect compared to the 16 AMI patients without the stem cell therapy (Bartunek et al. 2005). However, it also increased the incidence of coronary events. In another study, the researchers compared intracoronary infusion of CD133+ cells from bone marrow and peripheral blood with the standard therapy (five patients each group) (Colombo et al. 2011). It was found that LVEF and wall motion score index remained stable for all groups, but the infarct-related myocardial blood flow was only increased in the bone morrow group after 1 year. In addition, infarct size and summed rest score decreased most significantly in the bone marrow-treated group. Overall, some studies such as BOOST, TOCARE-AMI, FINCELL, REGENERATE-AMI, and REPAIR-AMI demonstrated improvement in global and regional left ventricular function after the bone marrow cell therapy. Nonetheless, there are also many trials, i.e., BONAMI, LEUVEN-AMI, HEBE, ASTAMI, BOOST-2, TIME, LateTIME, SWISS-AMI, and REGENT, which failed to detect significant functional improvement. Furthermore, in the systematic review and metaanalysis conducted by Fisher et al. in 2015, the authors recovered 41 randomized controlled trials (RCT) that compared the safety and efficacy of autologous bone marrow cells with no cell therapy group and found that there is insufficient evidence to prove the advantages of applying autologous bone marrow cells in MI patients (Fisher et al. 2015). These discrepancies might be due to the differences in the cell preparation technique, the number of cells administered, cell composition, and timing of cell administration.

Heart Failure The FOCUS-CCTRN trial studied the safety of transendocardial injection of autologous BMMCs in end-stage ischemic heart disease patients (Perin et al. 2012). Results indicated that the mononuclear cells were well-tolerated, and the treatment showed a positive effect on myocardial perfusion and contractility. The investigators reported that the cell therapy resulted in a slight improvement in patients’ LVEF but did not reduce LVESV or increase maximal oxygen consumption. In addition, the clinical trial also showed a positive correlation between the degree of LVEF improvement and the percentage of CD34+ and CD133+ cells. Specifically, every 3% increase in CD34 or CD133 cells was associated with an increase in LVEF of 3–5.9%. These critical findings suggested that the cellular composition does affect the clinical effectiveness of stem cell therapy. In 2016, Fisher et al. published a systematic review and meta-analysis that included 38 randomized controlled trials (RCTs) and 1907 participants. The authors concluded that BMSCs therapy could reduce mortality and improve the cardiac function of patients with chronic ischemic heart disease and heart failure, albeit with the low quality of the collected evidence (Fisher et al. 2016). The BMSCs used in these RCTs included BMSCs, BMMCs, CD 133+ cells, CD34+ cells, aldehyde dehydrogenase-bright cells, bone marrow aspirate

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concentrate, and G-CSF mobilized and cultured circulating mononuclear proangiogenic cells. Interestingly, bone marrow stem cells have been cardiomyogenically induced for the treatment of heart failure clinically. The C-CURE trial used cardiopoietic stem cells (cardiomyogenic differentiated BMSCs) to treat patients with chronic heart failure (Bartunek et al. 2013). The 2-year follow-up showed that cell therapy is safe. In addition, the efficacy measurement at 6 months showed that LVEF increased, LVESV decreased, and 6-min walk distance improved significantly compared to the control group. Instead of expanding the cells in normoxic conditions like most studies, Butler et al. cultured the allogeneic BMSCs in hypoxic condition and used the cell to treat nonischemic cardiomyopathy (Javed et al. 2017). Treatment with ischemia-tolerant BMSCs is safe and significantly improved the 6-minute walking distance and Kansas City Cardiomyopathy Questionnaire clinical summary score compared to the control group. Apart from bone marrow stem cells, umbilical cord-derived mesenchymal stem cells (UC-MSCs) also have been used to treat heart failure. The RIMECARD trial applied allogeneic UC-MSCs to treat 15 patients with heart failure (Bartolucci et al. 2017). The cell therapy group demonstrated significant improvement in LVEF, New York Heart Association (NYHA) functional class, and Minnesota Living with Heart Failure Questionnaire (MLHFQ) score. In a separate study, Zhao et al. administered UC-MSCs to treat chronic systolic heart failure. The authors reported improved 6-minute walk distance and LVEF improved besides significantly reduced mortality rate compared to the control group (Zhao et al. 2015). In 2015, Menasché et al. reported the first clinical case of using human embryonic stem cell (ESC)-derived CPCs to treat severe heart failure (Menasche et al. 2015). The patient showed improvement in NYHA functional class and LVEF. More importantly, no adverse event was reported.

Refractory Angina Refractory angina (RA) is a chronic condition (3 months) characterized by angina in the setting of coronary artery disease (CAD). Currently, RA cannot be controlled by conventional therapeutic interventions, angioplasty, coronary artery bypass surgery, or a combination of the two methods mentioned above. Hence, Losordo et al. conducted a phase II, randomized, controlled clinical trial involving 26 centers (167 patients) in the United States to treat patients with chronic RA with autologous stem cells (Losordo et al. 2011). In this study, patients received either low dose (1  105 cells/kg) or high dose (5  105 cells/kg) of G-CSF mobilized autologous CD34+ stem cells through the intramyocardial route. The 6-month and 12-month follow-up showed that only the low-dose group recorded a significant reduction in angina frequency and improvement in exercise tolerance. Similarly, the RENEW trial and clinical trial by Wang et al. that also administered autologous CD34+ stem cells showed a reduction in angina frequency, improvement in exercise tolerance, and improvement in the Canadian Cardiovascular Society (CCS) class compared to the placebo group (Povsic et al. 2016, Wang et al. 2010).

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Mathiasen et al. found benefit in treating patients with CAD and RA using BMSCs (Mathiasen et al. 2013; Haack-Sorensen et al. 2013). The results from the 3-year observation period showed that the cell therapy reduced the frequency of hospital admission, improved the exercise time, lowered the CCS class, increased the Seattle Angina Questionnaire (SAQ) scores, and exhibited long-term safety with no adverse effect. Other investigators assessing the use of bone marrow-derived stem cells to treat RA also reported encouraging results (Beeres et al. 2006; Vicario et al. 2004; Tse et al. 2007; van Ramshorst et al. 2009; Pokushalov et al. 2010). The PROGENITOR trial and REGENT-VSEL trial applied transendocardial administration of autologous bone marrow CD133+ cells to treat patients with RA (Wojciech et al. 2017, Pilar et al. 2014). The results showed that cell therapy reduced frequency of angina and improved the CCS class in the PROGENITOR trial. Still, fewer promising findings were reported in the REGENT-VSEL trial that found no significant difference in single-photon emission computed tomography (SPECT) score, left ventricular function, and CCS class compared to the control group. In a recent clinical trial (MyStromalCell trial), Qayyum et al. reported the intramyocardial administration of autologous adipose-derived stromal cells (ASCs) to treat 40 patients with RA with another 20 patients in the control group receiving normal saline (Qayyum et al. 2019). Patients treated with ASCs demonstrated improved metabolic equivalents, CCS class, and NYHA class compared to the control group during the 3-year observation period. In addition, the ASCs-treated group was able to maintain the exercise capacity, while deterioration was recorded in the control group. In the ATHENA trial that also used ASCs to treat RA, the authors found that more patients demonstrated improvement in HYHA class and CSS class than the placebo group (Henry et al. 2017).

Limitations of Stem Cell Therapy Despite significant progress in clinical translation of cell therapy in heart disease over the past decade, many uncertainties remain regarding the most efficacious cell type, cell dosage, and route and timing of cell administration (Haider and Ashraf 2005). Adding to the complexity, there is growing evidence showing that stem cells harvested from elderly patients do not produce the same benefits from healthy donors (Haider et al. 2018). Collectively, these issues highlighted the need to investigate further the mechanisms underlying stem cell survival, plasticity, and functionality. The most critical question yet to be answered is to find an ideal type of stem cells to treat heart diseases. For this purpose, it is crucial to understand the mechanisms of each type of stem cells in affecting the myocardial performance and also in modulating different cardiac pathologies. Different types of cells might be needed for diverse cardiac pathologies. Thus far, bone marrow-derived stem cells have been widely used clinically, and they have been proven safe and beneficial under certain circumstances. However, the cells’ regeneration potential is controversial. CSCs can

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be patient-specific, but the tissue collection procedure is highly invasive, and the culture procedure needs further optimization, especially the upscaling process. An alternative option is to use the cardiomyogenic differentiated MSCs. In fact, the C-CURE trial has used the cardiomyogenic differentiated BMSCs to treat heart failure (Bartunek et al. 2013). Another key factor for successful cell-based therapy is determining the optimal route of delivery. Cells can be injected intravenously, intracoronary, or directly into the myocardium, each with distinct pros and cons. Cell engraftment at the injury site majorly depends on the delivery strategy. Generally, cell engraftment improved through intramyocardial injection under direct vision at the injury site. Scaffoldbased cell delivery strategy is gaining popularity as it promotes the rate of cell survival and integration of transplanted cells in the hostile host environment besides ensuring site-specific delivery (Kc et al. 2019). In addition, the cells also can be expanded and transplanted as a monolayer or multilayer cell sheet (Guo et al. 2020). This method of cell delivery reduces the need for the transplanted cells to migrate long distance to home into the injured myocardium to participate in the repair process (Meluzin et al. 2006; Nogueira et al. 2009). Kanelidis et al. in their metaanalysis of preclinical and clinical studies found that intramyocardial injection of stem cells through catheter-based transendocardial stem cell injection provided more benefits than the intravenous route in terms of infarct size reduction and LVEF improvement (Kanelidis et al. 2017). A few clinical trials have examined the effects of different cell dosages for cardiac regeneration (Meluzin et al. 2006). Averse to the findings of Meluzín et al. that found that the high-dose treatment is more effective in treating MI, Losordo et al. reported that the low-dose cell therapy gave better results in a patient with RA. The discrepancy could be due to their use in different cardiac pathologies and diverging routes of cell administration. For example, more cells are needed when using the intravenous and intracoronary routes of cell delivery compared to intramyocardial transplantation due to lower cell engraftment and vast distribution in nontargeted organs besides massive cell death post engraftment. Preconditioning protocols are being developed and optimized to support donor cell survival (Haider and Ashraf 2012) via various mechanisms including induction of pro-survival microRNAs (Kim et al. 2009). Lastly, stem cells isolated from different individuals might vary in cellular composition, characteristics, and functionality. These variations contributed to the outcome disparity. Quality of the cell preparation remains an ultimate determinant of the outcome and success of cell-based therapy (Haider 2017). It is well known that human stem cells become less viable and dysfunctional with age and individuals with chronic diseases (Efimenko et al. 2015; Shahid and Haider 2016). Such conditions may alter the effectiveness of stem cell therapy. Therefore, the efficacy of autologous stem cells used in previous studies to treat older patients with heart disease may be inconsistent. Thus, the considerations mentioned earlier should be taken into account while interpreting the data of previous studies and designing future research for heart diseases besides developing strategies to restore reparability characteristics of the aging cells (Igura et al. 2011).

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Future Perspectives Supported by the advancement and introduction of new technology, various pharmacologic and genetic strategies are being developed to improve the currently available cell-based therapy for heart disease. These efforts include combination with gene therapy and biomaterial as well as application of stem cell-derived extracellular vesicles.

Combination with Gene Therapy Recent advancements in high-precision genome-engineering CRISPR/Cas9 system have allowed the researchers to design gene therapy to up- or downregulate the expression of cardiac, vascular, or immune system-relevant gene(s) which is/are abnormally expressed and permanently correct disease-causing mutations in the adult cardiomyocytes. However, despite the tremendous promise, most studies encountered difficulties translating gene therapy to the clinical setting. For example, the recent CUPID2 trial that applied gene therapy (AAV1/ SERCA2a) showed low efficacy in heart failure patients despite having encouraging results during the experimental preclinical studies (Greenberg et al. 2016). Discouraging results were also reported in the STOP-HF trial (Chung et al. 2015). Other gene-based innovations such as lineage reprogramming sound promising in theory. However, such intervention could introduce ectopic cardiomyocyte formation, and the activity of the transferred genes is not wellestablished. Thus, a novel combinatorial approach of gene therapy and stem cell treatment may prove to be most feasible and efficacious (Haider et al. 2011). The combination therapy offers the advantage that cells can be genetically engineered ex vivo before transplantation, offering a safer alternative and more precise control of gene expression than gene therapy alone (Jiang et al. 2006; Haider et al. 2008). Another potential strategy involves genetically engineered cell grafting using a mixture of physiologically relevant cell types, including stem cells, cardiomyocytes, and neuronal, vascular, and immune cells (Hosseini et al. 2018). These cells serve as vehicles to deliver the gene(s) or even microRNAs of interest to the heart and provide a more comprehensive regenerative strategy compared to each cell type without genetic modulation (Kim et al. 2012a, b).

Exosomes Exosomes are membrane-bound vesicles produced by cells and contain a variety of factors, including nucleic acids, lipids, and proteins which are considered to be primarily responsible for intercellular transfer of bioactive molecules. More recently, exosomes-based research has gained intense interest as a fast-emerging cell-free therapy approach for many diseases, including heart disease (Haider and Aslam 2018).

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The systematic review and meta-analysis conducted by Zhang et al. found that MSCssecreted exosomes improved the heart function in experimental animal model of myocardial ischemia-reperfusion injury (Zhang et al. 2016). Similarly, CSC-secreted exosomes were also reported to promote heart regeneration in experimental mouse model of MI (Ibrahim et al. 2014). Adamiak et al. reported that induced pluripotent stem cell (iPSC)-secreted exosomes are more potent than iPSCs in myocardial reparability in vivo (Adamiak et al. 2018). Besides being equal or better than the original cells in cardiac protection, the use of exosomes is also safer by mitigating the risk of potential adverse effects observed in cell therapy, such as host immune rejection and tumor formation (Haider and Aramini 2020). These exciting results await future comprehensive clinical evaluations as most of the current findings are still at the preclinical stage.

Biomaterials Retention and engraftment of transplanted cells at the injury site are crucial to maintaining the therapeutic effects. Unfortunately, regardless of the route of administration, the majority of the cells do not reach (intravenous and intracoronary injection) or engraft (intramyocardial injection) at the damaged myocardium. The poor cell engraftment limited the therapeutic benefits of stem cell therapy. Therefore, scaffolds, either in the form of nanofibers, cell sheets, biodegradable hydrogels, or decellularized tissues, have been utilized to deliver and promote cell retention in the infarcted myocardium. The scaffolds can be made from various biodegradable biomaterials, such as the natural alginate, collagen, and Matrigel, as well as synthetic polycaprolactone (PCL) and poly-L-lactic acid (PLLA) (Reis et al. 2016). An ideal scaffold should be biodegradable and biocompatible having appropriate thickness and could provide mechanical support, besides being easy to handle and permitting precise placement. Currently, research is focused to develop an ideal scaffold for cardiac tissue engineering.

Conclusion The contemporary treatment options show limited success for the treatment of heart diseases. Stem cell-based therapy has emerged as a novel approach. Even though the quality and quantity of the available data, especially the clinical data, are still limited, the results reported thus far are promising. Further in-depth mechanistic studies are warranted in the future to understand how stem cells work and to optimize the treatment protocol to improve the safety and efficacy of stem cell-based therapy. A combination of stem cells with gene therapy and biomaterial might be able to create a synergistic effect and is an area that needs to be further explored. Also, more efforts are needed to develop the cell-free exosome therapy to ensure its safety and efficacy before being used clinically as an alternative for stem cell therapy.

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Cross-References ▶ Augmenting Mesenchymal Stem Cell-Based Therapy of the Infarcted Myocardium with Statins ▶ Considerations for Clinical Use of Mesenchymal Stromal Cells ▶ Mesenchymal Stem Cells for Cardiac Repair ▶ Molecular Signature of Stem Cells Undergoing Cardiomyogenic Differentiation ▶ Stem Cell Applications in Cardiac Tissue Regeneration ▶ Therapeutic Uses of Stem Cells for Heart Failure: Hype or Hope Acknowledgments This work was supported by research grants (FF-2019-450/1 and FF-2020038) from the Faculty of Medicine, Universiti Kebangsaan Malaysia.

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Tse H-F, Thambar S, Kwong Y-L, Rowlings P, Bellamy G, McCrohon J et al (2007) Prospective randomized trial of direct endomyocardial implantation of bone marrow cells for treatment of severe coronary artery diseases (PROTECT-CAD trial). Eur Heart J 28(24):2998–3005 van Ramshorst J, Bax JJ, Beeres SLMA, Dibbets-Schneider P, Roes SD, Stokkel MPM et al (2009) Intramyocardial bone marrow cell injection for chronic myocardial ischemia: a randomized controlled trial. JAMA 301(19):1997–2004 Vicario J, Campos C, Piva J, Faccio F, Gerardo L, Becker C et al (2004) Transcoronary sinus administration of autologous bone marrow in patients with chronic refractory stable angina: phase 1. Cardiovasc Radiat Med 5(2):71–76 Wang S, Cui J, Peng W, Lu M (2010) Intracoronary autologous CD34+ stem cell therapy for intractable angina. Cardiology 117(2):140–147 Wang Y, Chen X, Cao W, Shi Y (2014) Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol 15(11):1009 Ward MR, Abadeh A, Connelly KA (2018) Concise review: rational use of mesenchymal stem cells in the treatment of ischemic heart disease. Stem Cells Transl Med 7(7):543–550 Wojciech W, Tomasz J, Aleksandra M-W, Zofia P, Mirosław M, Wojciech R et al (2017) Effects of transendocardial delivery of bone marrow-derived CD133+ cells on left ventricle perfusion and function in patients with refractory angina. Circ Res 120(4):670–680 Wollert KC, Meyer GP, Lotz J, Ringes Lichtenberg S, Lippolt P, Breidenbach C et al (2004) Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364(9429):141–148 Wollert KC, Meyer GP, Müller-Ehmsen J, Tschöpe C, Bonarjee V, Larsen AI et al (2017) Intracoronary autologous bone marrow cell transfer after myocardial infarction: the BOOST-2 randomised placebo-controlled clinical trial. Eur Heart J 38(39):2936–2943 Yazdanyar A, Newman AB (2009) The burden of cardiovascular disease in the elderly: morbidity, mortality, and costs. Clin Geriatr Med 25(4):563–577 Yu B, Kim HW, Gong M, Wang J, Millard RW, Wang Y et al (2015) Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection. Int J Cardiol 182:349–360 Zhang H, Xiang M, Meng D, Sun N, Chen S (2016) Inhibition of myocardial ischemia/ reperfusion injury by exosomes secreted from mesenchymal stem cells. Stem Cells Int 2016:4328362 Zhao XF, Xu Y, Zhu ZY, Gao CY, Shi YN (2015) Clinical observation of umbilical cord mesenchymal stem cell treatment of severe systolic heart failure. Genet Mol Res 14(2):3010–3017

Mesenchymal Stem Cells for Cardiac Repair The Clinical Perspective

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Abdullah Murhaf Al-Khani, Mohamed Abdelghafour Khalifa, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BM-Derived MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and Characterization of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important Characteristics of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation Potential of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trophic Functions of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory Characters of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical Studies with MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small Experimental Animal Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Large Animal Experimental Models: Translational Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs in the Clinical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cardiovascular diseases (CVDs), particularly acute and chronic ischemic heart disease (IHD), are the primary cause of morbidity and mortality worldwide. The contemporary pharmacological and invasive revascularization strategies, i.e., percutaneous coronary angiography (PCA) and coronary artery bypass grafting (CABG), reduce mortality and could only provide symptomatic relief. Cell-based therapy has emerged as a novel breakthrough strategy to ensure angiomyogenic repair of the ischemically damaged heart via the generation of neomyocytes and biological bypassing to restore regional blood flow. In this regard, bone marrow (BM)-derived mesenchymal stem cells (MSCs) have shown promise and progressed to advanced phases of clinical assessment. MSCs are one of the wellA. M. Al-Khani · M. A. Khalifa Sulaiman AlRajhi Medical School, Al Bukairiyah, Kingdom of Saudi Arabia K. H. Haider (*) Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_11

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studied and characterized cell types in vitro and various small experimental animal models and during the translational animal models for safety and reparability. They are currently the most used cells for cell-based cardiovascular therapy with excellent safety profiles, immunomodulatory properties, paracrine action, and differentiation potential. With overwhelming success, researchers have advanced their efforts to bring MSCs a step closer to their eventual routine use in the clinics. This chapter summarizes the advancement of MSCs from in vitro characterization to the clinical phase and discusses their future perspective. Keywords

Bone marrow · Cell therapy · Heart failure · Infarction · In vitro · Ischemia · Large animal models · Mesenchymal · MSCs · Small animal models · Stem cells Abbreviations

6-MWT AMI Ang-1 BM Brdu CFU-F CVDs eGFP FGF GLP-1 Glut HSCs IGF-1 IHD ISCT LAD LinLV LVEF MDCT MHC miR MSCs M-SPECT NPY RBCs SAE TGF-b TUNEL

Six-minute walk test Acute myocardial infarction Angiopoietin-1 Bone marrow 5-Bromodeoxyuridine Colony-forming units-fibroblastic Cardiovascular diseases Enhanced green fluorescent protein Fibroblast growth factor Glucagon-like peptide-1 Glucose transporter Hematopoietic stem cells Insulin-like growth factor-1 Ischemic heart disease International Society for Cell Therapy Left anterior descending Lineage negative Left ventricle Left ventricular ejection fraction Multi-detector computed tomography Major histocompatibility complex MicroRNA Mesenchymal stem cells Myocardial single-photon emission tomography Neuropeptide Y Red blood cells Serious adverse events Transforming growth factor-b Terminal deoxynucleotidyl transferase dUTP nick-end labeling

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VEGF WNT

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Vascular endothelial growth factor Homologous of wingless and Int-1

Introduction Cardiovascular diseases (CVDs) are the leading cause of mortality globally (WHO 2017) as they account for nearly 836,546 deaths in the USA alone, which is equivalent to one out of every three deaths (AHA-2018 Statistics at a glance). Among the CVDs, ischemic heart disease (IHD) is a major clinical challenge that causes morbidity and mortality in patients in acute and chronic forms. Thrombotic coronary occlusion due to atherosclerotic rupture leads to acute myocardial infarction (AMI) due to compromised blood supply to the affected region of the heart, thus causing massive cardiomyocyte death (Falk et al. 1995). Although the number of surviving patients, their quality of life post-AMI, and the rate of rehospitalization due to recurrence of AMI episodes improved due to advancements in contemporary revascularization techniques and better pharmacological management, these interventions only provide symptomatic relief to the patients. The infarcted myocardium undergoes a series of detrimental events as part of the intrinsic repair process. This involves forming a noncontractile cicatricle tissue to replace the damaged myocardium besides undergoing geometrical changes in the myocardium to accommodate altered pressure-volume needs and overstretching of viable cardiomyocytes, especially in the peri-infarct region to sustain near-normal contractile function (Taggart 2012; Maron et al. 2006). Standard heart failure medical therapies suffer from a lack of potential to replenish the dead cardiomyocytes. Hence, the emerging strategy of cell-based therapy for myocardial repair and regeneration is revolutionary and exploits the exclusive reparability and differentiation potential of stem cells into structurally and functionally competent neomyocytes and neovascular structures to restore the lost myocardial function (Kwon et al. 2010; Haider et al. 2010a). Given their optimal characteristics and ease of availability, bone marrow (BM)-derived mesenchymal stem cells (MSCs) indeed provide an attractive source of cells for heart cell therapy. This chapter gives an overview of the progress of BM-derived MSCs from in vitro (Table 1) to preclinical studies in small and large animal models focusing on their safety and efficacy leading to the published randomized placebo-controlled clinical trials in the human subjects.

BM-Derived MSCs BM comprises two main lineages of stem/progenitor cells, i.e., hematopoietic stem cells (HSCs) and MSCs (Haider and Ashraf 2005). MSCs were first described by Friedenstein et al. in 1968 as the fibroblast-like colony-forming units (Friedenstein et al. 1968) and later substantiated and characterized by many other research groups (Bobis et al. 2006; Deng et al. 2008; Luu et al. 2007; Bhat et al. 2021). Such characterization is of great significance to ensure uniformity of cell characteristic prepared from various labs (Stroncek et al. 2020) and to ensure quality preparation

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Table 1 Summary of some of the studies reporting in vitro differentiation of BM-derived MSCs Author/year Wakitani et al. (1995)

Cell type Rat BM-derived MSCs

Method of differentiation 5-Azacytidin treatment

Makino et al. (1999)

Murine BM-derived MSCs

5-Azacytidin treatment

Rangappa et al. (2003)

Human MSCs

Human MSCs and human cardiomyocytes were mixed at a 1:1 ratio in smooth muscle 2 media

Antonitsis et al. (2007)

Human BM-derived MSCs

Second passaged cells were treated with 10 microM 5-azacytidine for 24 h

Main findings 5-Azacytidine treatment at 24 h after seeding of twice-passaged MSCs in vitro culture. After an exposure of 24 h, long, multinucleated myotubes developed in some of the dishes on days 7–11 after treatment Nearly 30% of the cells connected with adjoining cells after 1 week, formed myotube-like structures, began spontaneously beating after 2 weeks, and beat synchronously after 3 weeks. Expressed ANP and BNP, stained positive for anti-myosin, anti-desmin, and antiactinin. Revealed a cardiomyocyte-like ultrastructure. Also expressed MEF-2A and MEF-2D Differentiated hMSCs from the coculture expressed myosin heavy chain, beta-actin, and cTnT detected by PCR. Immunostaining also showed myosin heavy chain and cTnT. Only beta-actin expression was observed in the hMSCs incubated with conditioned media without serum MSCs treated with 5-azacytidine became stick-like morphology, connecting with adjoining cells forming myotube-like structures, running in a parallel fashion. Immunohistochemically positive for myosin (continued)

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Table 1 (continued) Author/year

Cell type

Method of differentiation

Ramkisoensing et al. (2012)

Human adipose tissue MSCs and amniotic membrane MSCs with or without Cx-43

Ten-day coculture with neonatal rat ventricular cardiomyocytes

Hou et al. (2013)

Rat BM-derived MSCs

BMP-2 and 5-azacytidine treatment

Liu et al. (2013)

Rat BM-derived MScs

Combined treatment with 5-AZA (10 μmol/L), Ang-II (0.1 μmol/L), PFT-α (20 μmol/L), and BMP-2 (10 μg/L)

Main findings heavy chain and vimentin. The mRNAs of alpha-cardiac actin, beta-myosin heavy chain, and cTn-T were also expressed Functional cardiomyogenic differentiation, based on action potential recordings, occurred only in control fetal AM hMSCs. Cx45 overexpression in Cx43 knockdown fetal AM hMSCs restored their ability to undergo cardiomyogenesis (1.6%
0.4%, n ¼ 2500) in coculture with neonatal myocytes. Gap junctional coupling is required for differentiation of fetal AM hMSCs into functional CMs Combined treatment with BMP-2 and 5-AZA significantly improved the cardiac differentiation with fewer cell damage effects as compared to either of the them alone, and combined treatment was safer and effective method of induction in vitro. Expression of cTnI and Cx-43 was used as differentiation markers Development of cardiomyocyte-like cells expressing cTnT, cTnI, and Cx43 suggesting that the combination of inductors improved rate of differentiation. Total potassium current level (continued)

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Table 1 (continued) Author/year

Szaraz et al. (2017)

Cell type

BM-derived MSCs and UC-perivascular cell MSCs

Method of differentiation

Coculture on feeder layer

Main findings and calcium transient in PFT-α cardiomyocytelike cells was also higher in the differentiated cells Increased expression of cardiomyocyte markers (i.e., MEF2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43) showed aggregate-based contracting cells in vitro

ANP atrial natriuretic peptide, BNP brain natriuretic peptide, BM bone marrow, CMCs cardiomyocytes, cTnT cardiac troponin T, Cx-43 connexin 43, MEF2c myocyte enhancer factor 2C, FACS fluorescence-activated cell sorting, MSCs mesenchymal stem cells.

for clinical use of the cells (Trivedi et al. 2019). In the published data, MSCs have also been reported as mesenchymal stromal cells, mesenchymal progenitor cells, multipotent mesenchymal stromal cells, BM stromal cells, BM-derived MSCs, multipotent stromal cells, mesenchymal precursor cells, and medicinal signaling cells (Caplan 1991; Bobis et al. 2006; von’t Hof et al. 2007; Deng et al. 2008; Samsonraj et al. 2017). MSCs are a heterogeneous group of cells that constitute only 0.001–0.01% of the stem/progenitor population in the BM (Haider 2006; Wilson et al. 2019). They, therefore, necessitate extensive amplification in vitro culture to achieve a sufficient number for cell therapy applications (Lennon and Caplan 2006). Although MSCs were first isolated from the BM more than 50 years ago (Friedenstein et al. 1970), there are no unique surface markers for their definite identification from other cells (Haider 2018). More recently, the International Society for Cell Therapy (ISCT) has suggested standardized criteria to define human MSCs, which includes (1) adherence to the plastic surface under standard culture conditions; (2) the expression of CD73, CD90, and CD105 and lack of CD34, CD45, HLA-DR, CD14 or CD11b, and CD79a or CD19 membrane surface molecules; and (3) tri-lineage differentiation potential to adopt osteoblasts, adipocytes, and chondroblasts under a defined set of culture conditions in vitro. The ISCT standardized norms for the characterization of MSCs have been instrumental in removing the inconsistencies regarding the nomenclature as well as biological characteristics of MSCs. It is important to mention that MSCs from different species and even from various tissue sources cultured under a different set of conditions may differ in the expression of surface markers (Boxall and Jones 2012; Jones and Schäfer 2015) as well as in the number of isolated cells (Sullivan et al. 2015; Yoshimura et al. 2007) and their efficacy (Shariatzadeh et al. 2019). Moreover, the innate expression levels of a set of surface markers are not a guarantee of MSC homogeneity. Besides other cell types, MSCs are an integral part of the HSC niche in the BM and essentially instrumental in offering a unique microenvironment for the HSCs

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(Morrison and Scadden 2014). Together with the endothelial cells and megakaryocytes, MSCs provide proximity to the HSCs and contribute to the maintenance of niche homeostasis by providing instructive cues for their quiescence and functional activity (Schepers et al. 2015; Asada et al. 2017). These MSCs have been identified as CD45-Nestin+, contain all CFU-fibroblastic activity associated with HSCs, and respond to adrenergic stimulation (Méndez-Ferrer et al. 2010).

Isolation and Characterization of MSCs Other than the BM, MSCs have been isolated from many other adult- as well as fetus-associated tissues, including adipose tissue, peripheral blood, lung, marrow spaces of a long bone, synovial fluids, muscle, placenta, umbilical cord, cord blood, periodontal ligaments, and dental pulp (Aust et al. 2004; Smiler et al. 2008; He et al. 2007; Griffiths et al. 2005; Tuli et al. 2003; Fan et al. 2009; Gay et al. 2007; Jackson et al. 2010; Anker et al. 2004; Miao et al. 2006; Corrao et al. 2013; Ong et al. 2014; Erices et al. 2000; Mareschi et al. 2001; Camilleri et al. 2016) (Fig. 1).

Fig. 1 Some of the adult- and fetus-associated tissue sources of MSCs used in experimental or clinical studies

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The lineage-tracing studies strongly propose that progenitor cells of the MSCs come from around the blood vessels (capillaries, arteries, and veins), thus pointing their perivascular origin (Seo et al. 2004; Corselli et al. 2010). Besides other parameters, the quality of MSC preparation is determined by their isolation procedure and expansion in the culture conditions (Haider 2018). The three steps needed for isolation and purification of MSCs from the BM include their separation from the nonnucleated RBCs by density gradient centrifugation, adherence to the plastic surface, and removal of monocytes by trypsinization. Generally, mechanical and enzymatic dissociation of the tissue has their respective advantages, but combining both of the approaches may enhance the yield of MSCs as compared to the enzymatic digestion alone as it results in loss of extracellular matrix that increases their time of adherence and low yield (Mushahary et al. 2018). Expansion of plastic adherence is the most employed way of obtaining and expanding the MSCs. Given their 3D habitat in the niche, the 2D in vitro culture expansion of MSCs may lead to loss of their progenitor potency and function. A paradigm shift in MSC culture in vitro uses 3D culture conditions that closely mimic their natural habitat (Hoch and Leach 2014). Besides improving their proliferation rate, paracrine activity, and differentiation potential, more recent studies have shown that 3D culture conditions promote the expression of pluripotency genes (Zhou et al. 2017). Elucidating the underlying mechanism, it has been attributed to the relaxation of the cytoskeleton due to more conducive culture conditions. The commonly employed 3D methods include the hanging-drop approach, scaffold-free method, spin or rotate wall vessels, and fabricated membrane culture methods (Bartosh et al. 2010; Zhang et al. 2015; Miyagawa et al. 2011). As discussed earlier, MSCs are generally characterized by their capacity to form colonies, renew themselves, express surface markers, and differentiate into multilineages (Friedenstein et al. 1974). The MSC colonies show heterogeneous morphological characteristics ranging from fibroblastoid- to spindle-shaped or from large-flattened to small-round cells. The first recognized multipotent stromal precursor cells from the BM were the colony-forming unit-fibroblastic (CFU-F), which are mainly composed of primary BM-derived MSCs. After further proliferative expansion in culture, they constitute MSCs/stromal cells, and in vitro they give rise to colonies during their initial growth (Pittenger et al. 1999). CFU-F is the efficiency of self-renewal assessed by the rate of colony formation that is a routinely employed standard approach to characterize MSCs. Additionally, research labs from all over the world have diverse sets of antigens for characterization, and there is no consistency in the use of cell surface antigens for the isolation of MSCs. There is no one universal marker that precisely identifies MSCs. This divergence in surface marker expression besides extremely low-frequency presence in the tissues renders it challenging to identify MSCs in vivo. From among the wide array of surface antigens expressed by MSCs, CD105, CD73, and CD90 are reckoned as the primary markers, which are expressed on more than 95% of MSCs, while the expression of CD105, CD90, and CD73 is not completely specific to undifferentiated multipotent MSCs as some of these markers are also expressed by vascular cells, smooth muscle cells, and mature stromal cells

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such as fibroblasts (Dominici et al. 2006). On the contrary, cultured MSCs do not express CD45, CD34, CD14, CD11b, CD19, and HLA-DR. Additionally, MSCs expressing STRO-1, CD146, SSEA-4, CD271 (NGFR), and MSC antigen 1 (MSCA-1) have been identified but with little significance as markers of identification and purification (Andersen et al. 2011; Jones et al. 2006). Nestin, a neural stem cell marker, has also been reported as a selective marker for BM-MSCs (Nombela-Arrieta et al. 2011; Méndez-Ferrer et al. 2009, 2010).

Important Characteristics of MSCs Differentiation Potential of MSCs Friedenstein was the first researcher who described that MSCs could differentiate into mesodermally derived cells (Friedenstein et al. 1974). Henceforth, it was shown that MSCs can cross lineage restriction and transform into morph functionally competent osteogenic, adipogenic, chondrogenic, vasculogenic, and myogenic phenotypes (Piersma et al. 1985; Caplan 1986; Wakitani et al. 1995; Kopen et al. 1999). This inherent property of multipotentiality may be accentuated in the presence of various factors such as ascorbic acid, dexamethasone, bone morphogenetic proteins (BMPs), WNTs, fibroblast growth factors (FGFs) besides other heparin sulfate-sensitive morphogens, and growth factors (Bhakta et al. 2012; Bramono et al. 2012; Dombrowski et al. 2013; Helledie et al. 2012; Ling et al. 2010; Teplyuk et al. 2009). While dexamethasone, indomethacin, insulin, and isobutylmethylxanthine generally support adipogenic differentiation (Scott et al. 2011), the presence of ascorbate, insulin, transferrin, selenic acid, and TGF-β promotes chondrogenesis (Johnstone et al. 1998; Mackay et al. 1998; Barry et al. 2001). Similarly, treatment with 5-azacytidine induces the cardiomyogenic differentiation of BM-derived MSC in vitro (Makino et al. 1999; Fukuda 2001; Antonitsis et al. 2007; Ullah et al. 2021). Please refer to summary of studies reporting in vitro differentiation of MSCs (Table 1). Other factors included in the culture medium, i.e., antibiotics, serum, and growth supplements, to support cell stability and proliferation may significantly interfere with the undifferentiated expansion of MSCs (Riis et al. 2016; Pountos et al. 2014; Gharibi and Hughes 2012; Lee et al. 2001). On the same note, cellular and subcellular level preconditioning approaches have been developed to promote survival signaling, cell proliferation, differentiation, and paracrine activity of the preconditioned cells (Haider et al. 2010b; Lu et al. 2010, 2012; Suzuki et al. 2010; Haider and Ashraf 2010; Kim et al. 2009; Afzal et al. 2010). More recently, the focus has shifted to the use of pharmaceutical-grade human plasma derivatives and platelet lysate to promote MSC proliferation (Diez et al. 2015; Haider 2017). In vitro expansion of MSCs is also imperative during the use of autologous cell therapy due to the limited availability from the tissue source, especially from the aging patients as aging impairs their proliferation and differentiation potential (Kretlow et al. 2008).

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Haider and colleagues have already reported that the doubling time of BM-derived MSCs from aging rats (24 months of age) was significantly higher than the young donor MSCs (Jiang et al. 2007, 2008; Haider et al. 2008). The harvest of MSCs was meager, and it took a significantly more extended time for the aging BM cells to adhere to the plastic surface. Moreover, when the cells got adhered to the plastic surface, they were stickier and required longer time to get dislodged from the tissue culture plates during trypsinization. In an attempt to support their proliferation in vitro culture, Igura and colleagues have reported that transgenic overexpression of neuropeptide Y (NPY) receptors and subsequent treatment with NPY5 significantly enhances MSCs’ rate of proliferation (Igura et al. 2011). Other strategies to enhance MSC proliferation include concomitant transgenic overexpression of Ang-1 and Akt with downstream involvement of miR-143, a critical regulator of cell proliferation (Lai et al. 2012). One needs to understand that culture-expanded colonies of MSCs show limited differentiation potential that ranges from bi-potency (osteogenic and chondrogenic lineages) to monopotency (Muraglia et al. 2000; Pevsner-Fischer et al. 2011; Russell et al. 2010). The reason for this variation is not well understood, but it may be due to the epigenetic adaptations to the culture conditions.

Trophic Functions of MSCs Various stem cell research groups now support the “paracrine hypothesis” and attribute the therapeutic benefits of MSCs with their capability to release trophic factors with or without their ability to undergo differentiation (Lei and Haider 2017). MSCs are also capable of generating a reparative microenvironment with their paracrine secretions rich in bioactive molecules, including chemokines, cytokines, morphogens, growth factors, microvesicles, and microRNAs (Kordelas et al. 2014; Lai et al. 2010; Amable et al. 2014; Eirin et al. 2014, 2016; Haider and Aramini 2020). Besides vasculogenic and myogenic differentiation potential and immuneregulatory properties, paracrine activity is an essential characteristic of MSCs that tips them as one of the choice cells for cell-based therapy. Various research groups have provided evidence that the MSCs can maintain the growth, viability, and multipotent status of HSCs in long-term cocultures which lacked growth factor supplementation by secreting these trophic factors (Spees et al. 2016; Dexter et al. 1977; Dexter and Spooncer 1987; Queensberry et al. 1989; Wu et al. 2013). Although the paracrine hypothesis has gained popularity and acceptance as one of the important underlying mechanisms of cell therapy and has led to the novel strategy of cell-free therapy (Haider and Aziz 2017), there is no comprehensive uniform list of paracrine factors released from MSCs. The paracrine activity of MSCs is sensitive to the signals from their microenvironment. Hence, the composition of the paracrine secretions is influenced by a multitude of factors, including physical factors, i.e., hypoxia, stretch, pulsed focused ultrasound, electrical stimulation, and heat-shock treatment (Kusuma et al. 2017; Lei and Haider 2017; Antebi et al. 2018; Razavi et al. 2020; Parate et al. 2020), and chemical cues, i.e., vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and IL-6

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(Jeanmonod et al. 2018; Wei et al. 2013). Haider and colleagues have already reported that exposure to intermittent cycles of anoxia-reperfusion during preconditioning of the cells significantly altered their paracrine activity (Kim et al. 2009, 2012a). A marked upregulation of various growth factors and HIF-1α-related miRs including miR-107 and miR-210 in the preconditioned cells was observed (Kim et al. 2012b). Besides physical and chemical manipulation, MSCs have also been genetically modulated to accentuate their paracrine activity (Jiang et al. 2006; Haider et al. 2008; Ahmed et al. 2010; Haider et al. 2011).

Immunomodulatory Characters of MSCs Bartholomew and colleagues were the first to describe the ability of allogenic MSCs to modulate immune responses in a baboon model of skin allograft (Bartholomew et al. 2002). Immunomodulatory characters of MSCs may be due to their hypoimmunogenic nature, ability to alter the T-cell response, and immunosuppression of the local microenvironment while modulating angiogenesis, apoptosis, and cell proliferation (Atoui and Chiu 2012; Faiella and Atoui 2016; Hong et al. 2012; Marfy-Smith and Clarkin 2017). Although the immune-regulatory properties of MSCs remain less well defined, the general perception is that MSCs can modulate immune cells’ functions by cell-to-cell contact and by secretory mechanisms involving the paracrine release of bioactive molecule factors. Moreover, MSCs express low levels of major histocompatibility complex (MHC) class I and lack MHC class II and co-stimulatory molecule B7 and CD40 ligand (Devine and Hoffman 2000; Majumdar et al. 2003; Tse et al. 2003). They can also interfere with the normal B-cell function by the T-cell suppression (Castro-Manrreza and Montesinos 2015). Besides their anti-apoptotic and anti-inflammatory functions, MSCs interact with tumor cells via paracrine signaling to increase the risk of metastasis (Costanza et al. 2017; Lacerda et al. 2015; Ye et al. 2012; Ridge et al. 2017).

Preclinical Studies with MSCs Small Experimental Animal Studies MSCs are genetically stable and less susceptible to malignant transformation (Izadpanah et al. 2006). They have been extensively characterized in various small and large experimental animal models for safety and reparability. Since the early report of heart cell therapy using lineage-negative (Lin-) BMCs expressing enhanced green fluorescent protein (eGFP) in a mice model of coronary artery ligation, it was observed that the transplanted cells repopulated the infarcted myocardium with neomyocytes by 9 days after treatment. The neomyocytes were also interspersed by vascular structures, a which contributed to the recovery of global cardiac function (Orlic et al. 2001). Similar observations were later reported in a study which used human BM-derived MSCs for transplantation in an adult

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murine heart (Toma et al. 2002). These encouraging data lead to a plethora of experimental studies in vitro as well as in the preclinical settings to support the safety and effectiveness of BM-derived MSCs for myocardial repair and paved the way for their further investigations in the human patients. A direct comparison of BM-derived MSCs and MNCs in a porcine model of chronic IHD showed superiority of the former over the latter cells in terms of improvement of systolic function (van der Spoel et al. 2015). A meta-analysis of the translational studies shows that BM-derived MSC-based cell therapy is safe and effective in preserving ischemic heart function including LVEF (van der Spoel et al. 2011). The proposed mechanism is multifactorial and involves stimulation of angiogenesis and neovascularization (Psaltis et al. 2008; Haider et al. 2008), neomyogenesis (Nagaya et al. 2004; Haider 2006), paracrine effects (Mirotsou et al. 2007), reduction of fibrosis (Molina et al. 2009; Jin et al. 2020), immunomodulation (Van den Akker et al. 2013; Hamid and Prabhu 2017), and stimulation of endogenous cardiac stem cells (CSCs) to proliferate and participate in the repair process (Hatzistergos et al. 2010; Table 2).

Large Animal Experimental Models: Translational Studies Large experimental animal studies are considered a critical step forward in establishing novel treatment approaches for diseases. The data generated from these studies are more relevant for translation to humans and hence greatly influence the advancement of novel treatments to the clinical phase of assessment. Given that stem cell-based therapy has already entered into the clinical phase of evaluation for cardiovascular applications, the support from the large experimental animal models is of utmost significance in translating the continuum of safety and efficacy data for a rationale designing the clinical trials (Harding et al. 2013). However, the need for large animal models has been generally ignored by the researchers due to their high cost, complexity, labor-intensive nature, and less suitability for the mechanistic understanding of cell-based therapy. Amid these challenges, which have restricted them to less than optimal usage to treat specific cardiac pathologies, the debate for an ideal translational model for modeling of cardiac pathologies continues unabated. However, as a fundamental principle, the body-to-heart weight ratio of the experimental animal should be comparable to that of humans to yield reliable simulation besides giving due consideration to other factors such as age appropriateness, etc. (Milani-Nejad and Janssen 2014; Haider 2018). A wide array of large experimental animal models, i.e., pigs, dogs, sheep, monkeys, etc., have been developed to model CVDs in general and assess stem cells’ safety and regenerative potential. However, experimental pig models of myocardial injury have been preferred over the other models and are primarily classified as open and closed-chest models with their respective advantages and limitations (Munz et al. 2011). Some of the typical translational studies using stem cells for myocardial repair and regeneration and their outcome have been summarized in Table 3.

Cell type Allogenic BM-MSCs with or without VEGF overexpression (VEFGMSCs)

Allogeneic

Author/year Wang et al. (2006)

Noiseux et al. (2006)

Intramyocardial injection

Mode of delivery Intramyocardial injection of VEGF MSCs and cytokine therapy

12

Sample size 48

AMI

Model type AMI

Mice

Animal Mice

Table 2 Summary of preclinical trials utilizing BM-derived MSCs in the small animal models of heart diseases

Mesenchymal Stem Cells for Cardiac Repair (continued)

Main findings Extensive regeneration and survival of VEGFMSCs in and around the infarcted heart. Blood vessel density significantly increased in VEGFMSCs as compared to other groups in periinfarct area. Fibrosis was significantly reduced with improved LV contractile function. LV systolic and diastolic functions were well preserved in VEGFMSCs as indicated by +dP/dt, -dP/dt, and Tau (glantz) MSC engraftment within infarcted myocardium was transient but significantly enhanced by Akt-MSCs fusion with CMs and was observed but was infrequent. Also a low rate of cardiomyogenic differentiation of MSCs was observed. MSC-Akt decreased infarct size at 3 days and restored early cardiac function. These observations confirmed that paracrine mechanisms mediated by MSC are responsible for CM survival and Akt could alter the secretion of various cytokines and growth factors

10 281

Cell type Syngeneic

Allogeneic

Author/year Boomsma et al. (2007)

Derval et al. (2008)

Table 2 (continued)

Patch system

Mode of delivery Intravenous injection

107

Sample size 25

Chronic

Model type AMI

Murinae

Animal Mice

Main findings Two weeks after coronary occlusion, there was a significant drop in LV systolic pressure, dP/dt (max), dP/dt(min), ESPVR, and E (max) and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSCs 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size or end-diastolic volume. Injected MSCs pre-labeled with fluorescent paramagnetic microspheres were observed scattered in non-infarcted myocardium. Flow cytometry of whole heart digests after intravenous injection of MSCs labeled with either fluorescent microspheres or fluorescent PKH26 dye showed that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart than those from non-infarcted controls This study showed that cell therapy improved angiogenesis and cell survival in the scar but only MSCs exhibited the capacity to invade the

282 A. M. Al-Khani et al.

Intramyocardial injection

First and fifth passage naïve BM MSCs

BM-MSCs overexpressing Mir-1, naïve, or null vector transduce MSCs

Jin et al. (2011)

Huang et al. (2013)

Intramyocardial injection

GCSF mobilization of BM cells

GCSF mobilization of and intramyocardial injection of BM-MPCs

Dai et al. (2008)

80

45

96

AMI

AMI

AMI

Mice

Mice

Mice

Mesenchymal Stem Cells for Cardiac Repair (continued)

scar. No evidence of myocardial or vascular regeneration from progenitor cells. Engraftment of the progenitors/unfractionated BMC mix increased repopulation and thickness of the scar LVEF and LVFS improved in MPC-treated animals as compared to control animals which received the medium. The infarcted area was also significantly smaller in MPC transplanted hearts than that in control hearts. MPC injection significantly promoted GFP+ cells homing from BM LacZ or β-gal in the hearts from the MI þ first passage MSC group was greater, leading to increased LV wall thickness, significant reduction in fibrotic area, and significantly improved LVEF than MI þ fifth passage BM-MSC group on week 6 after cell transplantation The transplanted MSCs were able to differentiate into cardiomyocytes in the infarcted zone. Cardiac function in the MSCs, MSCs-null, and MSCsmiR-1 groups was significantly improved compared to the PBS group (p < 0.01 or p < 0.001).

10 283

Cell type

Allogenic

Author/year

Liao et al. (2017)

Table 2 (continued)

Intravenous injection

Mode of delivery

54

Sample size

N/A safety study

Model type

Mice

Animal

However, most significant improvement highest rate of myocyte differentiation was observed in miR-I-expressing MSCs Clinical doses of BM-derived MSCs induced mild and reversible coagulation, which increased BM-derived MSC lung embolism and clearance. Anticoagulation treatment by heparin (400 U/kg) prevented BM-derived MSC-induced coagulation and the acute adverse effects of large-dose BM-derived MSC infusion efficiently. Heparin treatment decreased BM-derived MSC lung embolism and enhanced migration and maintenance of BM-derived MSCs to target organs in cell therapy. Based on an experimental colitis model, it was confirmed that heparin treatment enhanced the effect of BM-derived MSC therapy efficiently to reduce mortality, prevented weight loss, suppressed inflammation reaction, and alleviated tissue injury

Main findings

284 A. M. Al-Khani et al.

AD-MSCs

99mTc-exametazime labeled allogenic rat BM-MSCs

Syngeneic

Chen et al. (2020)

Barbash et al. (2003)

Davani et al. (2003)

Intramyocardial injection

Intra-left ventricular injection vs. intravenous injection

Tailorable hydrogel (ColT gel) or intramyocardial

16

39

150

AMI

2, 10, and 14 days after transient coronary artery occlusion

AMI

Rat

Rat

Mice

Mesenchymal Stem Cells for Cardiac Repair (continued)

The Col-Tgel created a suitable microenvironment for long-term retention of ADSCs in an ischemic area and enhanced their cardioprotective effects, significantly increased LVEF at 4 weeks after MI, and decreased LV end-systolic diameter but did not significantly decrease the LV end-diastolic diameter at 4 weeks after MI compared to PBS treatment Comparison of intra-left ventricle, right ventricle, and intravenous routes. Drastically low uptake of the intra-left ventricular injected cells in the lungs and high uptake in the heart compared to intravenous injected cells. Histological evidence of cells in the infarct and peri-infarct regions on day 7 after intra-LV injection of cells Thirty days after implantation, immunofluorescence studies showed engrafted cells expressing a smooth muscle phenotype (alpha SM actin+), as similarly observed in culture. Other engrafted cells lost their smooth muscle phenotype and acquired an endothelial phenotype (CD31+). Furthermore, vessel density was augmented in the MPC

10 285

Cell type

Syngeneic

Allogenic vs. syngeneic MSCs

Author/year

Nagaya et al. (2004)

Imanishi et al. (2008)

Table 2 (continued)

Intramyocardial injection

Intravenous injection

Mode of delivery

119

70

Sample size

AMI

AMI

Model type

Rat

Rat

Animal

Main findings group compared to the control group. After 30 days, echocardiography showed an improvement in LV performance in MPCs than the control group The engrafted MSCs were positive for cardiac markers: Desmin, cTn-T, and connexin43. Some of the transplanted MSCs were positive for von Willebrand factor and vascular structures. Capillary density increased after MSC transplantation. Infarct size was significantly smaller in MSC group than control group (24+/2 vs. 33 +/ 2%, P < 0.05). MSCs decreased LV end-diastolic pressure and increased LV maximum dP/dt (both P < 0.05 and control) Both syngeneic and allogeneic MSC transplantations were useful for AMI, increased VEGF, and blood vessel density. The donor MSCs disappeared rapidly but became a trigger of VEGF paracrine effect. One day after cell transplantation, transient increase in IL-1 beta and MCP-1 in recipient

286 A. M. Al-Khani et al.

Xenogeneic BM-MSCS or fibroblasts

Allogenic rat MSCs with or without IGF-1 overexpression (IGF-1MSC)

Allogeneic

Atoui et al. (2008)

Haider et al. (2008)

Enoki et al. (2010)

40

78

Intramyocardial injection

Intramyocardial injection

90

Intramyocardial injection

Chronic

LAD ligation//AMI

AMI

Rat

Rat

Rat

Mesenchymal Stem Cells for Cardiac Repair (continued)

hearts were enhanced in allogenic MSCs, with macrophage infiltration at the injection site Xenogeneic transplanted human BM-derived MSCs survived in the rat heart for more than 8 weeks without the use of immunosuppressants. The implanted MSCs significantly contributed to the improvement in ventricular function and attenuated LV remodeling. No immune cell infiltration, characteristic of immune rejection, was noted in MSC transplanted cells Elevated myocardial IGF-1, pAkt, and SDF-1 in IGF-1MSCs. Massive mobilization and homing of ckit+, MDR1+, CD31+, and CD34+ in IGF-1 MSCs. Extensive angiomyogenesis in the infarcted heart with significantly improved LVEF and LVFS One month after MI, rat hearts were injected with MCs in the presence or absence of 10 μg/ml IGF-1 with or without PI3K inhibitor, 5 μM LY294002. IGF-1 significantly increased engraftment of MSCs between 6 h and 3 days after transplantation associated with the increase in stromal cell-derived

10 287

Cell type

Allogenic MSC overexpressing mir-210 or preconditioned

Human cells (xenogenic engraftment)

Author/year

Kim et al. (2012a)

Armiñán et al. (2010)

Table 2 (continued)

Intramyocardial injection

Intramyocardial injection

Mode of delivery

90

48

Sample size

AMI

AMI

Model type

Rat

Rat

Animal

Main findings factor-1α in the infracted LV. IGF-1-MSCs significantly increased neovascularization and inhibited CM apoptosis 3 days and 1 month after MSC transplantation. This improved LV function 1 month after MSC transplantation. LY294002 abrogated all of the beneficial effects of MSC transplantation with IGF-1 Induction of miR-210 in MSCs, preconditioned or transfected, promoted cell survival postengraftment in the infarcted heart. Moreover, direct transfer of pro-survival miR-210 from miR-MSCs to host cardiomyocytes led to functional recovery of the ischemic heart. Improved LVEF, attenuated infarct size, and reduced fibrosis Both cell types induced an improvement in LV cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, MSCs were more effective for the reduction of infarct size and prevention of ventricular

288 A. M. Al-Khani et al.

Allogeneic

Allogenic

Li et al. (2012)

Wang et al. (2012)

Intramyocardial injection

Intramyocardial injection/intravenous injection

18

36

AMI

AMI

Rat

Rat

Mesenchymal Stem Cells for Cardiac Repair (continued)

remodeling. Scar tissue was 17.48 +/ 1.29% in the CD34 group and 10.36 +/ 1.07% in the MSCs group (p < 0.001 in MSCs vs. CD34). Moreover, unlike MSC, CD34 (+)-treated animals showed local inflammatory infiltrates in LV wall that persisted until 4 weeks after transplantation The cardiac function of the intramyocardial group increased significantly compared to the intravenous and control groups at day 7. In intramyocardial group, labeled cells were visualized in the infracted heart by serial MRI, and the intensity by OI was significantly higher on day 1. In the intravenous group, the heart signals were significantly attenuated by dual-modal tracking at two time points Every week for 6 weeks, hearts from three rats injected with MSCs were harvested to observe single CMs. Although each week numerous round MSCs were found in the hearts of animals treated with MSCs, beating CM-like cells labeled with PKH26 were observed by the sixth week of observation. The contractility of CM-like cells

10 289

Cell type

Allogenic

Author/year

Flynn et al. (2012)

Table 2 (continued)

Intramyocardial injection

Mode of delivery

17

Sample size

AMI

Model type

Rat

Animal

was the same to that of the unlabeled contractile native CMs at the sixth week and that of the normal group (10.71
1.59 vs. 11.09
3.42 vs. 11.21
2.16, p > 0.05). The contractility of CM-like cells was greater than cells both from the first week (10.71
1.59 vs. 7.37
3.47, p < 0.01) and the second week (10.71
1.59 vs. 8.08
3.11, p < 0.05) which was associated with significantly increased LVEF Assessed 28 days after MI, the delivery of MSCs 24 h post-MI did not improve LVEF (p ¼ 0.19) and did not prevent the decline in LVEF observed in the absence of cell therapy (p ¼ 0.17). The administration of unrestricted somatic stem cells also did not improve LVEF (p ¼ 0.11) but prevented a further decline in LVEF (p ¼ 0.001). The infarct area (p ¼ 0.2), apoptosis (p ¼ 0.07), and angiogenesis (p ¼ 0.09) did not differ between groups

Main findings

290 A. M. Al-Khani et al.

Allogenic

Skeletal myoblasts or BM-MSCs

Allogeneic

Chen et al. (2019)

Zhu et al. (2009)

Xu et al. (2010)

Intravenous

Intra-arterial (LAD)

Intramyocardial injection

46

45

14

AMI

AMI

AMI

Rabbit

Rabbit

Rat

(continued)

Coculture showed that IL33-MSCs reduced T-cell proliferation and enhanced CD206+ macrophage polarization. Echocardiography showed LVEF was enhanced in IL33-MSC-injected rats compared with vector-MSC-injected rats. Postmortem analysis of rat heart tissue showed reduced fibrosis and less inflammation in IL33-MSCinjected rats Four weeks after the cell transplantation, LVEF was significantly improved in all cell treatment groups compared to control. Neovascularization was observed in both of the treatment groups. There was no significant difference between the two cell types The cardiac improved significantly in the US + microbubble + MSC treatment group. The number of capillaries stained by HE in US + microbubble + MSC group was much greater than that of the other groups. US + microbubblemediated supply of MSCs increased the level of VEGF in ischemic myocardium

10 Mesenchymal Stem Cells for Cardiac Repair 291

BM autologous preconditioned (2% oxygen culture) MSCs

Tanaka et al. (2016)

Cell sheet-based delivery

Mode of delivery Intramyocardial

N¼5 per group

Sample size 42

Chronic MI

Model type AMI

Rabbit

Animal Rabbit

Main findings Transplanted GFP-positive MSCs were enriched with time in the periinfarct border zone with differentiation potential into three major cell types of the heart, including cardiomyocytes, endothelial cells, and smooth muscle cells, and there was significant augmentation of microvascular density Preconditioned autologous BM-derived MSC sheets were implanted into a rabbit old MI model. Implantation of BM-derived MSC sheets increased angiogenesis in the peri-infarcted area and decreased the infarcted area, leading to LVEF improvement. Importantly, the therapeutic efficacy of the preconditioned BM-derived MSC sheets was higher than that of standardly cultured sheets

ADSC adipose-derived stem cells, AMI acute myocardial infarction, BM bone marrow, BMC bone marrow concentrate, CMs cardiomyocytes, ESPVR end-systolic pressure-volume relationship, GCSF granulocyte colony-stimulating factor, LV Left ventricle, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, MPC mesangiogenic progenitor cells, MSCs mesenchymal stem cells, VEFG vascular endothelial growth factor

Cell type Autologous

Author/year Rahbarghazi et al. (2014)

Table 2 (continued)

292 A. M. Al-Khani et al.

Allogenic MSCs

Allogenic

Autogenic

Amado et al. (2005)

Quevedo et al. (2009)

Schuleri et al. (2009)

Surgical-anterior thoracotomy

Transendocardial injection

Intramyocardial injection

15

10

14

Chronic

Chronic

Acute

Model type Chronic

Porcine

Porcine

Porcine

Animal Porcine

(continued)

Main findings Extensive survival and engraftment of MSCs at the site of cell graft. Expression of muscle-specific proteins after 2 weeks. Contractile dysfunction attenuated at 4 weeks in cell-treated animals as compared to controls. Attenuation of LV remodeling Significant long-term retention of the injected cells at 2 months after delivery. Gross pathology showed significant reduction in fibrosis and near normalization of systolic and diastolic cardiac function as compared to the placebo-treated animals Long-term MSC survival, engraftment, and tri-lineage differentiation after transplantation into chronically scarred myocardium. MSCs showed capacity for cardiomyogenesis and vasculogenesis which contribute, at least in part, to their ability to repair chronically scarred myocardium Autologous MSCs’ safety and efficacy in a heart failure model. Showed substantial structural and functional reversal remodeling. Support clinical trials of MSC therapy

Author/ year Shake et al. (2002) Sample size 14

Table 3 Summary of preclinical trials utilizing BM-derived MSCs in the large animal models of heart diseases

Mode of delivery Intramyocardial injection

Mesenchymal Stem Cells for Cardiac Repair

Cell type Autologous MSCs

10 293

Allogenic

de Jong et al. (2014)

Intracoronary infusion

Intracoronary, NOGA-guided transendocardial, or surgical

Intramyocardial injection

Autologous

Autologous

Myocardial injections

Mode of delivery

Allogeneic

Cell type

van der Spoel et al. (2012)

Schuleri et al. (2011) Cai et al. (2016)

Author/ year

Table 3 (continued)

36 with moderate and 33 with severe infarct

19

24

22

Sample size

Acute

Acute

Acute

Chronic

Model type

Porcine

Porcine

Mini-swine

Porcine

Animal

in patients with chronic ischemic cardiomyopathy MSC-based therapy led to reduction in infarct size, whereas infarct size increased in non-treated animals Twenty animals survived full length of experiment. PET-CT showed better 18F-FDG uptake in the celltransplanted LV segments. Improved global cardiac function. Molecular studies showed mTOR activation signaling and improved LV function due to paracrine activity Direct comparison of MSCs with BM-derived mononuclear cells and effect of repetitive injections. At 4 weeks, LVEF improved in MSC-treated animals, but endpoint analysis showed no significant difference in efficiency after surgical injections at 8 weeks Encapsulated MSCs (eMSCs) transduced to express glucagon-like protein-1. Three incremental doses of eMSCs or Ringer’s solution without cells. LVEF increased more (9.3%) in

Main findings

294 A. M. Al-Khani et al.

Autologous

Adipose tissue allogeneic MSCs overexpressing IGF/HGF

Allogenic naïve or Akt-transduced MSCs

Sheu et al. (2015)

Guadalupe et al. (2016)

Lim et al. (2006)

Intracoronary infusion

Intramyocardial injection

Surgical

36

14

25

Acute MI

Acute MI

Acute MI

Porcine

Porcine

Porcine

Mesenchymal Stem Cells for Cardiac Repair (continued)

animals with severe infarction. Vessel density increased threefold in the infarct zone 30% more in the border zone Baseline LV injection fraction and LV chamber size did not differ among the five groups. By day 60, LVEF was highest in group 1 and lowest in group 2, significantly higher in group 5 than that in groups 3 and 4, and significantly higher in group 4 than that in group 3 Treatment with paMSC-IGF-1/HGF (1:1) vs. the other groups had a reduced inflammation in some sections analyzed. This also enhanced angiogenesis in ischemic myocardium. Although indices of cardiac function did not show significant improvement, cell retention and IGF-1 overexpression were confirmed within the myocardium Highest LV systolic function improvement and reduction in infarct size were observed by M-SPECT in Akt-transduced MSCs than naïve MSC- or vehicle-treated animals. Akt-MSCs survived better in the periinfarct zone observed by FISH

10 295

Autologous Pre-treated MSCs

Autologous

Mathieu et al. (2009)

Cell type Allogeneic

Bartunek et al. (2007)

Author/ year Silva et al. (2005)

Table 3 (continued)

Intramyocardial injection

Intramyocardial injection

Mode of delivery Intramyocardial injection

24

30

Sample size 20

Chronic

Chronic

Model type Chronic

Canine

Canine

Animal Canine

Main findings Mean LVEF was significantly higher in treated dogs at 60 days. WBC and CRP levels were similar in both groups. CK-MB and troponin-I increased from baseline to 48 h and then returned to baseline. There was a trend toward reduced fibrosis and more vascular density in treated group. MSCs co-localized with endothelial and smooth muscle cells but not with myocytes Pre-treated MSCs with cardiomyogenic factors significantly increased their myogenic differentiation and increased LV wall thickness by week 12. The biological ex vivo cardiomyogenic specification of adult MSCs is feasible and increases in vivo cardiac differentiation as well as the functional recovery Direct comparison of BM-derived MSCs and mononuclear cells. Treatment with latter were superior in giving sustained improvement in wall motion score index, reduction in infarct size, improved end-systolic elastance, and improved regional systolic function. VEGF-3 levels

296 A. M. Al-Khani et al.

Allogenic MSCs +bFGF

Autologous

Allogenic male donor naïve or VEGFexpressing BM-MSCs

Wang et al. (2015)

Zhao et al. (2012)

Locatelli et al. (2015)

Intramyocardial injection

Surgical

Retrograde coronary infusion

24

10

32

AMI

Acute

Subacute

Ovine

Ovine

Canine

(continued)

increased in border zone leading to more vascular density in mononuclear cell-treated animals Retrograde coronary venous bFGF infusion enhanced engraftment and differentiation capacity of MSCs, recovery cardiac function, improved LVEF, attenuated remodeling, and increased vascular density Significant attenuation of CM hypertrophy with the normalization of the hypertrophy-related signaling proteins PI3Kα, PI3Kγ, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (p-ERK) in the adjacent zone of the MSC-treated group versus the MI-alone group One month posttreatment with MSCs overexpressing VEGF, infarct size (magnetic resonance) decreased by 31% vs. pre-treatment. LVEF recovered to almost its baseline value and marked decrease in end-systolic volume. Moreover, the treatment induced significant capillary and arteriolar proliferation, which reduced subendocardial fibrosis. These data were in comparison with naïve MSC-injected animals

10 Mesenchymal Stem Cells for Cardiac Repair 297

Hu et al. (2016)

Author/ year Rabbani et al. (2017)

Allogenic hypoxia cultured

Cell type Autologous

Table 3 (continued)

Intramyocardial injection

Mode of delivery Intramyocardial injection

49

Sample size 18

Acute

Model type Acute

Cynomolgus monkey

Animal Ovine

Main findings Echocardiography revealed a significant improvement in the LVEF compared with the control group (p value 3  1010 particles per μg of protein and low-purity EVs present a ratio of 2  109 to 2  1010 particles per μg of protein (Webber and Clayton 2013). The EV particle-protein ratio could be changed according to culture conditions or isolation methods. 3D-culture can increase the EV release from a single cell but decrease the ratio of particles per μg of protein than 2D-culture (Haraszti et al. 2018; Cha et al. 2018b). Several groups have identified the proteome of MSC-EVs using various proteomic approaches. They have suggested that proteins in MSC-EVs have sufficient biochemical potency for disease pathogenesis or regenerative therapy (Xing et al. 2020; Lai et al. 2012; Kim et al. 2012; Angulski et al. 2017; Anderson et al. 2016; La Greca et al. 2018). Kim et al. characterized the MSC-EVs proteome and reported that EVs contain several candidate proteins that may mediate potential therapeutic effects such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibronectin, integrins, and various proteins associated with signaling pathways (Wnt, TGF-β, and RAS-MAPK pathways) (Kim et al. 2012). Another group demonstrated that MSC-EVs contain growth factors i.e., glial cellderived neurotrophic factor, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) and angiogenic factors (i.e., hepatocyte growth factor, Ang1, HES1, and S1P), which promote tissue repair and regeneration (Hu et al. 2015; Zhu et al. 2014). More recently, Xing et al. performed proteomic analysis of EVs released from adipose-derived MSCs and reported that the EV proteins participated in several enriched pathways such as the MAPK, VEGF, and Jak-STAT signaling pathways, which are related to tissue repair (Xing et al. 2020). Anderson et al. demonstrated that angiogenic signaling proteins in MSC-EVs increased when MSCs were exposed to ischemic conditions such as PDGF, EGF, FGF, and most notably nuclear factor-kappaB (NF-κB) signaling pathway proteins, suggesting that MSCs could release EVs containing robust pro-angiogenic paracrine effectors under pathological conditions (Anderson et al. 2016). Notably, some surface proteins of EVs, i.e., tetraspanins, integrins, extracellular matrix (ECM) proteins, immunoglobulins, proteoglycan, and lectins, play an essential role in the interaction of EVs with the plasma membrane of target cells. This interaction results in the EVs uptake or regulation of various signaling pathways in the target cells (Sheldon et al. 2010; Lam et al. 2020; Rana et al. 2012; Mulcahy et al. 2014).

32

Mesenchymal Stem Cell-Extracellular Vesicle Therapy in Patients with Stroke

959

Other Cargos Lipids are essential molecular components of EVs because they make up the lipid bilayer membrane that protects the encapsulated material. However, information regarding the composition and function of lipids in EVs is limited. Membranous lipids have many roles, such as EV markers, trafficking or stabilizing EVs, transporting membranous lipids to recipient cells, transferring bioactive lipids or enzymes to recipient cells, and waste disposal (Skotland et al. 2020). Recently, Barzegar et al. showed that MSC-EVs promoted neuroprotective effects in an animal model of stroke in cholesterol or lipid-dependent manner (Barzegar et al. 2020). Several studies have suggested an association between mitochondrial dysfunction and brain impairments after stroke, and hence, transplantation of healthy mitochondria can be a promising approach in stroke (Hayakawa et al. 2018). MSCs control intracellular oxidative stress by targeting mitochondria. Wang et al. showed that EVs containing mitochondria were released from MSCs and engulfed by recipient cells by fusion (Wang et al. 2018a).

Isolation, Dosage, Mode of Application, and Biodistribution In EV studies, two main dosing strategies used are the number of EVs and protein concentration. However, because protein concentration does not correlate with EV number readout (as assessed by NTA), this concentration is not valid for determining EVs dosing (Lobb et al. 2015). Different EVs isolation methods can yield samples with up to an eight-fold difference in protein content relative to EVs number from the same source material (due to co-isolating contaminating proteins) (Kennedy et al. 2021). Although the EV number yield can vary with isolation technique, it is more reliable than protein as a surrogate (Kennedy et al. 2021). However, it is observed that NTA, the most widely used method for measuring the EVs number, may show variation in EV numbers up to 25% (Vestad et al. 2017). The optimal time and mode of EVs application should be studied in stroke patients. Most recovery occurs in the first few months following a stroke, with only minor additional measurable improvements occurring thereafter. The levels of chemokines, trophic factors, and related miRNAs increase markedly in the infarcted brain during the acute phase of stroke and decrease over time. Such changes in the brain microenvironment may significantly affect the biodistribution of EVs and the degree of recovery and neurogenesis/angiogenesis after administering EVs therapeutics in stroke patients. The brain capillary endothelium forms BBB that prevents the passage of 100% of large-molecular neurotherapeutics and more than 98% of all small-molecular-sized drugs into the brain (Pardridge 2003). Hence, compared with the amount of MSC-EVs required to treat patients with topical diseases or other systemic illnesses with a local application, a more considerable amount of MSC-EVs may be required to treat stroke patients (especially in the chronic phase when the BBB is closed). BBB manipulation (e.g., with use of mannitol) may enhance endogenous repair

960

O. Y. Bang et al.

mechanisms following stroke by allowing entry of paracrine factors (e.g., trophic factors and EVs) more easily into the brain (Borlongan et al. 2004). At 24 h after systemic application, EVs preferentially accumulate in the liver, spleen, and lung and at low but detectable levels in the kidney, heart, and brain. The average half-life of EVs in the circulation is short (between 2 and 20 min in mice) (Wiklander et al. 2015; Gudbergsson et al. 2019). The biodistribution of EVs is dosedependent, and effected by the route of administration, and parent cell source of EVs (Wiklander et al. 2015). The biodistribution study using fluorescence-labeled MSC-EVs and MSCs in a rat model of stroke revealed that although most MSCs got trapped within the lung immediately after injection, the amounts of MSC-EVs in the infarcted hemisphere increased in a dose-dependent manner and were rarely found in the lung and liver overtime (Moon et al. 2019). Another analysis of EV treatment in a stroke rat model revealed that circulating EV levels did not differ with the dose (for the tested doses). Both low and high doses of EVs improved recovery after stroke (Otero-Ortega et al. 2020).

Production of EV Preparation An optimal manufacturing process would have the following attributes: high capacity for mass production, closed system with defined disposable components, purity with high yield, serum-free cell culture conditions, and Good Manufacturing Practice (GMP) compliance (Reiner et al. 2017). Many different cell culture media are used to produce EVs, such as serum-supplemented media, serum-free media, and EVs-free/reduced serum-supplemented media. Prior elimination of EVs from fetal bovine serum is crucial; commercial exosome/EVs-depleted serum is expensive and maybe imperfect. Hence, various methods to deplete EVs are being investigated, such as the ultrafiltration method or the use of xeno-free and chemically defined media (Kornilov et al. 2018). The heterogeneity of EVs and their cargo may increase or decrease during the production of EVs. Further in-depth studies are required to curtail the heterogeneity of EVs and increase the levels of the therapeutic components of EVs in clinically feasible ways for stroke patients (Xin et al. 2017b; Cha et al. 2018b; Wang et al. 2018b; Harting et al. 2018; Domenis et al. 2018). MSCs heterogeneity (donor variation) because of the origin of MSCs or the conditions of donors (e.g., age and diseases) can be minimized with optimal culture conditions or with the use of a working cell bank (Costa et al. 2021).

Isolation of Prepared EVs Contamination of non-EVs material is by far the greatest variable associated with the isolation method, with significant potential for the non-specific effects of EV-based therapeutics. Moreover, EVs from stem cells exhibit heterogeneity in terms of size, and EVs of a different size may show differential effects when used as therapeutics. Differential EVs isolation methods can yield samples with varying degrees of

32

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contaminating proteins and therapeutic cargo proteins/miRNAs/lipids, although the material source is the same. Various techniques that include (but are not limited to) ultracentrifugation, PEG precipitation, size exclusion chromatography, and tangential flow filtration are available for the isolation of EVs. However, each method has advantages and disadvantages; hence, no reliable method for the isolation of EVs is available (Reiner et al. 2017). A recent survey performed by the ISEV revealed that ultracentrifugation and density-gradients are included in the most commonly used protocols for EVs isolation and purification. Moreover, size-exclusion chromatography and tangential flow filtration are being increasingly used recently (Royo et al. 2020). In addition, Watson et al. suggested GMP-compatible methods for clinical-scale production, purification, and EVs isolation (Watson et al. 2018).

Recent Advances in EV Therapeutics The clinical translation of EVs-based therapeutics is impeded by some practical issues such as heterogeneity in both extensive therapeutic cargo and surface configuration, which could lead to uncontrollable or lower therapeutic efficacy and low yield. This may be problematic for a stable supply of potent EVs-based medicinal products, especially at the onset of phase III clinical trials and on the market scale (Gimona et al. 2017). To date, thanks to great strides in understanding complicated EVs physiology, numerous bioengineering methodologies are developed to address these challenges and help support the onward clinical advances. This section will discuss the current state-of-the-art bioengineering technologies that have been designed to augment the therapeutic potency and production yield of stem cellderived EVs. Since EVs are secreted products of cells, there have been various attempts in research to genetically or biochemically modulate parental cells’ phenotypes to influence the yield and therapeutic potential of the resulting EVs. MSC-EVs have often been reported as an efficient promoter of angiogenesis, as they incorporate a multitude of angiogenic factors. Tao et al. reported that the proangiogenic ability of MSC-EVs is further enhanced by genetic modification of MSCs to overexpress miRNA-126, one of the primary angiogenic mediators both in vitro and in vivo (Tao et al. 2017). Kang et al. reported that EVs derived from MSCs overexpressing CXC chemokine receptor 4 significantly promoted angiogenesis in a rat model of myocardial infarction and protected neonatal cardiomyocytes apoptosis in vitro (Kang et al. 2015). In the stroke field, engineering MSCs to overexpress specific therapeutic proteins or RNAs increased their efficacy after stroke (Xin et al. 2017b). A wide variety of molecules and culture methods prime MSCs and modify their EVs accordingly. For example, preconditioning of sub-lethal stimuli can trigger an adaptive response of MSCs to injury or damage. Moon et al. showed that the cultivation of MSCs with either serum of stroke patients or treatment with ischemic brain extracts could increase MSCs’ restorative properties and EVs release. These data suggested that signals from an ischemic brain could affect the efficacy of MSCs

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and MSC-EVs while stimulating EVs release from MSCs (Moon et al. 2018, 2019). Similar findings were reported by Lee’s research group (Lee et al. 2016). Treatment with ischemic brain extract and MSCs-conditioned medium upregulated miRNAs and proteins that modulate tissue repair pathways (Moon et al. 2019; Lee et al. 2016). Thrombin-preconditioning of MSCs increased EVs yield and enriched their therapeutic payload of interest (Sung et al. 2019). It is widely accepted that hypoxic culture conditions similar to the bone marrow microenvironment (i.e., 0.1–2% O2) are beneficial to MSCs as they exhibit adaptive cell response to the injury sites. MSCs culture under hypoxic conditions with or without serum deprivation amplified the secretion of EVs, enriched therapeutic payload (e.g., miRNAs), and improved their efficacy in experimental tissue injury models (Zhang et al. 2012; Bian et al. 2014; Wang et al. 2018b, 2020; Park et al. 2018). Pro-inflammatory priming of MSCs renders EVs release with enhanced anti-inflammatory properties (Harting et al. 2018; Domenis et al. 2018; Hyland et al. 2020). Exogenous supplementation of bioactive molecules (e.g., growth factors, cytokines, and chemicals) in MSCs culture could influence the cellular biosynthesis machinery, and thus, regulate the payload of MSC-EVs and yield procurement (Choi et al. 2019; Woo et al. 2020). For example, erythropoietin (100 IU/mL) supplementation of MSCs culture medium resulted in a significantly higher EVs production yield compared with the untreated control group; moreover, the secreted EVs were rich in anti-apoptotic miRNAs, i.e., miR-299, miR-499, miR-302, miRNA-200, and demonstrated greater therapeutic efficacy in experimental renal injury models both in vivo and in vitro (Wang et al. 2015). Lopatina et al. reported that MSCs stimulated by PDGF supplementation (20 ng/mL) increased the EV secretion rate and augmented proangiogenic capacity (Lopatina et al. 2014). With ever-increasing information about EVs biogenesis mechanisms, key modulators and mechanisms associated with EVs secretion have been identified. Modification of specific molecular pathways in EVs biogenesis increase EV production (Phan et al. 2018). Recent studies have reported the activation of EVs biogenesis during membrane blebbing (P2X7 receptor, phospholipase D2), or multivesicular body fusion with the plasma membrane (Rab GTPase, SNARES) can increase the secretion of EVs, leading to an increased yield (Phan et al. 2018; Colombo et al. 2014; Qu and Dubyak 2009; Urbanelli et al. 2013; Rao et al. 2004; Hsu et al. 2010; Ostrowski et al. 2010; Laulagnier et al. 2004). The homing ability of MSC-EVs towards ischemic and/or inflamed regions aids in delivering their payload more specifically to injury sites, contributing to maximizing therapeutic efficacies and minimizing systemic effects (Gudbergsson et al. 2019). These abilities of EVs (tissue tropism and cell-selective fusion) are attributed to the EVs membrane proteins determined by the phenotype of the parental cell (Peinado et al. 2012). However, recent findings revealed that after systemic injection, most of EVs are hardly free from the first-pass effect after accumulation in the liver, spleen, and lungs (Di Rocco et al. 2016). Therefore, the technologies to design and incorporate the targeting ligands of interest on the surface of EVs are valuable for enhancing the biodistribution and disease-targetability of EVs, eventually increasing their therapeutic efficacy (Kim et al. 2020; Man et al. 2020). One simple approach is

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the direct insertion of hydrophobic molecules on the phospholipid bilayer of the EVs membrane for the hydrophobic attraction. Kim et al. reported the direct incorporation of aminoethyl anisamide-PEG onto the surface of EVs loaded with the anticancer drug paclitaxel to target lung cancer cells (Kim et al. 2018). The results showed enhanced accumulation of the functionalized EVs in lung cancer tissues after systemic injection, thereby improving the therapeutic outcomes. Kooijmans et al. used an alternative approach; fusogenic micelles conjugating EGF were successfully fused with EVs derived from platelets or Neuro2A cells without any configurational alterations in the size, morphology, and protein composition. The surface-modified EVs showed improved tumor-specificity and had longer stay in the blood circulation after systemic delivery in vivo (Kooijmans et al. 2016). Shear stress at the physiological level contributes to the homeostasis of multiple tissues and organs in vivo, especially the tissues influenced by the presence of interstitial fluid flow or blood flow (Arora et al. 2020). Differentiation of MSCs into osteogenic, cardiogenic, chondrogenic, adipogenic, and even neurogenic lineages can be induced by varying different shear-stress conditions (0.01–2 Pa) (Arora et al. 2020). Shear stress can enhance the immune regulatory function of MSCs (Diaz et al. 2017). Likewise, the release of EVs from cells occurs inherently in response to shear stress at physiological or pathological levels in vivo. Therefore, as a bioinspired means to physically influence EVs production, controlled shear stress provided to the MSCs culture is considered an effective strategy to increase the yield of EV production and regulate its therapeutic composition (Piffoux et al. 2019). Hence, hollow fiber bioreactor technology that allows steady medium perfusion through massively bundled-up hollow microfibers provides great promise for MSCs-EVs production. This is because MSCs would constantly be under the controlled shear stress of a laminar flow condition. The maximum surface area would be available for cell seeding suitable for scaled-up culturing of MSCs (Colao et al. 2018). Furthermore, continuous medium perfusion can provide several vital practical benefits during MSC culture including: (1) the adequate mass transfer of oxygen, nutrients, and metabolites during the long-term culture period; (2) facilitate monitoring and controlling of culture parameters to maintain a well-defined MSC phenotype. These changes circumvent the unexpected alterations in the derivative EVs. Finally, retain the EVs product within a confined volume of the culture compartment during culture, which could yield more concentrated EVs in the conditioned medium (Cha et al. 2018a; Piffoux et al. 2019; Colao et al. 2018). Besides mimicking the physiological features of EVs, the challenges in scalingup the production of EVs are required be addressed for clinical applications. An automated cell culture platform based on the hollow fiber bioreactor technology would be one of the promising strategies for the scalable manufacturing and bioprocessing of therapeutically viable EVs products. This allows the large-scale production and prolonged culture of MSCs without phenotypic alterations and limited passage windows (Mendt et al. 2018). Concurrently, it provides MSCs with the controlled shear stress environment to modify and/or augment the therapeutic potential and yield of secreted EVs.

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Many studies have reported that the intrinsic ability of MSCs to secrete a variety of therapeutic molecules is difficult to reproduce in vitro. This is because the natural 3D-interactions between cells and either ECMs or other neighboring cells are readily disrupted in conventional monolayer culture conditions, wherein the individual cells encounter limited 2D-borders (Placzek et al. 2009). Therefore, such conventional culture platforms are vastly problematic to reach the clinical-scale production of therapeutic EVs and demanding countless batches of MSCs with significant impact on labor, time, and cost. Given the lack of the physiologically relevant phenotype of MSCs resulting from the 2D-culture condition, the configuration of secreted EVs could be driven far from the natural compositions that MSC-EVs would have in the pathological conditions in vivo (Man et al. 2020). Accordingly, Nalamolu et al. reported that the treatment of EVs from 2D-cultured MSCs failed to improve the survival rate and adversely influenced recovery after stroke (Nalamolu et al. 2019). Such limitations of the monolayer culture have necessitated the development of 3D-culture platforms that closely mimic body’s physiological microenvironment, which induces close cell-cell interaction and ensures improved cellular communication with highly cumulated signaling molecules (Cha et al. 2017). Numerous recent studies have reported that the formation of 3D MSC-aggregates can create a microenvironment akin to that in vivo wherein the phenotype and innate properties of MSCs are highly preserved (Bartosh et al. 2010; Frith et al. 2010). In a previous study, a novel 3D-culture platform using the microwell-array system was developed for the large-scale culture of 3D-MSCs spheroids. This 3D-system prevented cell loss, significant cost-saving without wasting expensive cell material, achieving highly reproducible and precisely controlled cell-size and cell number of the MSC spheroids (Cha et al. 2017). Furthermore, a simple and effective 3D-bioprocessing method has been developed using micro-well culture system for scalable production of therapeutically effective MSC-EVs. Moreover, 3D-culture increased the production of MSC-EVs enriched with angiogenic and neurotrophic factors (cytokines and miRNAs, respectively) approximately 100 folds more than the ones derived from 2D-culture system and minimized the uncertainty in cellular behaviors due to heterogeneous spheroid sizes (Cha et al. 2018b). Stem cells detect ECM-derived mechanical cues that are conveyed through the cytoplasmic compartment and cause phenotypic changes as gene and/or protein expression profiles are inherently influenced (Engler et al. 2006). A previous study showed that varying stiffness of culture substrate influenced the secretory profiles of MSCs. For example, it significantly enhanced the secretion of pro-angiogenic factors (Abdeen et al. 2014). These data imply that different mechanical cues provided through the microenvironment can alter the fate of MSCs and their secretome profile. Our research group has investigated the effects of 3D-physical interactions between MSCs and culture matrix upon regulating the therapeutic compounds of MSC-EVs (unpublished data). In this study, MSCs encapsulated in GelMA hydrogels of varying stiffness from 9 to 21 kPa showed

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differential gene expression profiles with the retention of innate characteristics of MSCs. The substantial upregulation of angiogenesis-related genes was observed besides higher mechanical properties. This data showed enhanced angiogenic capacity of the MSCs-derived EVs that was also confirmed by tubulogenic assay using human umbilical vein endothelial cells. On the other hand, the interventions of 3D-biomaterial scaffolds can be supportive in regulating MSC-EVs. In a traumatic brain injury model, MSC-EVs cultured in the 3D-collagen scaffold exhibited a twofold increase in EVs secretion and functional recovery of traumatic brain injury due to neurogenesis and angiogenesis compared with MSC-EVs cultured in the 2D-culture system (Zhang et al. 2017). Native (including decellularized tissues), as well as synthetic polymerbased scaffolds, can be used as 3D-microenvironment to regulate cellular attachment, growth, migration, differentiation, and secretome profile (Phan et al. 2018). Based on the average EVs yield in previous studies, the estimated number of EVs secreted by 2D-cultured MSCs was approximately 500 EVs per cell, which was significantly higher (up to 100 folds) from the 3D-culture system (e.g., microwell culture or bioreactor system) (Kordelas et al. 2014; Cha et al. 2018b; Mendt et al. 2018). Some researchers in the bioengineering field have focused their efforts to address the limitations in scalability and cost-effectiveness of the onerous purification steps and low production yield from the current EVs manufacturing protocols. The new concept of EVs bioprocessing is based on phospholipids’ spontaneous self-assembly, the significant component of the membranous organelles and structures in a cell. The cells in defined culture conditions are subjected to physical processes, such as sonication and serial mechanical extrusions through filters of reducing pore sizes ranging from 1 μm to 100 nm and custom-made devices with hydrophilic microchannels or centrifuge modules, to disassemble them into nano-sized vesicles (Goh et al. 2017). The cell-derived nanovesicles (CDNs) may encapsulate endogenous cytosolic substances and therapeutic molecules of interest that are exogenously loaded during the plasma membrane self-assembly process. Moreover, molecular engineering techniques can modify cell membrane proteins to equip CDNs with customized ligands on their surfaces to specifically recognize the target disease sites (de Jong et al. 2019). Han et al. reported that a >300 times higher yield of CDNs than the amount of naturally secreted EVs could be obtained during MSC cultivation. Moreover, these CDNs possess attributes similar to those of the parental cells, MSCs (Han et al. 2019). These biofabricated CDNs have also demonstrated better skin wound healing potential in an experimental mouse model than the naturally secreted MSC-EVs. Most recently, successful preclinical outcomes with the use of CDNs were reported in regenerative medicine (Man et al. 2020). However, this EVs manufacturing approach has serious concerns regarding regulatory compliance, particularly regarding safety and quality control issues. These concerns emanate due to the high possibility of containing and shuttling undesirable factors such as genomic DNA compounds, unwanted metabolic molecules, and signaling molecules related to cell death.

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Conclusions and Perspective Unlike MSCs-based therapy, MSC-EVs therapy is still in the process of development. Currently, there are no Food and Drug Administration (FDA)-approved EVs-based therapeutic products. However, the use of MSC-EV therapy is rapidly expanding and could be a promising therapy for severe stroke patients as MSCsbased therapies have already been tested in preclinical and clinical trials. Compared with MSCs-based therapy, EVs-mediated therapy has unique advantages in terms of safety, biodistribution, stability, and off-the-shelf approaches for acute ischemic stroke. MSC-EVs therapy has advantages over conventional drugs or protein/ RNA-delivery systems because MSC-EVs contain therapeutic payload with heterogeneous functions for recovery after stroke. To date, the efficacy of MSC-EVs therapy has not been tested in stroke patients. An early phase clinical trial investigating the safety of allogeneic MSC-EVs in five patients with acute ischemic stroke is ongoing (clinicalTrial.gov. Identifier: NCT03384433). Our research group is also planning to conduct a phase 1/2a randomized trial on allogeneic Wharton’s Jelly MSC-EV therapy (Stem Cell Extracellular Vesicle In Acute stroke trial). For successful clinical translation of EVs-based therapeutics, quality management and establishment of standard operating procedures for EV therapeutics as well as optimization of EV cargo and time/ dose/mode of EV application for stroke patients are required.

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Bruna Arau´jo, Rita Caridade Silva, Sofia Domingues, Anto´nio J. Salgado, and Fa´bio G. Teixeira

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs’ Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs’ Biological Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Pitfalls of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretome Derived from MSCs as Cell-Free-Based Therapeutic Strategy . . . . . . . . . . . . . . . . . . . . Composition and Characterization of MSCs’ Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Action and Principal Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pre-clinical Approaches of Mesenchymal Stem Cell Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wound Healing and Cartilage Repair and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kidney and Lung Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Central Nervous System Pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Traumatic Brain Injury (TBI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spinal Cord Injury (SCI) and Ischemic Stroke (IS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurodegenerative Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Approaches Based on Mesenchymal Stem Cell Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . The Benefits and Barriers of MSCs’ Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improvements Needed for Secretome Application as a Cell Transplantation-Free Tool . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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B. Araújo · R. C. Silva · S. Domingues · A. J. Salgado Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal F. G. Teixeira (*) Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_46

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Abstract

Over the last decade, the use of stem cells has remarkably been proposed as a regenerative tool, and within it, mesenchymal stem cells (MSCs) have emerged as a promising therapeutic option. As a consequence, they currently represent an effective tool in the treatment of several diseases due to their tissue-protective and tissue-reparative properties. Based on these MSCs’ regenerative potentialities are the secretion and release of trophic molecules and vesicles, nowadays known as stem cell secretome. Notably, MSCs’ secretome itself is starting to be considered a potential active pharmaceutical component, in which its vesicular portion has been revealing promising characteristics to be used as a drug delivery system, thereby opening an opportune window to the specific release of drugs, peptides, or specific agents to targeted damaged areas. Therefore, the use of MSCs’ secretome as a whole or its components per se has demonstrated remarkable advantages over cell transplantation procedures, with no adverse effects, thereby indicating that it can be used as a source of bioactive agents that can be efficiently stored and transported as a ready-to-use biocomponent. Thus, on the scope of the present chapter, we intend to provide an overview of the application of MSCs’ secretome as a therapeutic strategy to the regenerative medicine field. Keywords

Mesenchymal stem cells · Reparative properties · Secretome · Therapeutic strategy · Regenerative medicine Abbreviations

AD AKI ALI AMPK Ang-1 ARDS ASCs Bcl-2 BDNF bFGF BM BSCB circRNAs CKD CM CNS CSCs CXCR4 ECM

Alzheimer’s disease Acute kidney injury Acute lung injury 5' Adenosine monophosphate-activated protein kinase Angiopoietin-1 Acute respiratory distress syndrome Adipose-derived stem cells B-cell lymphoma 2 Brain-derived neurotrophic factor Basic fibroblast growth factor Bone marrow Blood-spinal cord barrier Circular RNAs Chronic kidney disease Conditioned medium Central nervous system Cardiac stem cells C-X-C chemokine receptor type 4 Extracellular matrix

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Mesenchymal Stem Cell Secretome: A Potential Biopharmaceutical. . .

ECs EG-VEGF EVs FGF-BP FPHL GAP-43 GvHD hAMSCs HD HGF hMSCs IGF IL IS ISCT JNK KGF lncRNAs MAPK mHtt miRNAs MMP-13 MRCTs mRNA MS MSCs mTOR MVBs NFκB NGF NO NT-3 OA PD PDGF PEDF PGE2 PI3K PK1 RNAseq SCI SDF-1 TBI TGF-ß1 TIMP3

Endothelial cells Endocrine gland-derived vascular endothelial growth factor Extracellular vesicles Fibroblast growth factor-binding protein 1 Female pattern hair loss Growth-associated protein 43 Graft-versus-host disease Human adipose tissue-derived MSCs Huntington's disease Hepatocyte growth factor Human mesenchymal stem cells Insulin-like growth factor Interleukin Ischemic stroke International Society for Cellular Therapy c-Jun N-terminal kinase Keratinocyte growth factor Long noncoding RNAs Mitogen-activated protein kinase mutant Huntingtin microRNAs Matrix metallopeptidase 13 Massive rotator cuff tears Protein-coding messenger Multiple sclerosis Mesenchymal stem cells Mammalian target of rapamycin Membrane of multivesicular bodies Nuclear factor kappa B Nerve growth factor Nitric oxide Neurotrophin-3 Osteoarthritis Parkinson’s disease Platelet-derived growth factor Pigment epithelium-derived factor Prostaglandin E2 Phosphoinositide 3-kinases Pyruvate kinase 1 RNA sequencing Spinal cord injury Stromal cell-derived factor-1 Traumatic brain injury Transforming growth factor ß1 Tissue inhibitor of matrix metalloproteinase-3

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TSG VEGF VWF

Tumor necrosis factor-stimulated gene Vascular endothelial growth factor Von Willebrand factor

Introduction In normal conditions, the organs and tissues of our body have limited regenerative capacities, which indicate that in case of failure of those systems, life-threatening conditions can arise. Moreover, many diseases result from the interface between complex mechanisms, including cellular impairments and tissue or organ dysfunctions. Among them, difficulties in treatment are prevalent since classic strategies display an inability to efficiently stimulate vital mechanisms of tissue regeneration, repair, and renewing and to promote the reversion of cell loss (Hoda Elkhenany et al. 2020). Over the past decades, the field of stem cell-based research has gained particular interest under the scope of novel regenerative/repairing therapeutic opportunities, as it appears to target several disorders and public health issues for which current medical and surgical solutions are insufficient. From a spectrum of several stem cell populations, mesenchymal stem cells (MSCs) have become a pivotal population in developing promising therapies involving the maintenance and repair of adult tissues and organs.

MSCs’ Roots During the 1960s, it was with the trailblazing work of Friedenstein and colleagues that MSCs were first isolated from rodent bone marrow (BM) and characterized as a rare population of adherent clonal non-hematopoietic precursors, capable of differentiating into mesodermal-derived cell types (Caplan 1991; Friedenstein et al. 1968). These pioneer observations sparked a significant degree of curiosity and led to the elaboration of heterogeneous procedures in isolation and cultivation of them among laboratories. For this reason, the International Society for Cellular Therapy (ISCT) established, in 2006, minimal criteria to classify a cell population as MSCs, namely, the (1) plastic adherence when maintained under standard culture conditions, (2) phenotypic expression of specific surface antigens (CD73, CD90, CD105) and concomitant absence of hematopoietic markers (CD14, CD34, CD45, and human leucocyte antigen-DR), and (3) tri-lineage mesenchymal differentiation toward osteoblasts, adipocytes, and chondroblasts in vitro (Dominici et al. 2006). However, ever since, numerous reports have confirmed that MSCs are not only present in the bone marrow and have now been isolated from a variety of non-marrow tissues using different protocols, including the placenta, skeletal muscle, adipose tissue, umbilical cord, Wharton’s jelly, peripheral blood, lung, liver, dermal tissue, and even brain (Chamberlain et al. 2007; Venkataramana et al. 2010; Phinney 2007; Praveen Kumar et al. 2019). Notably, various studies have revealed that, despite their uniformly marked

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profiles and similar cellular phenotypes, MSCs derived from different tissues exhibit a differentiation potential broader than initially thought (Phinney 2007; Harrell et al. 2019). These data indicate that under specific niches, conditions, and cellular microenvironments, these cells can “transdifferentiate” at different rates due to plasticity and modify their biological and functional characteristics accordingly (Praveen Kumar et al. 2019; Squillaro et al. 2016; Paul and Anisimov 2013). Moreover, epigenetic findings in MSCs from distinctive tissues determined the existence of substantial differences in gene expression patterns, transcriptome/proteome, and functionality that were associated merely with the tissue source (Phinney 2007; Le Blanc and Davies 2018). Thereby, when applying different protocols for their isolation and cell culture expansion, MSCs could generate cells of neuroectodermal and endodermal origin, including neuron-like cells, hepatocytes, pancreatic islet-like cells, cardiomyocytes, and alveolar and gut epithelial cells (Harrell et al. 2019; Squillaro et al. 2016; Teixeira et al. 2013; Trohatou and Roubelakis 2017; Chen et al. 2004). Additionally, besides the heterogeneity observed among MSCs from different sources, there are also variances after acquiring them from individual donors (Andrzejewska et al. 2019). Overall, these assumptions support the idea that MSC benefits organ and tissue repair due to their multipotency to generate cells of the aimed tissue and substitute damaged resident cells (Squillaro et al. 2016).

MSCs’ Biological Properties MSCs were primarily used for musculoskeletal regeneration and wound healing. Nonetheless, and acknowledging their benefits afar from tissue repair, they have been primarily used in numerous experimental, pre-clinical, and clinical models, involving organ transplantation, cancer, rheumatic diseases, autoimmune diseases, inflammatory disorders, spinal cord injuries, acute ischemic stroke, diabetes, neurodegenerative disorders, microbial infections, myocardial infarction, and so forth (Ferreira et al. 2018; Serra et al. 2018; Bai et al. 2016; Hu and Li 2018). Given that, a comprehensive study of the molecular and biological properties that define the MSCs is critical (Bai et al. 2016). One of the most common features of MSCs is, as previously mentioned, their undifferentiated self-renewal ability along with multi-lineage differentiation potential that significantly influences tissue homeostasis (Eleuteri and Fierabracci 2019; Mendes-Pinheiro et al. 2020). In contrast, these cells present further multifunctional characteristics, which embrace not only immunomodulation and homing capability but also pro-angiogenic, antioxidant, antimicrobial, neuroprotective, antitumorigenic, anti-apoptotic, and chemoattractive effects (Squillaro et al. 2016; Bai et al. 2016; Murphy et al. 2013; Spees et al. 2016). Given the immunomodulatory functions of MSCs, it has been reported that they can switch their profile from an innate to acquired immune response or vice versa, either by endorsing pro-inflammatory events if the level of inflammatory cytokines is low or by negatively regulating the immune response upon an inflammation, often relying on the

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context, local microenvironment, and disease status (Murphy et al. 2013; Harrell et al. 2019; Andrzejewska et al. 2019; Song et al. 2020). Likewise, MSCs can interact with immune cells via cell-to-cell contact and interfere with their proliferation, activation, and function. Of note, peripheral blood mononuclear cells (monocytes), neutrophils, B and T (including regulatory T cells) lymphocytes, natural killer cells, macrophages, and dendritic cells are all possible targets of the interference of these cells (Harrell et al. 2019; Song et al. 2020; Gao et al. 2016). Connecting to this effect, the homing capability of MSCs centers its attention on their migratory behavior and capacity to navigate and reach damaged tissue as feedback to a combination of cytokines (Nitzsche et al. 2017). For instance, considering its potential of tumor inhibition and tropism, it is also worth mentioning that MSCs can be loaded with chemotherapeutic agents and successfully deliver their payload in site-directed manner to the tumor sites (Kwon et al. 2019). Nevertheless, the majority of the previously mentioned potentialities are the outcome of the paracrine activity-based mechanisms of these cells. Indeed, it has been considered that the array of bioactive factors they secrete in response to the local environment constitutes the mechanism by which MSCs assist many of their beneficial effects.

The Pitfalls of MSCs Notwithstanding the promising results obtained in the clinical trials, MSC-based therapies are not considered a standard of care at the clinic, facing several obstacles to their applicability (Squillaro et al. 2016; Zaher et al. 2014). Firstly, there is an obvious lack of standardized procedures regulating cell culture, ex vivo expansion, cryopreservation, and differentiation, which affects MSCs’ properties and, as a consequence, leads to stemness attenuation and replicative senescence (Praveen Kumar et al. 2019; Squillaro et al. 2016; Kwon et al. 2019; Kandoi et al. 2018). Also accompanying this issue is the large variability in cell quality, due to the usage of different donors and their tissues, known as donor heterogeneity (Squillaro et al. 2016; Teixeira and Salgado 2020; Gao et al. 2016). Equally, regarding transplantation, a significantly high number of cells are required to have a notable effect (Teixeira and Salgado 2020; Vizoso et al. 2017). Posterior to this procedure, not only do MSCs exhibit a low survival rate (Hu and Li 2018) but also do not commonly become a part of the injured site, a detail previously revealed by cell tracking analysis (Teixeira et al. 2013). Furthermore, hurdles in defining a correct therapeutic rationale, including determining optimal dosage type, administration frequency, mode of infusion, and effective delivery route, are likewise points to consider when selecting these cells for transplantation. Given the drawbacks mentioned above, more recently, the use of MSCs per se has been reconsidered, and in its place, a direct application of their secreted trophic factors is being evaluated as part of fast-emerging cell-free therapy approach (Gnecchi et al. 2005; Haider and Aslam 2018), since those might be the essential foundations required to create effective cellular strategies.

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Secretome Derived from MSCs as Cell-Free-Based Therapeutic Strategy The plethora of protective bioactive factors secreted by MSCs is known as stem cell secretome or conditioned medium (CM). These paracrine factors released by a cell, tissue, or organism into the extracellular space can promote their repair and regeneration (Mendes-Pinheiro et al. 2020). Secreted molecules play a valuable contribution in crosstalk communication between cells and the surrounding tissues. Therefore, the MSCs’ secretome-based therapies are considered as an appealing proposal to be used in several domains of regenerative medicine (Haider and Aziz 2017). Like MSCs, the secretome profile depends on the MSCs’ source and culture conditions used to expand the cells (Yin et al. 2019). The factors and their concentration used may diverge depending on cellular and preparation parameters and by intrinsic and extrinsic environment conditions (Kehl et al. 2019; Bundgaard et al. 2020). Owing to that dissimilarities, the secretome composition may be manipulated to obtain the an optimal bioproduct profile for a specific therapeutic application and to identify the regenerative mechanisms of distinct tissue types/origins (Chang et al. 2021).

Composition and Characterization of MSCs’ Secretome The secretome composition is determined by the conditioned medium after MSCs culture in vitro. It can be divided into two distinct fractions – the soluble fraction (essentially proteins and soluble factors, such as cytokines) and the vesicular fraction (Pinho et al. 2020) (Fig. 1). A plethora of protective bioactive factors classified as growth factors, chemokines, cytokines, microRNA, hormones, free nucleic acids, lipid mediators, extracellular vesicles (EVs), and other small molecular weight signal cues constitute the secretome composition, creating a microenvironment suitable for cellular repair and regeneration (Ranganath et al. 2012; Beer et al. 2017; Hu et al. 2020). Those molecules influence the crosstalk communications between cells and the surrounding tissue to stimulate the recruitment, proliferation, and differentiation of the endogenous cells. Regarding EVs, these are secreted by the MSCs transporting active molecules and genetic information to target cells. They contain growth factors, cytokines, miRNA, and mRNA that trigger several biological responses in the target area (Rani et al. 2015). More recently, it has been accepted that when used alone, the EVs may provide a similar or enhanced therapeutic advantage compared to the cells secreting the secretome (Phelps et al. 2018).

The Soluble Fraction: Cytokines and Growth Factors Regarding the soluble fraction of MSCs’ secretome, the most physiologically relevant biomolecules secreted include basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF), which are involved in immunomodulation, cell migration, development, and regulating apoptosis; vascular endothelial growth factor (VEGF) which is a critical regulator of angiogenesis, immunomodulation,

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Fig. 1 The possible sources and composition of MSCs’ secretome. When exposed to different conditions and in response to pathological processes, MSCs secrete a plethora of factors for the repair and regeneration of the host tissue. The therapeutic benefits are determined by the secretome composition. The secretome is divided into soluble fraction (cytokines and factors) and vesicular fraction (extracellular vesicles)

and cell survival; transforming growth factor ß1 (TGF-ß1) which targets immunomodulation, cell growth, proliferation, and differentiation; tumor necrosis factor-stimulated gene (TSG-)6, prostaglandin E2 (PGE2), and galectins 1 and 9 that are also associated with immunomodulation and anti-inflammatory functions (Noone et al. 2013; Madrigal et al. 2014; Gieseke et al. 2013); and finally insulinlike growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and interleukin 6 (IL-6) that are mainly angiogenic and immunomodulatory (Martín-Martín et al. 2019; Phelps et al. 2018). Genetic modification of MSCs with IGF-1 or the concomitant overexpression of pro-survival and pro-angiogenic factors, i.e., Akt and angiopoietin-1, significantly enhanced the paracrine activity of the cells and supported angiomyogenic repair of the infarcted heart in experimental animal model (Haider et al. 2008; Jiang et al. 2006).

The Vesicular Fraction: Extracellular Vesicles Extracellular vesicles are important for carrying components from donor cells to recipient cells serving as intercellular mediators of communication (Marote et al. 2016). They are phospholipid membrane-bound particles that contain biological material (DNA, RNA), bioactive lipids, and proteins. However, EVs’ composition

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depends on the MSCs’ source; the donor-related factors such as age, health condition, etc.; genetic and epigenetic memory of the cells; the pathological state of the cells; etc. (Raposo and Stoorvogel 2013). The EV is a general term for various vesicles secreted by MSCs and include exosomes, microvesicles, and apoptotic bodies (Raposo and Stoorvogel 2013). Their characterization is based on the size, origin, and markers. For instance, exosomes (30–200 nm) originate from the internal budding of the membrane of multivesicular bodies (MVBs), a subset of endosomes that contains membrane-bound intraluminal vesicles. Afterward, it is released into the extracellular environment, subsequent to fusion with the plasma membrane. Therefore, their small size simplifies the transfer through blood and to other biological fluids. Exosomes are classified by CD9, CD63, and CD81, proteins Alix, and TSG101-positive expression. In terms of characterization, microvesicles (50–1000 nm) sprout directly from the plasma membrane and are identified by CD40 marker, integrin, and selectin-positive expression. Finally, apoptotic bodies (500–2000 nm) include fragments of dead or dying cells and are characterized by the presence of histones and annexin V-positive staining (Phelps et al. 2018) (Fig. 1). Thus, considering the paracrine signaling as the primary therapeutic mechanism of MSCs, many promising in vivo studies have reported the use of MSCs’ secretome in toto or in the form of fractionated MSC-derived EVs as a promising therapeutic approach. Recent studies have evidenced that exosomes may be primarily responsible for the therapeutic effects of the MSCs’ secretome (Shao et al. 2017; Nakamura et al. 2015; Furuta et al. 2016). However, the benefits of EVs have been only been attributed to the cytokines and growth factors but also to the RNAs and miRNAs, which play an essential role in gene expression regulation and the surrounding cell/ tissue’s microenvironment (Shao et al. 2017).

The Vesicular Fraction: Coding and Non-coding RNAs In addition to the protein fraction of the secretome, MSCs also secrete proteincoding messenger (mRNA) and non-coding RNAs such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) via their EVs, which can regulate the various cellular functions and activities, i.e., cell cycle, metabolism, migration, inflammation, and angiogenesis. The mRNA component can be transported and delivered to the recipient cells to cause changes in their protein or gene expression profile (Phelps et al. 2018). The miRNAs are important constituents of the cell secretome and contribute essentially in providing the therapeutic effects of the secretome in terms of repair and regeneration of the injured tissue. They are associated with the expression of proteins related with apoptosis and cell survival, stem cell differentiation, hematopoiesis and vascular development (miR-23), insulin secretion, cell growth (miR-125b), angiogenesis (miR-29), and immune response (Zhang et al. 2017; Wang et al. 2017; Ferguson et al. 2018; Tsukita et al. 2017). On the other hand, the lncRNAs are involved in chromatin organization, gene transcription, mRNA turnover, protein translation, assembly of macromolecular complexes, etc. (Ng et al. 2012; Gezer et al. 2014; Kim et al. 2017). Also, the circRNAs have shown the capacity to take advantage of miRNAs and control their

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function as regulators of mRNA stability and/or translation (Kim et al. 2017). Still, in EVs, they could constitute a potential biomarker in the disease processes as well as therapeutic targets as they play a pivotal role in diverse cellular processes and cell fate determination (Kim et al. 2017; Bao et al. 2016).

Mechanisms of Action and Principal Effects Cell secretome-based therapy has some distinct advantages over the cell-based therapy approach, rendering their use a promising therapeutic strategy. Autocrine or paracrine effects of the MSCs rather than direct engraftment and tissue differentiation play an essential role in tissue repair. Due to the excellent pro-proliferative and anti-apoptotic effects and given their potent trophic properties, MSCs’ secretome has emerged as a prospective therapeutic tool for numerous clinical applications. Indeed, MSCs’ secretome targets some major biochemical processes, providing anti-inflammatory, pro-angiogenic, immunomodulatory, anti-fibrotic, and antiapoptotic effects, besides promoting cell proliferation and supporting migration and homing-in and retention of the inherent stem/progenitor cells to the site of injury for participation in the ongoing repair process (Chang et al. 2021; Hu et al. 2020; Xia et al. 2019).

Pre-clinical Approaches of Mesenchymal Stem Cell Secretome The presence of trophic factors and exosomes offered by MSCs’ secretome has expanded its utility as a cell-free therapy. In recent years, a large number of preclinical studies have been reported that show that the administration – either systemic or local – of the secretome from MSCs derived from different tissue sources possess distinct therapeutic benefits when applied for the treatment of different diseases, e.g., inflammatory and degenerative diseases of hepatobiliary, respiratory, skeletal, gastrointestinal, cardiovascular, and nervous systems. Treatment with MSC-derived conditioned medium successfully reverted the characteristic damaging phenotypes (Vilaça-Faria et al. 2019) (Fig. 2). Herein, we will briefly dissect the distinctive in vitro and in vivo experimental approaches in animal models wherein MSCs’ secretome was applied.

Wound Healing and Cartilage Repair and Regeneration Under different experimental conditions, MSCs’ secretome has shown its proficiency to promote wound closure and healing in diverse types of lesions. A study by Heo et al. demonstrated that upon administration of human adipose tissue-derived MSCs’ (hAMSCs’) secretome into an experimental rat excisional wound model, there was a significant angiogenic response evidenced by increased vascular density,

Mesenchymal Stem Cell Secretome: A Potential Biopharmaceutical. . .

Fig. 2 Summary of the encouraging diverse therapeutic benefits of MSC-derived secretome in distinct disorders and lesions. Pre-clinical studies suggest that the secretome and its components have the capability to enable the regeneration of the injured brain, spinal cord, heart, bone, kidney, and lungs by immunomodulating the injury microenvironment and protecting the tissues, targeting different cell types and mechanisms

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wound closure, and proliferation/infiltration of immune cells, mostly in response to IL-6 and IL-8 (Heo et al. 2011; Makridakis et al. 2013). On the same line of thoughts, another report in an experimental rat model of dry eyes revealed that following injury, the secretome was able to enhance epithelial regeneration and reduce mRNA expression of corneal macrophage inflammatory cytokines, such as protein-1α (MIP-1α) and TNF-α (Vizoso et al. 2017; Bermudez et al. 2015). Moreover, when exploring healing strategies for massive rotator cuff tears’ (MRCTs’) lesions in an experimental rat model, our research group has reported that treatment with hMSC-derived secretome successfully reduced the fatty degeneration and atrophy of the muscles. Molecular studies revealed that hMSC-derived treatment increased pigment epithelium-derived factor (PEDF) and follistatin expression (Sevivas et al. 2017). Analogously, SCs also have the capacity to accelerate the formation of bony scars. Using umbilical cord-derived secretome, several studies have shown its capability to promote skin wound healing with the formation of fewer scars, through the stimulation of macrophage and endothelial migration homing-in and retention at the site of the injury, myofibroblast differentiation, and expression of extracellular matrix (ECM) genes (Li et al. 2017; Jackson et al. 2012; Baez-Jurado et al. 2019). Similarly, published data suggest using MSCs’ secretome for osteoarthritis (OA), wherein chondrocytes exhibit a damaging role in the cartilage degeneration that contributes to the progression of the disease. Using in vitro experimental models, researchers have found that treatment with MSC-derived CM or its derived extracellular vesicles (exosomes) decreased the inflammatory phenotype of OA chondrocytes by downregulating the expression of inflammatory cytokines (TNF-α, IL-1, IL-6, nitric oxide (NO)) and enhancing the production of immunosuppressive IL-10. These cellular and molecular changes contributed toward the anti-inflammatory and chondroprotective effects of the CM treatment (TofiñoVian et al. 2018; Chen et al. 2018). These positive consequences of the secretome in OA were similarly confirmed in vivo. Using experimental animal models of OA, MSC-sourced secretome administration enhanced the formation of new tissue and increases type II collagen synthesis (Zhang et al. 2016a). Similarly, treatment with MSC-derived extracellular vesicles stimulated endogenous cartilage repair and regeneration, primarily associated with specific exosomal miRNAs to re-establish to basal level (homeostasis) of metabolism in the proliferating chondrocytes (Liu et al. 2018a, b; Sun et al. 2019; Toh et al. 2017; Harrell et al. 2019). Additionally, it has also been observed that cell-derived secretome showed the ability to: 1. Reduce hypertrophy and de-differentiation of chondrocytes in culture via the secretion of hepatocyte growth factor (HGF), which may be noteworthy for other osteoarticular disorders since it could offer chondroprotection (Maumus et al. 2013) 2. Induce bone regeneration (as observed in rabbit’s mandibles) after surgical lesions (Linero and Chaparro 2014)

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Cardiovascular Diseases Although there has been an evolution in treatment options, cardiovascular diseases continue to be major causes of morbidity and mortality globally (Squillaro et al. 2016). Hence, in recent years, MSC-sourced secretome has been used for cardiac regeneration as the fast-emerging cell-free therapy approach (Lei and Haider 2017). Experimental animal studies have shown that secretome treatment per se improved left ventricular function, heightened myocyte nuclear density and neovascularization, and reduced apoptosis/fibrosis in the ischemic heart (Baez-Jurado et al. 2019; Shabbir et al. 2009; Dai et al. 2007). Nonetheless, the cardioprotective effects of secretome-based treatment have been attributed to the MSC-derived EVs that affect the cellular targets, i.e., cardiomyocytes, endothelial cells (ECs), and cardiac stem cells (CSCs) (Suzuki et al. 2016; Zhang et al. 2016b; Silva et al. 2017). Actually, and by using experimental animal models of cardiovascular dysfunction, several studies showed that MSC-derived exosomes could notably reduce the infarct size and enhance global cardiac function (Harrell et al. 2019; Suzuki et al. 2016; Silva et al. 2017; Lai et al. 2010). Arslan et al. in one of such cases demonstrated that exosome-treated animals showed suggestive conservation of the left ventricular geometry and the contractile performance through the re-establishment of myocardial bioenergetics, a reduction in oxidative stress, and activation of the PI3K/Akt pro-survival signaling pathway (Harrell et al. 2019; Arslan et al. 2013). Explicitly considering the cardiomyocytes, various studies have shown that the administration of MSC-derived exosomes increased the survival and proliferation of cardiomyocytes by inhibiting apoptotic signaling pathways and stimulation of autophagy (Harrell et al. 2019; Ju et al. 2018). For example, Liu et al. reported that MSC-derived exosomes promoted the autophagy process by upregulating the AMPK/mTOR and Akt/mTOR signaling pathways (Harrell et al. 2019; Liu et al. 2017). Similarly, Cui et al. showed that the protection of cardiomyocytes against apoptosis was due to an increased expression of anti-apoptotic protein, Bcl-2, and concomitant downregulation of pro-apoptotic Bax protein expression. In addition, they also observed suppression of caspase-3 activity in response to exosome treatment (Harrell et al. 2019; Cui et al. 2017). Also, treatment with exosomes successfully supported microvascular regeneration (Harrell et al. 2019; Gong et al. 2019; Ma et al. 2018). Supporting these findings, the delivery of specific microRNAs presented the capacity to modulate CSCs, by either stimulating their proliferation and migration or increasing their capacity of self-renewal (Harrell et al. 2019; Zhang et al. 2016b). Furthermore, the cardiac benefits of MSC-derived exosomes were associated with the inhibition of the inflammatory reaction. Indeed, extracellular vesicles contributed to the regulation of immune cell function, controlling macrophages’ polarization, and diminishing the influx of inflammatory cells into treated hearts (Harrell et al. 2019; Silva et al. 2017; Sun et al. 2018). This is accomplished by delivering exosomal payload of miRNAs to the heart, the composition of which may be altered by exogenous manipulation of the cells of their origin (Haider and Aramini 2020).

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Kidney and Lung Injuries Some of the other models in which MSCs’ secretome has displayed ameliorative benefits include chronic kidney disease (CKD) and acute kidney injury (AKI). Regarding CKD, the administration of MSC-derived secretome promoted marked (reno)protective effects, which were revealed by a decrease in glomerular damage and hypertension besides improved glomerular endothelial regeneration and genome integrity preservation via active DNA repair (Van Koppen et al. 2012). Likewise, the same research group observed in vitro that treatment with secretome improved endothelial cell migration and angiogenesis, followed by a reduction in tubular inflammation and fibrosis, which indicated an anti-inflammatory and anti-fibrotic effect. These data were consistent with the previously published studies in other experimental disease models (Van Koppen et al. 2012). For example, Togel et al. attributed the renoprotective effects of the secretome to various growth factors, including VEGF, HGF, and insulin-like growth factor (IGF) (Tögel et al. 2009). Nevertheless, reports in AKI experimental models have encouraged the use of MSC-derived microvesicles for kidney protection (Gatti et al. 2011; Zhou et al. 2013b; Bruno et al. 2012). In fact, Gatti et al. showed that one-time administration of microvesicles suppressed apoptosis and boosted tubular epithelial cell proliferation, which considerably attenuated renal function impairment (Gatti et al. 2011). Some other research groups have confirmed that the pro-survival effects of secretome were mostly ascribed not only to anti-apoptotic effects but also to an amelioration of oxidative stress (Zhou et al. 2013b, Bruno et al. 2012). Parallel to this, lungs’ MSC-derived exosomes have been reported in experimental animal models of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), asthma, and idiopathic pulmonary fibrosis (Squillaro et al. 2016; Harrell et al. 2019). Considering ALI and ARDS, there is an obvious lack of therapeutic options to prevent/treat injury and/or promote lung repair. For example, in vivo data have shown that secretome administration successfully attenuated lung inflammation and activation of macrophage to an M2 “healer” phenotype, partially influenced by the presence of IGF-I (Ionescu et al. 2012). In another report, researchers found out that ALI treatment with MSC-derived vesicles mostly targeted the immunomodulatory properties of angiopoietin-1 (Ang-1) that successfully led to a reduction in inflammation and vascular stabilization (Tang et al. 2017). In experimental ALI models, treatment with microvesicles significantly reduced pulmonary edema and decreased the influx of both inflammatory cells, proteins, and bacteria, in a keratinocyte growth factor (KGF)-dependent manner (Zhu et al. 2014; Monsel et al. 2015), thereby opening new therapeutic opportunities for this kind of disorders.

Central Nervous System Pathologies Cerebral homeostasis is preserved through complex and cohesive interactions between distinctive cell types, comprising neurons and glial cells. In the central nervous system (CNS) pathologies, the physiology of these cells gets highly changed, leading to

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impairments in their function and altering the protection and balance in the nervous tissues. Usually, brain disorders display typical biological hallmarks such as an intensification of reactive oxygen/nitrogen species production, protein aggregation and denaturation, secretion of apoptotic factors, metabolic alterations, mitochondrial dysfunction, and, as an outcome, huge percentages of neuronal and glial cell death populations. Since CNS lacks the capability of self-repair, or at least limited self-repair, it is challenging to design treatments that could prevent the progression of injury and concomitantly halt the cognitive and motor/non-motor functional decline during pathologies, such as traumatic brain injury (TBI), ischemic stroke, spinal cord injury, and neurodegenerative diseases (Teixeira et al. 2013; Baez-Jurado et al. 2019; Mendes-Pinheiro et al. 2020). In this regard, MSCs’ secretome has shown its potential as a therapeutic tool to brain tissue recovery and repair, owing to its capability to trigger and/or modulate endogenous neuro-restorative processes, such as neurogenesis, angiogenesis, and inflammation (Teixeira et al. 2013; Makridakis et al. 2013; Pinho et al. 2020; Mendes-Pinheiro et al. 2020).

Traumatic Brain Injury (TBI) TBI is a global health challenge that arises through external mechanical forces triggering injury and disrupting normal brain function (Pinho et al. 2020). Two main physiopathological phases are generally involved in this disorder, englobing firstly the mechanical impairments (disruption of the blood-brain barrier and disseminating axonal damage) and, secondly, chronic inflammation due to the excessive production of pro-inflammatory cytokines, mitochondrial breakdown followed by oxidative stress, and excitotoxicity (exaggerated glutamate levels) (Baez-Jurado et al. 2019; Muhammad 2019; Mendes-Pinheiro et al. 2020). As a consequence of these molecular events, patients develop physical, cognitive, and emotional deficits. Therefore, the use of MSCs’ secretome is being considered as a promising therapeutic opportunity to treat TBI, demonstrating potential modulatory effects in the lesioned microenvironment. Indeed, during the acute phase of the injury, treatment of TBI with secretome containing neurotrophic factors (NGF, BDNF, NT-3) supported rats’ neurological repair and reduced apoptosis (Kim et al. 2010). Moreover, the same authors have shown that TBI-treated rats showed improvement in motor function and cognitive performance, essentially due to an increase in VEGF and HGF expression, which was correlated with neurogenesis in the damaged tissue areas (Chang et al. 2013; Chuang et al. 2012). In an experimental acute TBI model, MSC-secreted soluble factors were significantly modified the expression of pro- and anti-inflammatory cytokines and modulated the serum levels of chemokines due to their immunomodulatory properties (Galindo et al. 2011). Similarly, Pischiutta et al. concluded that upon amniotic mesenchymal stromal cell-secreted metabolite administration, brain slices of TBI mice developed protective effects, including the promotion of M2 microglia polarization and neuronal rescue (Pischiutta et al. 2016). Additionally, in a study using a new in vitro experimental model of traumatic brain-like injury, researchers have found that hMSC-derived secretome could

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preserve cell morphology and their polarity index to increase wound closure, migration, and proliferation and control the oxidative stress by decreasing superoxide production (Torrente et al. 2014). Comparably, using adipose-derived stem cells’ (ASCs’) secretome, Tajiri et al. suggested that the secretome could support/boost endogenous repair mechanisms by improving motor/cognitive behavior but also by avoiding cortical and hippocampal damage. More importantly, they found that the major players involved in this efficacy were the lncRNAs (Tajiri et al. 2014). More recently, in an SH-SY5Y model of TBI, apart from diminishing neuronal cell death, the therapeutic application of ASC-derived secretome significantly increased mitochondrial function and reduced inflammatory cytokine IL-1β (Kappy et al. 2018). Consistent with the published data, RNA sequencing (RNAseq) revealed that hMSCs’ secretome administration normalized the expression of 49% of the genes disrupted by TBI, especially in specific pathways that were involved in immune cell signaling and infiltration, energy metabolism, receptor-mediated cell signaling, and neuronal plasticity and remodeling (Darkazalli et al. 2017). In an attempt to understand the underlying molecular mechanism, different groups showed that proteins such as Wnt3a and tissue inhibitor of matrix metalloproteinase-3 (TIMP3) had a role in the promotion of cerebral endothelial adherent junction integrity, neuroprotection, and neurocognitive function (Zhao et al. 2016; Gibb et al. 2015). Having all this in mind and following in the same direction, the use of the extracellular vesicles secreted by MSCs has gained particular interest in TBI. Indeed, treatment with MSC-derived EVs is described as a potential promoter of neurological recovery through either of the following mechanisms: (i) Modulation of inflammation-related pathways, in which they shifted microglia polarization and prevented reactive astrogliosis (ii) Inhibition of pro-apoptotic proteins and oppositely an increase in the expression of the anti-apoptotic ones (iii) Restoration of myelination deficits and white matter microstructure (iv) A significant increase in the neurogenesis of endothelial cells, neuroblasts, and mature neurons in the lesion site (v) Rescue of the sensorimotor function (specially learning and motor performance), opening new routes for future clinical translation (Williams et al. 2019; Cantinieaux et al. 2013; Thomi et al. 2019)

Spinal Cord Injury (SCI) and Ischemic Stroke (IS) Numerous studies have indicated that in addition to (neuro)protection, MSCs’ secretome may also support neural regeneration (Teixeira et al. 2013; Pinho et al. 2020; Cantinieaux et al. 2013; Martins et al. 2017; Cizkova et al. 2018; Park et al. 2010; Tsai et al. 2018; Haider et al. 2015; Guo et al. 2016). In SCI, the lesion causes the loss of neurons and glial cells, leading to high-level inflammation, demyelination, and pain (Teixeira et al. 2013; Pinho et al. 2020). MSC-derived secretome has already shown a neuroprotective role against apoptosis-activated macrophages

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(pro-inflammatory M1 microglia toward inflammation-resolving M2 cells) and pro-angiogenic activity in vitro (Cantinieaux et al. 2013). Additionally, it has also been demonstrated that MSCs’ secretome impacts axonal function, promoting their outgrowth through the action of trophic factors such as BDNF, HGF, and VEGF (Martins et al. 2017; Cizkova et al. 2018) and leading to the decrease of oxygenglucose deprivation-induced cell damage (Park et al. 2010) and intensification of neuronal connections (Tsai et al. 2018). Cantinieaux’s group reported that treatment with BM-MSCs led to an evident locomotor recovery in an experimental animal model. These results were supported by the pro-angiogenic tissue-protective effects of a cytokine cocktail (VEGF-A, VEGF-C, osteopontin, EG-VEGF/PK1, TAL1A, MMP-13, and FGF-BP, among others) present in the secretome (Cantinieaux et al. 2013). Corroborating with these results, studies have shown that injecting secretome into lesioned animals promotes neuroprotection, attenuation of cavity formation, and preservation of the spinal tracts. These observations correlated well with the recruitment of CD68+ cells with a concomitant reduction in oxidative stress and creation of an anti-inflammatory environment at either the lesion or parenchyma site (Haider et al. 2015; Guo et al. 2016). More recently, different groups have observed an overall motor recovery in SCI rats after treatment together with an increase in tissue sparing and axon density at the lesion site, which were remarkably correlated with the presence of GAP-43-positive axons, upregulation of Olig-2 and HSP70 protein levels, activation of autophagy, and lower levels of inflammatory cytokines (IL-2, IL-6, and TNF-α), respectively (Cizkova et al. 2018; Tsai et al. 2018). The mere administration of the vesicular fraction, including MSC-derived exosomes, contribute to the direct neuroprotective/neuroregenerative effects in experimental SCI models. Different research groups have reported that treatment with exosomal fraction in vivo successfully modulated the microenvironment of spinal cord lesions by delivering anti-inflammatory and pro-angiogenic factors (Lankford et al. 2018; Liu et al. 2019). It has been evidenced that MSC-derived exosomes suppressed inflammation via the direct generation of immunosuppressive M2 macrophages (Lankford et al. 2018) and decreased TNF-α, Interleukin (IL)-1α, and IL-1β, inhibited nuclear translocation of NF-κB, and increased IL-10 secretion, thereby leading to neurotoxic A1 astrocyte suppression (Liu et al. 2019; Huang et al. 2017). Also, the attenuation of neuronal cell apoptosis was observed due to abrogation of pro-apoptotic proteins (Bcl-2-associated X protein, Bax, activated caspase-3, and caspase-9) and an upregulation in the levels of anti-apoptotic ones (i.e., Bcl2; B-cell lymphoma 2), which correlated well with axonal regeneration, glial scar suppression, mitigation of the lesion size, and a better functional behavioral recovery (Huang et al. 2017; Lankford et al. 2018; Liu et al. 2019). In a similar approach, Dong et al. and Zhou et al. attributed the modulatory effects to the miRNA content of exosomes claiming that both MSC-derived EVs miR-21-5p and miR-133b are able to activate distinct signaling mechanisms, thus influencing positively the treatment of SCI (Zhou et al. 2019; Li et al. 2018). Curiously, in a comparative study of secretome versus vesicles, Lu et al. have reported that the administration of BMSC-derived EVs could sustain the integrity of the blood-spinal cord barrier (BSCB) by increasing the total number of pericytes in the barrier

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throughout the suppression of their migration (via the downregulation of NF-κB p65 signaling) (Lu et al. 2019). Similarly, the effects of treatment with MSC-derived secretome have also been observed in ischemic stroke (IS) conditions. IS is caused by a reduction in blood supply that leads to a multicell signaling phenomenon englobing the death of endothelial and neuronal cells, inflammation (with prominence for microglia activation), and white matter pathophysiology (Pinho et al. 2020; Vizoso et al. 2017; Xing et al. 2012). When applied in IS models, Huang et al. reported the secretome competence to block astrocytic cell death, raising their metabolism through the action of IGF-1 and BDNF (Huang et al. 2015). Moreover, they correlated these outcomes to the inhibition of p38 MAPK and JNK signaling pathways (Huang et al. 2015). Similar to other pathologies addressed already, the therapeutic potential of MSC-derived exosomes has been shown in the context of IS as well. Besides functional recovery, exosome treatment post-injury provided an appropriate external milieu for successful brain remodeling since it enhanced axonal density and synaptophysin-positive staining, thereby providing long-term neuroprotection due to higher angioneurogenesis levels and immunosuppression (Xin et al. 2013; Lee et al. 2016a; Doeppner et al. 2015). In line with this, Xi and colleagues have shown that upon MSC-derived exosome treatment, the presence of neuroblasts and endothelial cells in ischemic regions was significantly increased, demonstrating MSC-derived exosomes as key players contributing toward neuronal differentiation (Xin et al. 2013). Additionally, a novel role of EVs has been reported in the myelination processes in IS. The authors have reported that after IS, EVs could restore white matter integrity by interfering in the axonal sprouting and growth, oligodendrocyte formation, tract connectivity, and remyelination (Otero-Ortega et al. 2017).

Neurodegenerative Disorders Multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are the most common and well-known brain neurodegenerative pathologies with a higher incidence in the world’s population (BaezJurado et al. 2019; De Pedro-Cuesta et al. 2015; Cannon and Greenamyre 2011). Although different pharmacological treatments and surgical procedures improve the quality of life for these patients, long-term clinical recovery is not achieved. Hence, the progressive degeneration continues, leading to the consequent loss of neurons (Baez-Jurado et al. 2019). In search of alternative treatments for these brain disorders, stem cell-free strategy using stem cells’ secretome is also being considered (Drago et al. 2013). Various studies (both in vitro and in vivo) have described MSC-derived secretome as a stimulator of neurotrophic factor release (i.e., BDNF, a factor commonly affected in the majority of neurodegenerative disorders) and neuronal survival pathways (Vizoso et al. 2017) that allow the prevention of cell death (Drago et al. 2013; Fu et al. 2006) and promote neuronal cell survival, differentiation, and proliferation of pre-existing cells (Mita et al. 2015; Kim et al. 2015;

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Moraes et al. 2012; Teixeira et al. 2015; Teixeira et al. 2017). The authors have also observed induction of endogenous neurogenesis and synaptic activity in the main affected areas (e.g., the hippocampus in AD or the substantia nigra in PD) (Kim et al. 2015; Teixeira et al. 2017; Cova et al. 2010; Wang et al. 2010). Secretome treatment also reduced the accumulation of protein aggregates such as alpha-synuclein in PD (Oh et al. 2017), misfolded tau protein (AT tau) and β-amyloid plaques in AD (Zilka et al. 2011; Kim et al. 2012), and mutant Huntingtin (mHtt) in HD (Lee et al. 2016b), which per se are focal hallmarks of these diseases. Lastly, cell-free therapy partially rescues the representative phenotype of the disability (Baez-Jurado et al. 2019; Pinho et al. 2020; Drago et al. 2013; Wang et al. 2010; Reza-Zaldivar et al. 2018; Sadan et al. 2009; Teixeira et al. 2020; Kim et al. 2014; Mendes-Pinheiro et al. 2020). MSCs’ secretome can incur behavioral improvements predominantly at the level of the motor and cognitive functions (Mita et al. 2015; Kim et al. 2014, 2015; Teixeira et al. 2015, 2017; Zilka et al. 2011; Vilaça-Faria et al. 2019; MendesPinheiro et al. 2019). At organelle and molecular levels, there is significant modulation of oxidative stress, mitochondrial dysfunction, apoptosis, inflammation, and the activation of ubiquitin-proteasome system (Mita et al. 2015; Kim et al. 2014, 2015; Lee et al. 2016b; Reza-Zaldivar et al. 2018; Jarmalavičiūtė et al. 2015). Of particular interest were the results reported by Mendes-Pinheiro et al. using an experimental PD rat model. The authors reported that the administration of MSC-derived secretome led to a more efficient response when compared to the MSCs’ transplantation in terms of preserving the dopaminergic system (MendesPinheiro et al. 2018). In another exciting study, Katsuda et al. explored the use of MSC-derived exosomes in the context of AD. The authors have observed that after MSC-derived exosome delivery to an experimental animal AD model, there was a remarkable decrease in the levels of brain-soluble Aβ plates, a fact that was attributed to the presence of neprilysin in exosomes (Katsuda et al. 2013). Concerning HD, intrastriatally transplanted bone marrow-derived MSCs integrated well in the host brain. They showed trophic effects through an increase in laminin, von Willebrand factor (VWF), stromal cell-derived factor-1 (SDF-1) α, and the SDF-1 receptor CXCR4, which in turn enhanced angiogenesis in the damaged striatum (Lin et al. 2011). Finally, in experimentally induced demyelinating models such as MS, secretome application was found to elicit an effect in the oligodendrogenic niche of the subventricular zone that led to oligodendrogenesis and functional remyelination (Cruz-Martinez et al. 2016).

Clinical Approaches Based on Mesenchymal Stem Cell Secretome As earlier discussed, pre-clinical experimental studies using MSC-derived secretome have given encouraging data, which has led to its applications in clinical settings. Nevertheless, the number of clinical trials around this topic is still scarce (Fig. 3). According to the US National Institutes of Health official database, only 45 secretome-based clinical trials have been reported as of February 21, 2021.

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Fig. 3 Representation of the percentages of MSCs’ secretome-based clinical trials. Data acquired from www.clinicaltrial.gov

Most of these trials were primarily aimed to assess the biomedical potential of either secretome or their derivatives (e.g., exosomes), addressing their safety, feasibility, and efficiency in improving the clinical outcomes of the participating patients. Secretome translation from the bench to the bedside has been mainly covering the treatment of conditions like graft-versus-host disease (GvHD), hair loss, COVID-19 pneumonia, diabetes, cancer, infertility, as well as lung, cardiovascular, bone and cartilage, skin, neurological, and autoimmune diseases (Fig. 3) (NCT04213248, NCT02192736) (Zhou et al. 2013a; Fukuoka and Suga 2015; Shin et al. 2015; Katagiri et al. 2016; Kordelas et al. 2014; Dahbour et al. 2017). Notably, the employment of secretome in COVID-19-associated pneumonia therapy has boomed in the preceding year, with seven clinical trials reported on http://www.clinicaltrials.gov (last access: 21/02/21). Herein, the conjoint idea relies on investigating the effects of intravenous injection/inhalation of secretome components (especially exosomes) for the treatment of patients with moderate/severe SARS-CoV-2 infection (NCT04753476; NCT04276987; NCT04602442; NCT04491240; NCT04398303; NCT04747574) and also to explore their tolerance in healthy volunteers (NCT04313647) (Abraham and Krasnodembskaya 2020; Bari et al. 2020). The administration of secretome in the form of conditioned medium in clinical context has been illustrated in patients suffering from severe alveolar bone atrophy, alopecia (or hair loss), MS, and skin injury. In the completed clinical studies, the use of ADSC-derived secretome in alopecia and female pattern hair loss (FPHL) could restore hair density, revealing itself as a novel therapy for hair regeneration (Fukuoka and Suga 2015; Shin et al. 2015). In the case of alveolar bone regeneration, upon MSC-derived secretome administration, patients needing bone augmentation revealed mineralization, early bone formation, and reduced inflammatory signs.

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In this first-in-human clinical study, researchers demonstrated the safety of secretome as a potential modulator of osteogenic regenerative medicine (Katagiri et al. 2016). Similarly, in an open-label prospective phase I/II clinical study, MSC-derived secretome was used for the first time to treat MS patients. The authors reported an improvement in all the measuring tests and observed a correlation between the decrease of the lesion and increased IL-6, IL-8, and VEGF contents in the secretome and concluded that the intervention was clinically safe and feasible (Dahbour et al. 2017). Currently, the focus has shifted more to the clinical applications of MSC-derived exosomes. Several ongoing clinical trials have already reported the benefits of exosome-based therapy (http://www.clinicaltrials.gov). For instance, a preliminary study showed that the application of a specific regimen of MSCs’ derivatives in a GvHD patient (with skin and intestinal tract complications) yielded a significant and sustainable improvement of symptoms by reducing the release of inflammatory cytokines and attenuating the ongoing inflammation in the gut and skin (Kordelas et al. 2014). Additionally, they noticed that the treatment was well-tolerated and remained stable for 5 months, denoting a long-lasting therapeutic influence of the exosomes (Kordelas et al. 2014). Furthermore, based on the available initial reports, it is predictable that further trials would be initiated soon. In light of the already completed human clinical studies exploiting secretome’s activity, it seems that they have established some degree of safety and viability. In fact, no data has been presented relevant to any adverse effects, demonstrating that the delivery (local or systemic) of MSCs secretome as whole and/or MSCs-derived exosomes in patients with distinct illnesses might be a secure and viable therapeutic approach in the future.

The Benefits and Barriers of MSCs’ Secretome In this chapter, we have shown that secretome-derived products are appropriate to significantly improve numerous pathophysiological mechanisms in experimental animal models of diseases as well as clinical trials approved by national agencies. Exploiting MSC-derived secretome and its insoluble derivatives (i.e., vesicles, exosomes) in experimental and clinical settings offers several benefits to the domain of stem cell-based approaches and regenerative medicine. Firstly, when using secretome, it is possible to solve some concerns related to the transplantation of living and proliferative cell populations, such as overcoming the low cell survival that follows the procedure, reducing the large number of cells needed, providing less immunogenicity and tumorigenicity, and decreasing the possibilities to alter its phenotypic and therapeutic potential as observed with MSCs’ expansion in vitro (Vizoso et al. 2017; Teixeira and Salgado 2020). Moreover, MSC-derived secretome also possesses advantages in terms of manufacturing, storage, management, and product shelf-life (Teixeira et al. 2016). Secondly, time and cost of expansion/ maintenance of cultured stem cells in dynamic culture conditions (e.g., bioreactors) can be greatly reduced. Thirdly, due to the nonliving profile of the secretome,

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this can be easily stored without the use of potential toxic cryoprotectant agents, packaged, and safely transported without losing its effectiveness, which represents a critical statement for the economic viability. Fourthly, the evaluation of secretome safety, dosage, and potency may be much simpler and analogous to the regular pharmaceutical compounds than the cells; and finally, being deemed as a ready-to-go product implies that it is immediately available for treatment (Baez-Jurado et al. 2019; Vizoso et al. 2017; Teixeira and Salgado 2020; D'angelo et al. 2020; Praveen Kumar et al. 2019). Nevertheless, although promising, the translation of MSC-derived secretome to clinical practice remains a challenge. Unfortunately, defining the complete biochemical composition of the secretome is difficult as it is altered by many factors and involves multiple mechanisms, which are responsible for its beneficial properties (Pinho et al. 2020; Vizoso et al. 2017; Teixeira and Salgado 2020; D'angelo et al. 2020; Mendes-Pinheiro et al. 2020). Actually, a relevant issue that remains elusive is the identification and characterization of the secretome-derived extracellular vesicles and their contents (Abraham and Krasnodembskaya 2020; Drago et al. 2013; Basu and Ludlow 2016). Moreover, when using secretome, it is often hard to measure the activity and half-life of its components, as well as to access their pharmacokinetics, bio-distribution, tissue transport, and protein stability (Pinho et al. 2020; Teixeira and Salgado 2020).

Improvements Needed for Secretome Application as a Cell Transplantation-Free Tool As soon as we understand and interpret the diverging data of using multiple conditions and platforms, we will be of guarantee a homogenous, scalable, and applicable secretome product (Teixeira and Salgado 2020). Undeniably, to attain a true impact, concomitant progress in multiple fields has to be achieved (Makridakis et al. 2013). Therefore, as a way to understand and boost secretome applications, a complete characterization of MSC-derived secretome becomes vital not only to identify the full scope of biochemical factors (with a special focus on active molecules that may be oncogenic) but also to clarify if the molecules released can target the cell and tissue dynamics. Likewise, it is also essential to elucidate the molecular mechanisms and pathways underlying the secretome-mediated effects (Marques et al. 2018; Mendes-Pinheiro et al. 2020). For instance, thorough proteomic analysis, next-generation metabolomics-driven approaches, and system biology techniques (Drago et al. 2013) may contribute immensely to understanding the secretome profile of cells cultured under diverse conditions. At the same time, regulatory requirements for manufacturing and quality control must still be implemented (Pinho et al. 2020; Vizoso et al. 2017). Standardized protocols for secretome production, storage, and transport should be employed to limit heterogeneity and enrich the predictability of secretome-derived products. Linked to this, it will also be crucial to define an optimal timing for secretome collection (Teixeira and Salgado 2020), as well as a bio-manufacturing protocol for collection (Abraham and

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Krasnodembskaya 2020; Vizoso et al. 2017). Another important step forward should be the modulation of secretome and/or EV composition to create an optimal biocomponent and to personalize it according to each patient’s condition. Finally, it is essential to highlight that before its application in clinical practice, additional studies should be performed paying attention to the treatment regimen, including the route and frequency of administration, the volume of injection, and dose application timeline (Praveen Kumar et al. 2019; Mendes-Pinheiro et al. 2020). Additionally, group-to-group data sharing and comparisons must be performed as a way to bring the field closer to clinical applications and, at the same time, help to understand secretome interactions with diverse pathologies in a global perspective (Abraham and Krasnodembskaya 2020) (Makridakis et al. 2013; Pinho et al. 2020; Teixeira and Salgado 2020; Mendes-Pinheiro et al. 2020). Doing so, this can lead to the rational design of multiple secretome strategies to tackle dissimilar pathophysiological deficits in a multifaceted way.

Conclusion Recently, MSCs’ secretome has emerged as a hopeful therapeutic tool to the regenerative medicine field. Apart from differences in their sources and composition, the secretome has been demonstrating therapeutic advantages compared to the classical cell-based approaches. Nevertheless, standardized parameters in the secretome production and its complete characterization are still missing, which somehow has delayed its consideration by the regulatory agencies and, consequently, its translation to the clinics. Nevertheless, the secretome is already implied in clinical trials, demonstrating promising effects on various tissues and a plethora of targeted diseases. Therefore, the challenge for the years to come will be to understand the precise impact of the secretome in a targeted cell/area and make it the most suitable therapy for an issue by modulating it according to the biological/therapeutic necessity.

Cross-References ▶ Adipose Tissue-Derived Regenerative Cell-Based Therapies: Current Optimization Strategies for Effective Treatment in Aesthetic Surgery ▶ Advances, Opportunities, and Challenges in Stem Cell-Based Therapy ▶ Current State of Stem Cell Therapy for Heart Diseases ▶ Direct Reprogramming Strategies for the Treatment of Nervous System Injuries and Neurodegenerative Disorders ▶ Extracellular Vesicles Derived from Mesenchymal Stem Cells ▶ Mesenchymal Stem Cell-Extracellular Vesicle Therapy in Patients with Stroke ▶ Mesenchymal Stem Cells

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▶ Sources and Therapeutic Strategies of Mesenchymal Stem Cells in Regenerative Medicine ▶ Stem Cell Applications in Cardiac Tissue Regeneration ▶ Therapeutic Uses of Stem Cells for Heart Failure: Hype or Hope Acknowledgments The authors want to acknowledge the financial support from Prémios Santa Casa Neurociências Prize Mantero Belard for Neurodegenerative Diseases Research (MB-282019). This work was supported by the European Regional Development Fund (FEDER), through the Operational Programme Competitiveness and Internationalization (POCI), and by national funds, through the Foundation for Science and Technology (FCT), under the scope of projects UIDB/50026/2020, UIDP/50026/2020, and POCI-01-0145-FEDER-029751. This chapter has also been developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This chapter has been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBI-POCI-01-0145-FEDER-022122).

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Exosome-Based Cell-Free Therapy in Regenerative Medicine for Myocardial Repair Khawaja Husnain Haider and Mustapha Najimi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-Based Therapy for the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-Free Therapy Approach for the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theranostic Role of Exosomes in the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostic Applications of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosomes as a Treatment Option for the Injured Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Exosomes in cardiovascular theranostics are gaining popularity due to their immense diagnostic and therapeutic potential generally ascribed to their specific payload and temporospatial release under a specific set of conditions. A recently published bibliometric research analyzing 1039 studies has shown a flourishing trend in cardiovascular medicine. Exosomes constitute an integral part of the paracrine secretions of stem/progenitor cells, which significantly contribute to intercellular communication by transferring signaling molecules between communicating cells. Interestingly, their payload can be modified by the physical,

K. H. Haider (*) Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] M. Najimi Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCLouvain), Brussels, Belgium e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_42

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pharmacological, or genetic manipulation of the parent cells from which they have been derived. Alternatively, more recent studies have developed protocols to load them with the payload of interest for specific therapeutic applications. Similarly, they are also released from various myocardial cells, including cardiomyocytes, in response to injuries with diverging payload profiles, which can be used for diagnostic purposes. This chapter discusses in-depth mesenchymal stem cell (MSC)-derived exosomes for their theranostic application in cardiovascular pathologies encompassing in vitro, small experimental animal models as well as translational and clinical studies. Keywords

Cell-free therapy · Exosomes · Heart · Infarction · Paracrine · Stem cells Abbreviations

AMI BM CABG CSCs CXCR4 DOXO ECs EPCs ESCs EVs GLUT GMP GP Hsp HUVECs IL IONP-MSCs iPSCs LV miR MSCs PCI PEP SDF-1α Sfrp-2 STG TIMP-2 TNF-1α

Acute myocardial infarction Bone marrow Coronary artery bypass grafting Cardiac stem cells Chemokine receptor-4 Doxorubicin Endothelial cells Endothelial progenitor cells Embryonic stem cells Extracellular vesicles Glucose transporter Good manufacturing practice Glycogen phosphorylase Heat shock protein Human umbilical vein endothelial cells Interleukin Iron oxide nanoparticle loaded MSCs Induced pluripotent stem cells Left ventricle MicroRNA Mesenchymal stem cells Percutaneous coronary intervention Purified exosome preparation Stromal-cell-derived factor-1α Secreted frizzled protein-2 Shear thinning hydrogel Tissue matrix metalloproteinase inhibitor-2 Tumor necrosis factor-1α

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Introduction Cell-Based Therapy for the Heart Based on encouraging data from small experimental animal models and translational studies, the use of stem-/progenitor-cell-based therapy has shown safety and feasibility in repopulating an infarcted myocardium with functionally competent neomyocytes and in reversing myocardial injury to preserve global contractile heart function. Notable and most extensively studied cell types used for cell-based therapy include bone marrow (BM)-derived mesenchymal stem cells (MSCs), BM-derived mononuclear cells, endothelial progenitor cells (EPCs), skeletal myoblasts, cardiac stem/progenitor cells (CSCs/CPCs), adipose tissuederived MSCs, embryonic stem cells (ESCs) and their derivative cardiomyocytes, and, more recently, induced pluripotent stem cells (iPSCs) and their derivative cardiomyocytes and EPCs. Each one of the cell types has specific advantages and limitations and is still being scrutinized to find an ideal cell type, which could offer the best compromise between limitations and advantages (Young and Schäfer 2015). Among all these cell types, MSCs have already progressed to the advanced phases of clinical assessment in diverse patient populations. According to a recently published systematic review, there are 1138 clinical trials have been registered on ClinicalTrials.gov, of which 60% are phase II while 30% are phase I trials with BM as the primary source of MSCs in most of the clinical trials and all of the trials reporting the safety of the procedure (Rodríguez-Fuentes et al. 2020). Irrespective of the cell type used, the stem-cell therapy approach is undoubtedly superior to contemporary treatment approaches in addressing the repair of the injured parenchyma of a cardiac architecture to limit the vicious progression of the disease process. It compensates for the loss of functioning cardiomyocytes, something not offered by any other treatment modalities. However, the further progression of the cell-based therapy approach is happening at a slower pace due to many factors encompassing the cell quality to meet the clinical-grade good manufacturing practice (GMP)-certified cell preparations with reproducible composition, to their biological characteristics, and uniform functionality, all of which remain problematic and time-intensive proposition (Bettonville et al. 2021). For further reading on the topic, please refer to ▶ Chap. 10, “Mesenchymal Stem Cells for Cardiac Repair,” contributed by AlKhani et al. in this volume, which provides a detailed account of the subject relevant to the experience of working with MSCs from small animal experimental models to large experimental animal models and in the randomized clinical trials in heart failure patients. Similarly, for the quality aspects of cell preparation, please refer to a recently published literature review, which discusses in depth the issue of the quality of cell preparation and the possible areas of improvement therein (Haider 2018).

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Cell-Free Therapy Approach for the Heart As discussed earlier, cell-based therapy has shown promise both in experimental animal models and clinical settings. Despite all the advantages, cell-based therapy has some limitations, which have significantly slowed its progress to the clinics. For example, the extensive death of donor cells, especially during the acute phase of engraftment; limited differentiation; the integration of the capacity of the transplanted cell; etc. severely hamper the desired outcome. Although various protocols have been tried and developed, we are still lacking in expertise to eliminate these issues in toto. For example, to overcome the issue of extensive donor cell death, protocols ranging from transient immunosuppression to the preconditioning of donor cells have been developed, tried, and reported, but the problem continues to persist (Haider et al. 2004; Xiao et al. 2004; Haider and Ashraf 2012). On the same note, the priming of cells through genetic modifications, cytokine and growth factor treatment, pharmacological and small molecule treatment, etc. has been investigated to enhance the cardiogenic differentiation of stem/progenitor cells (Noronha et al. 2019; Ullah et al. 2021); however, there are still many limitations on a clinical perspective. The paracrine activity of stem cells, which involves the release of both soluble and insoluble factors, constitutes an integral part of their therapeutic benefits post engraftment in the injured heart (Haider and Aziz 2017; Lei and Haider 2017). Based on the paracrine activity of stem cells, researchers have, more recently, started to focus on the cell-free therapy approach to avoid some of the potential limitations of cell-based therapy, such as difficulties in the clinical-grade manufacturing of cells, prevention of a likely tumorigenic potential, transmission of infection from the cell donor, immune rejection of the cells and cell-derived tissue graft, possible undesired lineage differentiation or no differentiation of the donor cells, etc. (Haider and Aslam 2018). Furthermore, the cell-free therapy approach also alleviates the need for in vitro cell expansion to achieve millions of cells needed for cell-based therapy. A modest outcome from the completed and ongoing clinical trials necessitates alternative approaches for a better therapeutic outcome (Haider 2018). As discussed earlier, the soluble and insoluble components of the cell secretome are rich in bioactive molecules (Rahimi et al. 2021). Although both secretome components have given encouraging results in the experimental animal models, the current focus is on extracellular vesicles (EVs) in general and exosomes in particular. The interest of researchers is directed toward exosomes in regenerative medicine, as may be judged by the fact that the keyword search on Pubmed yields more than 15,000 publications, during the last 5 years, primarily focused on their theranostic applications.

Theranostic Role of Exosomes in the Heart Although exosomes of plant and human tissue origin have progressed for clinical assessment, the use of human-tissue-derived exosomes is much more established in terms of their biological characterization, payload analysis, and GMP compliance.

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For their physical and biological characteristics and cell-specific payload, they are emerging as promising tools for cardiovascular theranostic applications (Wumei et al. 2021; Jayaraman et al. 2021). However, for clinical applications, exosomal preparations should conform to the minimum standards of GMP. Various research groups and drug manufacturing companies are coming up with their optimized protocols to achieve GMP in the manufacturing of exosomes to ensure high exosome yield with minimum levels of extraneous materials, i.e., xenogenic proteins, serumderived exosomes, etc. (Pachler et al. 2017; Andriolo et al. 2018). However, the Food and Drug Administration (FDA) has yet to approve any of these exosome preparations for routine clinical use. Many details about the characteristics, properties, and cargo of exosomes are also available through online sources and databases, like ExoCarta (http://www.exocarta.org), ExoBCD (https://exobcd.liumwei.org), Vesiclepedia (http://microvesicles.org), EVpedia (http://evpedia.info), exoRBase (http://www.ExoRBase.org), and exRNA Atlas (https://exrna-atlas.org), which provide bioinformatics-based information in a comprehensive and detailed manner (Sahoo et al. 2021). These online resources provide information seekers and researchers with an update on exosome and EV biology and an analysis of their payload.

Diagnostic Applications of Exosomes Like any other cell, cardiac cells, i.e., cardiomyocytes, telocytes, CPCs, etc., also release exosomes with specific payloads and functions (Vrijsen et al. 2016; Liao et al. 2021). For example, the profiling of cardiomyocyte-derived exosomes has revealed the presence of miR-208a, which gets transferred to the cardiac fibroblasts as a mechanism for cross talk between the two cell types to initiate myocardial fibrosis in the event of myocardial injury (Yang et al. 2016). Similarly, uptake of the injured cardiomyocyte-released exosomes by the transplanted bone marrow MSCs in the infarcted mouse heart has been shown to initiate cell-to-cell communication resulting in loss of the transplanted cell. This problem has significantly hampered the progress of cell-based therapy (Hu et al. 2018). These in vivo data were supported by MSCs cultured in vitro in the conditioned medium and exosomes derived from cardiomyocytes cultured under oxidative stress. Molecular studies revealed significantly high caspase-3 activation and decreased Bcl2/Bax in MSCs, which showed more elevated apoptosis under oxidative stress. On the other hand, Ribeiro-Rodrigues and team have demonstrated that cardiomyocytes cultured under ischemic conditions released exosomes, which had angiogenic potential when added to cultured endothelial cells (ECs) (RibeiroRodrigues et al. 2017). ECs cultured in the presence of H9C2 and primary cardiomyocyte-derived exosomes promoted EC proliferation, protection against oxidative stress, and enhanced tubulogenesis as compared to normal cardiomyocytederived exosomes. The profiling of exosomes from cardiomyocytes cultured under ischemic conditions showed that they were rich in metalloproteases and miR-143 and miR-222, which might be attributed to their enhanced angiogenic potential.

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In summary, the cardiomyocyte-released exosomes are integral mediators of their communication with other cells through the inclusion of specific payloads, such as heat shock proteins (Hsp)-20, 60, and 70, which regulate their apoptosis and survival; inflammatory factors IL-6 and TNF-a, which are responsible for cardiac remodeling; GLUT1, GLUT4, and glycolytic enzymes, which are involved in glucose transport and metabolism; and a long list of miRs, which regulate various cellular functions (Yu and Wang 2019). Given the significant role of EVs as mediators of intercellular communication, various research groups have focused their research on defining a role for cardiomyocyte-derived exosomes released as biomarkers of myocardial injury (Doran and Voora 2016). They have predominantly focused on the analysis of differential exosomal payload changes in response to myocardial injury. Recent advances are directed toward using a liquid biopsy approach, which involves sampling and the analysis of biofluids to detect the presence of exosomes with a specific payload for diagnostic applications (Valencia and Montuenga 2021). In the cells of the cardiovascular system, such as cardiomyocytes, ECs, smooth muscle cells, and other stem/progenitor cells in the heart, exosomes have a significant role in development, injury, and disease progression (De Freitas et al. 2021). A recently published systematic review has published the commonalities and differences of exosomes and their payloads in the six different types of cardiac-musclederived cells (Xu et al. 2019). In this regard, exosomal miR payload may have remarkable diagnostic and prognostic applications and is being exploited as a biomarker (Zamania et al. 2019). Moreover, the number of exosomes and the cell type of their origin significantly contribute to their diagnostic and prognostic value (Zarà et al. 2020; Tian et al. 2021). An example is the quantification of released EVs in response to doxorubicin (DOXO)-induced cardiac injury in an experimental mouse model (Yarana et al. 2018). The authors of the study observed a significant increase in the number of circulating EVs released in response to DOXO-induced injury. Moreover, the released EVs were rich in liver, heart, and muscle-related isoforms of glycogen phosphorylase (GP), while treatment with cardioprotective agents significantly reduced the EV payload of GPs. Beaumir et al. studied altered EV-miR payload as a potential biomarker of cardiotoxicity in nine client-owned dogs, which received five doses of DOXO therapy for sarcoma (Beaumier et al. 2020). The authors observed significant changes in at least ten EV-derived miRs, among which miR-502 showed a substantial increase while miR-107 and miR-146a recorded a considerable reduction in expression levels. Deddens et al. have reported spatiotemporal release characteristics of EVs in response to myocardial ischemia (MI)/reperfusion injury and profiled their miR payload in experimental murine and porcine models (Deddens et al. 2016). They observed the release of muscle-specific miRs (myomiRs), i.e., miR-1, miR-133b, miR-208b, and miR-499, in plasma after ischemia-reperfusion injury (miR-499 showed the highest levels in the plasma after MI injury). Their plasma levels were well correlated with cardiac Tn-I release, a well-established biomarker of myocardial injury (Haider and Stimson 1993). Interestingly, both miRs and exosomes appeared

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simultaneously in the plasma samples upon cardiac injury, and exosomes were found rich in miR-133b, miR-208b, and miR-499 but not miR-1. A position paper by the working group on the cellular biology of the heart provides an excellent review of literature on the topic for further reading (Sluijter et al. 2018).

Exosomes as a Treatment Option for the Injured Heart Exosomes derived from various stem/progenitor cells, i.e., ESC-derived cardiovascular progenitors, EPCs, iPSCs, adipose-tissue-derived MSCs, etc., have been extensively investigated for their therapeutic potential for myocardial repair (Barile et al. 2014; Cui et al. 2017; Chen et al. 2018; Kurtzwald-Josefson et al. 2020; Wu et al. 2020; Chung et al. 2020). A recently published systematic review based on the analysis of 24 experimental animal studies revealed that MSC-derived exosome treatment significantly improved cardiac function due to enhanced angiogenic response in the infarcted myocardium, besides reduced cell death (Meng et al. 2021).

Exosome in Experimental Animal Heart Models The cytoprotective effects of MSC-derived exosomes have been investigated in many recently published experimental animal studies on myocardial damage (Table 1). Liu et al. used exosomes derived from naïve MSCs to rescue ischemiareperfusion injury to neonatal cardiomyocytes in vitro and post intramyocardial injection in an experimental rat heart model of ischemia-reperfusion injury (Liu et al. 2017). The authors observed significantly attenuated myocardial injury. Molecular mechanistic studies in vitro showed internalization of exosomes by the cardiomyocytes and rescued them from H2O2 oxidative stress by inducing their autophagy via activation of AMPK/mTOR and Akt/mTOR signaling pathway (Liu et al. 2017). In a similar study, treatment with bone marrow MSC-derived exosomes revealed the repression of PTEN in H2C9 cells via miR-486-5p and the activation of PI3K/Akt signaling, which rescued the cells from hypoxia/reoxygenation injury (Sun et al. 2019). Zhao et al. have reported that MSC-derived exosomes via miR-182 mediated the polarization of M1 macrophages to M2 macrophages both in vitro and in vivo, with toll-like receptor 4 as its downstream target (Zhao et al. 2019). Besides other biomolecules, MSC-derived exosomes show a differential expression of both the negative, i.e., miR-130, miR-378, and miR-34, and positive, i.e., miR-29 and miR-24, regulators of cardiac function as their miR payload can be altered according to the intended outcome (Shao et al. 2017; Bellayr et al. 2017). Hence, MSCs have been manipulated in vitro to alter their miR payload for mircrining the injured heart using exosomes as carriers of the desired miRs (Haider and Aramini 2020). For example, MSCs have been genetically modified for myomiRs (myocardium-related miRs), i.e., miR-1, miR-133, miR-208, and miR-499, to enhance their cardiac differentiation (Huang et al. 2013; Lee et al. 2013; Neshati et al. 2018). Various research groups have assessed the effect of genetically modified MSC-derived exosomes for miR-125b expression on

I/M injection

Mice BM MSCsderived exosomes loaded with miR-132

Ma et al. (2018)

Intramyocardial injection

I/M

Exosomes derived for mouse BM-derived MSCs overexpressing GATA4

He et al. (2018)

Delivery mode I/V injection

Xiao et al. (2018) Human BM-derived MSCs

Source of exosomes BM MSC-derived exosomes

Author/year Sun et al. (2018)

MI by LAD ligation

MI

MI

Model type Doxorubicin-induced cardiomyopathy

Animal Main findings Mice Treatment with MSCs reduced cardiomyocyte apoptosis, reduced inflammatory cell infiltration as compared to the PBS-treated control group, and significantly reduced macrophage infiltration in the infarcted myocardium, JAK/STAT pathway activation. A significantly reduced infarct size and improved cardiac function were observed Mice BM-derived MSCs were transduced with a lentiviral vector encoding for GATA4 and cultured for exosome collection. Exosomes protected cardiomyocytes in vitro under hypoxic stress, while treating MSCs with exosomes increased cardiac marker expression and protected them against anoxic stress. It significantly increased LVEF and LVFS posttreatment in the infarcted rat heart, with increased blood vessel density, increased cardiac c-kit+ cell homing, and reduced cardiomyocyte apoptosis Mice There was a significant reduction in autophagic flux and death in neonatal cardiomyocytes, under hypoxia, cultured with MSC-derived exosomes and p53-Bnip3 signaling. These changes were abolished by exosomal inhibitor GW4869 and the inhibition of miR-125. In vivo treatment in a mice model of MI significantly improved infarct size and cardiac function, which were abolished by exosomes derived from MSCs treated with miR-125 Mice Internalization of miR-132-loaded exosomes by the incubated HUVECs with the reversal of its target gene RASA1 significantly increased tube formation. RASA1 was confirmed by luciferase assay as the downstream target of miR-132. Treatment of mice with infarcted hearts led to increased neovascularization in the peri-infarct area and preserved global cardiac function

Table 1 A summary of preclinical trials utilizing bone-marrow-derived MSCs in the small animal models of heart diseases

1014 K. H. Haider and M. Najimi

Spray formulation

Yao et al. (2021)

AMI

Ma et al. (2017)

Human umbilical cord- I/V injection MSCs with or without after coronary expressing artery ligation Akt-derived exosomes

MI

Shao et al. (2017) Rat BM-derived MSCs I/M

AMI

MI

I/M

Bian et al. (2014) Hypoxia-treated human BM-derived MSCs

MSC-derived exosomes

I/R injury

Zhao et al. (2019) Mouse BM-derived MSCs

Rats

Rat

Mice

Rat

Mice

Exosome-Based Cell-Free Therapy in Regenerative Medicine for. . . (continued)

MSC-derived exosomes were injected I/M immediately after I/ R injury. It suppressed inflammatory response. There was a significant reduction in systemic macrophages. There was a significant modification of M1 macrophages to M2 macrophages in vivo and in vitro with the vital role of miR-182 The study used the therapeutic efficacy of exosomes from hypoxia-treated human BM-derived MSCs. In vitro studies showed rapid internalization of exosomes by cultured HUVECs, which showed increased migration, proliferation, and tubulogenesis. I/M injection of exosomes significantly reduced fibrosis and infarct size and increased regional blood flow recovery due to angiogenesis. Overall, there was preserved cardiac function Designing, fabrication, and testing of a spray of exosomes in biomaterial – the treatment significantly improved cardiac function, reduced cardiac fibrosis, and promoted endogenous angiomyogenesis in the postinjury heart The study compared the efficacy of MSCs and their derived exosomes for myocardial repair. Treatment with exosomes significantly reduced myocardial fibrosis and inflammation and recovered cardiac function. In vivo studies showed MSCs and their derived exosomes had similar miRNA profiles. Also, exosome treatment of H2C9 increased their proliferation, protecting them against oxidative stress, and inhibited TGF-b-induced transformation of fibroblasts to myofibroblasts Endothelial cell function, i.e., proliferation, migration, and tubulogenesis, increased in vitro upon treatment with AktMSC-derived exosomes. Akt-exosomes were rich in PDGFD. Treatment of MI with Akt-exosome significantly improved cardiac function mediated by myocardial angiogenesis

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MSCs, I/M MSC-exosomes, exosomes from MSCs cultured in GW4869, & MSCs with antimiR-210

Cheng et al. (2020) MI

MI by LAD ligation

Model type Acute MI

Animal Main findings Rat I/M delivery of BM-derived MSC exosomes without or combined with I/V injection of atorvastatin-treated MSCs on day 1, 3, or 7. Echocardiography showed the most significant improvement in cardiac function, reduced infarct size in the combined treatment group receiving cells on day 3 after AMI. This was assigned to increased neovascularization, higher recruitment of MSCs, and reduced inflammatory response due to exosome treatment, which also reduced apoptosis of the recruited cells Rat I/M injection of MSC-derived exosomes in hydrogel enhanced their retention at the site of injection. There was a significant reduction of cardiomyocyte apoptosis and polarization of macrophages on day 3 after injection. At 4 weeks after delivery, scar thickness increased, heart function improved significantly in the hydrogel-exosometreated group of animals Rat The study determined the role of miR-210 in cardioprotection by exosomes derived from MSCs through a gain- or loss-of-function approach. MSC-derived exosomes significantly protected cardiomyocytes under hypoxia. Direct I/M injection of exosomes significantly reduced infarct size and area of fibrosis, with the significant role of miR-210 as a mediator of therapeutic outcome and AIFM3 as a downstream target

Abbreviations: AMI acute myocardial infarction, BM bone marrow, miR microRNA, I/M intramyocardial, I/V intravenous, LAD left anterior descending artery, MI myocardial infarction, MSCs mesenchymal stem cells, p53-Bnip3 B-cell lymphoma 2-interacting protein 3, PDGF-D platelet-derived growth factor

Three to five passages I/M injection Rat BM-MSC-derived 30 min after exosomes in alginate ligation gel

Lv et al. (2019)

Delivery mode I/V delivery of cells & I/M delivery of exosomes

Source of exosomes Atorvastatin-treated rat BM-derived MSCs & MSC exosomes

Author/year Huang et al. (2019)

Table 1 (continued)

1016 K. H. Haider and M. Najimi

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ischemia/reperfusion injury in the heart (Chen et al. 2019, 2020; Qiao et al. 2019), which was previously shown to reduce cardiomyocyte apoptosis due to ischemia/ reperfusion injury via tumor necrosis factor receptor-associated factor (TRAF)mediated activation of NF-kb (Xiaohui et al. 2014). The authors observed that intervention with miR-125b carrying exosomes increased cell viability and reduced apoptotic ratio with the concomitant downregulation of Bax and caspase-3 and the upregulation of Bcl2. Although the miR-centric explanation of the beneficial effects has gained vast acceptance among researchers in the field, some researchers have challenged this due to the low copy number of miRs present in the exosomes and attributed the beneficial effects to a protein-centric explanation based on the presence of enzymes, growth factors, etc. (Eisenstein 2020). Although the data from naïve MSC-derived exosomes have been encouraging, various strategies have been adopted to accentuate the therapeutic benefits of MSC-derived exosomes by manipulating their payload for delivery. This can be achieved by directly loading the payload of interest into the exosomes or alternatively by manipulating the cells from which the exosomes are to be derived. The latter strategy involves the physical, genetic, chemical, or pharmacological manipulation of the cells in culture to enhance the rate of exosome release as well as to modify their payload to achieve the desired benefits. For example, Bian et al. used hypoxia-treated MSC-derived vesicles of 100-nm size for the treatment of experimentally infarcted rat heart (Bian et al. 2014). The intramyocardial injection of vesicles significantly enhanced angiogenic response in the infarcted heart, which led to improved regional blood flow, reduced infarct size, and improved diastolic performance. The data were supported by in vitro studies during which vesicles were internalized by cultured human umbilical vein endothelial cells (HUVECs), which showed enhanced proliferation, migration, and tubulogenesis. The authors inferred that treatment with MSC-derived vesicles (a mix of exosomes and microvesicles) had cardioprotective effects through enhanced angiogenic response. To further dissect the mechanism, Wang et al. analyzed hypoxia-treated MSC-derived EVs for their payload and observed that the EVs were rich in miR-210 (Wang et al. 2017). The abrogation of miR-210 significantly abrogated EV angiogenic potential in vitro and in vivo in a murine heart model of acute myocardial infarction (AMI). It is pertinent to mention that hypoxamir-210, a member of the hypoxia-inducible factor1a-dependent miR family (Haider et al. 2009), is overly expressed in hypoxiapreconditioned MSCs, the transplantation of which resulted in an angiomyogenic repair of the infarcted rodent heart post engraftment, besides cytoprotection (Kim et al. 2009, 2012a, b). A summary of the exosome-based treatment strategy for myocardial infarction is provided in a recently published review of literature by Wang et al. (2021). Alternatively, the genetic manipulation of MSCs for the overexpression of various genes has been shown to significantly alter the payload of their derivative exosomes and hence their therapeutic benefits in treating infarcted myocardium. For example, Liu et al. genetically modified MSCs to overexpress macrophage migration inhibitory factor (MIF) and later cultured the cells for exosome collection (Liu et al. 2020). The treatment of neonatal cardiomyocytes with exosomes

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significantly reduced their apoptosis as well as mitochondrial fragmentation under serum-/nutrient-deprived culture conditions. The intramyocardial injection of exosomes significantly restored myocardial function in an experimental rat heart model of myocardial infarction and caused significantly reduced cardiomyocyte apoptosis in the recipient rat hearts. Ni et al. genetically modified MSCs with tissue matrix metalloproteinase inhibitor-2 (TIMP-2), a significant determinant of myocardial remodeling after MI (Ni et al. 2019). The genetically modified cells were cultured in vitro for exosome collection, which was then successfully used for treating experimentally infarcted rodent hearts. The intramyocardial injection of exosomes at multiple sites in and around the infarcted zone increased Akt expression, which was related to the highlevel expression of secreted frizzled protein-2 (Sfrp-2) in the TIMP-2 overexpressing MSC-derived exosomes, resulting in reduced cardiomyocyte apoptosis, decreased oxidative stress, and remodeling with the involvement of Sfrp-2/Akt signaling. Some of the other transgenes used to modify MSCs to obtain exosomes include Akt, SIRT1, GATA4, CXCR4, SDF-1α, etc. (Ma et al. 2017; Kang et al. 2015; He et al. 2018; Gong et al. 2019; Huang et al. 2020b). Most of these studies have reported a single transgene overexpression strategy in the cells. It would be interesting to characterize the cells for the payload profiling of their derivative exosomes after multitransgene modification to observe how the cells adjust for multitransgene overexpression in terms of exosome payload composition (Konoplyannikov et al. 2013). The strategy of pharmacologically manipulating MSCs to alter their derived exosomal contents has also been reported by many research groups. The primary advantage of pharmacologically conditioning MSCs is that the drugs used to condition the cells have already been assessed for human use. Huang et al. treated MSCs with atorvastatin, a commonly used lipid-lowering medication with pleiotropic functions, and characterized their derivative exosomes (Huang et al. 2020a). In vitro studies revealed enhanced EC survival, migration, and tubulogenesis, while the intramyocardial injection of exosomes significantly reduced infarct size and enhanced cardiomyocyte protection and angiogenesis. It inhibited the expression of TNF-1α and IL-1 in the peri-infarct zone in an experimental model of acute MI. The same researchers have also used a combinatorial approach involving concomitant treatment with atorvastatin-preconditioned MSCs and MSC-derived exosomes in a rodent model of AMI (Huang et al. 2019). Apart from atorvastatin, other pharmacological agents used to prime the cells to modify their exosome payload include oxytocin, pioglitazone, doxorubicin, curcumin, t-PA, etc. (Hu et al. 2021; Han et al. 2021). As an alternative to manipulating the cells, the noncellular manipulation strategy involves isolating the exosomes and their direct loading with the payload molecules of interest to tailor-made them according to their needs. Akin to cellular manipulation, the noncellular strategy also involves loading exosomes with various biomolecules, proteins, miRs, siRNA, etc. This is achieved by multiple well-established protocols based on sonication, electroporation, transfection, etc., while in some

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cases, the desired compounds are mixed and incubated with exosomes at room temperature (Fu et al. 2020). The other strategy being developed to enhance the therapeutic efficacy of exosomes is to increase the myocardial-directed/specific delivery of exosomes to treat myocardial infarction (Vandergriff et al. 2018). The conjugation of exosomes with cardiac homing peptides enhanced their homing into the ischemic myocardium after intravenous injection while drastically offsetting uptake by nontarget tissue (Vandergriff et al. 2018).

Exosome in Translational Experimental Studies Based on the encouraging data from the small experimental animal studies, exosomes derived from different stem/progenitor cells are being extensively studied for their therapeutic promise and reparability in translational animal models (Gallet et al. 2017; Adamiak et al. 2018; Maring et al. 2019; Potz et al. 2018). Most of these studies have reported the therapeutic promise of exosomes as good as the cell source of their derivation. Of these cell types, MSCs are the leading source of exosomes used in large animal experimental models (Spannbauer et al. 2020; Hade et al. 2021). In most experimental studies, as in small experimental animal studies, intramyocardial delivery has been used as the most preferred route of exosome delivery (Gallet et al. 2017; Collantes et al. 2017; Bayardo et al. 2021) (Table 2). However, given the invasiveness of the procedure, it allows a one-time administration of exosomes as an adjunct to routine coronary artery bypass grafting (CABG). As an alternative to a single-dose, one-time administration, researchers are optimizing protocols based on a multidosing strategy scheduled at different time points to achieve a better prognosis (Kisby et al. 2021; Charles et al. 2020). Charles et al. (2020) modified the treatment approach to intravenous bolus delivery of MSC-derived exosomes for 7 consecutive days, twice daily, in a porcine model of myocardial infarction (Charles et al. 2020). The systemic delivery significantly reduced the infacrt size on day 7 and 28 of observation using magnetic resonance imaging (MRI). Moreover, there was a significant increase in the indices of left ventricle (LV) function and reduced LV-wall thinning compared to the control group of animals. These data clearly show the importance of early and repeated injections of exosomes as a therapeutic approach in terms of safety and efficacy. In a recent study, Zhu et al. used a minimally invasive approach to deliver MSC-derived exosomes in hyaluronic acid gel via intrapericardial injection. The suspension of exosomes in the gel caused the slow release of exosomes from the injection site (Zhu et al. 2021). This approach helped increase the retention of the delivered exosomes, contributed to their slow release, and resulted in higher uptake by the cardiomyocytes. Moreover, hydrogel-based exosome delivery is translatable to the clinics and provides a safe alternative for repeated intravenous injections. The biodegradable and porous hydrogels also reduce the amount of exosome dose needed to achieve therapeutic benefits due to the reduced loss of the delivered exosomes from the delivery site. Some of the reported hydrogels used for exosomal

Source of exosomes iPSC-derived cardiomyocytes and MSC-derived exosomes

CDC-secreted exosomes

Human MSC-derived exosomes

CDC-derived exosomes

Author/year Bayardo et al. (2021)

Gallet et al. (2017)

Potz et al. (2018)

Nguyen et al. (2018)

I/M injection

I/M injection

I/C or I/M

Delivery Mode I/M

LAD occlusion for 150 min, followed by reperfusion for 4 weeks

Chronic myocardial ischemia by placement of ameroid ring around LCX

Acute and chronic MI

Model type Acute MI

Animal Main findings Porcine Transendocardial injection of exosomes derived from iPSCderived cardiomyocytes and MSCs in the preinfarct region – cardiac MRI at 2 and 4 weeks showed significantly improved LVEF and infarct size as compared to the control treated animals. Transcriptome data showed homeostatic preservation of energetics through hibernation of the injured myocardium Porcine I/C or I/M delivery of human cardiosphere-derived exosomes (or vehicle as control) 30 min after reperfusion – I/C were ineffective, but I/M delivery preserved LV function. In another set of animals, I/M injection of exosomes 4 weeks after MI led to significantly preserved LVEF and LV volumes as assessed by MRI, decreased scarring, decreased collagne contents and cardiomyocyte hypertrophy with a simultaneous in the blood vessel density Porcine Twenty-three Yorkshire pigs divided to receive control treatment (n ¼ 7) and exosome treatment (n ¼ 10) 2 weeks after the development of the animal model – there was a significant increase in blood vessel density in exosome-treated animals as compared to the control. There was a significant increase in phospho-mitogen-activated protein kinase/ mitogen-activated protein kinase ratio and phosphoendothelial nitric oxide synthase/endothelial nitric oxide synthase ratio in the exosome treatment group Porcine Two groups of control and exosome treatment by intramyocardial injection of placebo or CDC-derived exosomes at 4 weeks after ischemia/reperfusion injury – viability was improved and cardiac function was preserved in the exosome treatment. More importantly, the ultrastructure of the myocardial tissue was significantly preserved in the exosome treatment group

Table 2 Summary of translational studies using stem-cell-derived exosomes in large animal models of heart diseases

1020 K. H. Haider and M. Najimi

Twice daily I/V dose for 7 days

Minimally invasive intrapericardial injection

Charles et al. (2020) MSC-derived exosomes

MSC-derived exosomes in HA gel

MSC-derived exosomes

Cardiac-adiposetissue-derived or MSC-derived exosomes in peptide hydrogel

Zhu et al. (2021)

Yao et al. (2021)

Monguió-Tortajada et al. (2021) Direct placement of cardiac patch with exosomes

Spray formulation

Intrapericardial injection

CDC-derived exosomes

López et al. (2020)

MI

AMI

Normal heart

MI

LAD occlusion for 150 min, followed by reperfusion for 4 weeks

Exosome-Based Cell-Free Therapy in Regenerative Medicine for. . . (continued)

Porcine The animals in the treatment group received intrapericardial injection of exosomes during the early inflammatory phase after myocardial infarction. The authors observed significant changes in biochemical and immunological parameters. There was a significant increase in peripheral blood M2 macrophages. Overall, there was a significant reduction in intraperitoneal and peripheral blood leukocytes. These data show that exosomal treatment has immunomodulatory effects, especially on monocyte polarization. Also, exosomal treatment is proangiogenic Porcine There was a significant reduction in infarct size on days 7 and 28 of observation using MRI, as well as a significant increase in the indices of LV function and reduced LV-wall thinning as compared to the control group of animals Porcine The purpose of the study was to ascertain the fate of cells. Sizeable exosome retention in the heart was observed by fluorescent imaging. There was little change in blood chemistry indicators. Confocal microscopic images showed uptake of the exosomes by day 3 Porcine Designing, fabrication, and testing of a spray of exosomes in biomaterial – the treatment significantly improved cardiac function, reduced cardiac fibrosis, and promoted endogenous angiomyogenesis in the postinjury heart Porcine Treatment with exosome-embedded pericardial scaffold was placed over the infarcted myocardium integrated with the myocardium by day 6 after placement. There was a significant increase in angiogenesis and reduced macrophage T-cell infiltration in the damaged myocardium. The scaffold generated a vascularized niche for the infiltration of resident stem cells to participate in the repair process and modulated short-term post-ischemic inflammation

34 1021

Source of exosomes Cardiac-adipose-tissue derived exosomes, CDCs, or CDCs pretreated to release exosomes

Delivery Mode Intracoronary injection

Model type Porcine model of dilated cardiomyopathy

Animal Main findings Porcine Pretreatment of the cells to block exosome release abrogated the functional recovery of cell-based therapy. Even when exosomes were coinjected with these cells, they failed to recover functional benefits. Improved cardiac indices and reduced cardiac fibrosis were observed in animals treated with naive CDCs. CDC-derived exosomes were rich in proangiogenic and cardioprotective miRs, especially miR-146a-5p, but treatment with isolated exosomes did not recapitulate the beneficial effects. The study led to phase I safety assessment in five pediatric patients to provide a translational framework for the future use of CDCex-derived miRNAs as potential paracrine mediators as a therapeutic strategy

Abbreviations: AMI acute myocardial infarction, BM bone marrow, CDCs cardiosphere-derived cells, HA hyaluronic acid, I/C intracoronary, I/M intramyocardial, I/V intravenous, iPSCs induced pluripotent stem cells, LAD left anterior descending artery, LCX left circumflex, LVEF Left ventricle ejection fraction, miR microRNA, MI myocardial infarction, MSCs mesenchymal stem cells

Author/year Hirai et al. (2020)

Table 2 (continued)

1022 K. H. Haider and M. Najimi

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preparation include shear-thinning hydrogel (STG), PA-GHRPS, nanocomposite, and hydrogel patches (Chen et al. 2021). Monguió-Tortajada et al. have used a 3D-scaffold-based delivery of exosomes to create a vascularized niche in the infarcted myocardium to attract resident cells for participation in the repair process (Monguió-Tortajada et al. 2021). The authors of the study used cardiac-adipose-tissue-derived MSCs for the purification of exosomes, which significantly reduced proinflammatory cytokine production (i.e., IFN, TNF-1α, IL12) and promoted angiogenesis. Interestingly, within 6 days after implantation, the engineered scaffold carrying exosomes was found to integrate with the recipient infarcted myocardium efficiently. Recent advancement in the fast-developing exosome-based therapeutic approach is the site-directed delivery of exosomes, avoiding their spillover to the nontargeted tissues and organs (Barjesteh et al. 2021). The development of inorganic nanoparticle-loaded exosomes is gaining popularity in this regard. Lee et al. have recently reported using exosome-mimetic nanovesicle derived from iron oxide nanoparticle-loaded MSCs (IONP-MSCs) (Lee et al. 2020). These iron-oxidecontaining nanovesicles were magnetically guided to the infarcted porcine heart muscle, which significantly reduced the duration of the inflammatory phase, reduced apoptosis and fibrosis, and increased angiogenesis. Overall, the authors reported a significantly improved functional recovery of the infarcted heart and proposed the potential feasibility and target specificity of the treatment approach, besides alleviating the need for large quantities of exosomes. Moreover, with the encouraging data emanating from the translational studies, GMP compliance is the need of the hour to forward an exosome-based treatment approach to the clinics for therapeutic applications. Hence, attempts are underway to optimize protocols for the large-scale clinical-grade production of exosome preparation for human use in clinical settings (Andriolo et al. 2018).

Exosome in Clinical Studies Despite encouraging translational data from large animal studies and their advantages and superiority (i.e., safety and feasibility) over mother cells from which have been derived, little progress has been made in their use for myocardial repair and regeneration in clinical settings. As of the writing of this chapter (October 30, 2021), there are 112 clinical trials listed on www.clinicaltrials.org using exosomes. These trials are primarily about noncardiac conditions, including diabetes types I and II, stroke, chronic kidney disease, macular degeneration, cancer and cancer-associated conditions, COVID-19, acute respiratory distress syndrome (ARDS), etc. (Table 3). Moreover, some of the studies are intended to ascertain the diagnostic potential of the exosomes rather than the assessment of their therapeutic value. It is pertinent to mention here that though exosomes have tremendous therapeutic potential, it would be important that exosome preparations are clinical grade to ensure their safety for human applications. More recently, the FDA has cleared three IND (Investigational New Drug) applications based on PEP™ (Purified Exosome Preparations) for three different conditions, including wound healing (Phase 1b/2a study; ClinicalTrials.gov

Intradiscal injection of platelet-rich plasma (PRP) enriched with exosomes in chronic low back pain

Intra-articular injection of MSC-derived exosomes in knee osteoarthritis (ExoOA-1) Effect of UMSC-derived exosomes on dry eye in patients with cGVHD

ClinicalTrials. gov identifier: NCT04849429

ClinicalTrials. gov identifier: NCT05060107 ClinicalTrials. gov identifier: NCT04213248

NCT identifier Study title Therapeutic applications of exosomes in clinical trials ClinicalTrials. Role of exosomes derived from gov identifier: epicardial fat in atrial fibrillation NCT03478410 ClinicalTrials. Allogenic MSC-derived exosome in gov identifier: patients with acute ischemic stroke NCT03384433 ClinicalTrials. A pilot clinical study on the gov identifier: inhalation of MSC exosomes treating NCT04798716 severe novel coronavirus pneumonia ClinicalTrials. Evaluation of adipose-derived stem gov identifier: cell exosomes for the treatment of NCT04270006 periodontitis ClinicalTrials. Effect of plasma-derived exosomes gov identifier: on cutaneous wound healing NCT02565264 Biological: exosome

Biological: MSC-derived exosomes Biological: adipose-derived stem cell exosomes Other: plasma-derived exosomes

Cerebrovascular disorders Coronavirus

Periodontitis

Ulcer

Biological: exosomes (EVs)

Drug: umbilical MSC- derived exosomes

Osteoarthritis, knee Dry eye

Biological: platelet-rich plasma (PRP) with exosomes

Procedure: epicardial fat biopsy

Atrial fibrillation

Chronic low back pain, degenerative disk disease

Method of intervention

Disease

Zhongshan Ophthalmic Center, Guangzhou, Guangdong, China

Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan Anupam Hospital, Uttarakhand, India; Mother Cell Spinal Injury and Stem Cell Research, Anupam Hospital, India

Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Beni-Suef University, Banī Suwayf, Egypt; Cairo University, Egypt

Shahid Beheshti University of Medical Sciences, Tehran, Iran

Sheba Medical Center, Ramat Gan, Israel

Institution and location of the trial

Table 3 Clinical trials assessing the therapeutic and diagnostic significance of exosomes for different pathological conditions. (Data have been derived from ClinicalTrials.gov)

1024 K. H. Haider and M. Najimi

Procedure: sigmoidoscopy and biopsy, blood work

Other: exosome

Diagnostic test: ctDNA and exosome-combined detection

Diagnostic test: biomarker detection in different groups

MI

Pulmonary nodules

Lupus nephritis

Biological: low-, mild-, and high-dosage MSC-Exos administrated for nasal drip • Drug: EXO 1 inhalation • Drug: EXO 2 inhalation • Drug: placebo inhalation

Irritable bowel disease

Alzheimer’s disease

The safety and efficacy evaluation of allogenic adipose MSC-Exos in patients with Alzheimer’s disease Safety and efficiency of the method of exosome inhalation in COVID-19associated pneumonia

Biological: MSC-derived exosomes (MSC-Exo)

Other: sample collection

MHs

MSC-Exos promoting healing of MHs

Biological: MSC exosomes

• Covid19 • SARS-CoV-2 PNEUMONIA • COVID-19 Preeclampsia

T1DM

Effect of microvesicle and exosome therapy on β-cell mass in T1DM

ClinicalTrials. Exosome cargo from preeclampsia gov identifier: patients NCT04154332 ClinicalTrials. Plant exosomes +/ curcumin to gov identifier: abrogate symptoms of inflammatory NCT04879810 bowel disease Diagnostic applications of exosomes in clinical trials Differential expression and analysis ClinicalTrials. of peripheral plasma exosome gov identifier: NCT04127591 miRNA in patients with MI ClinicalTrials. Clinical study of ctDNA and gov identifier: exosome-combined detection to NCT04182893 identify benign and malignant pulmonary nodules ClinicalTrials. Urine exosomes to identify gov identifier: biomarkers for LN NCT04894695

ClinicalTrials. gov identifier: NCT02138331 ClinicalTrials. gov identifier: NCT03437759 ClinicalTrials. gov identifier: NCT04388982 ClinicalTrials. gov identifier: NCT04602442

Exosome-Based Cell-Free Therapy in Regenerative Medicine for. . . (continued)

Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

Shanghai Chest Hospital, Shanghai, China

Ethics Committee of Xinhua Hospital, Shanghai, China

University of Alabama at Birmingham, Birmingham, Alabama, United States University of Louisville, Louisville, Kentucky, United States

Ruijin Hospital affiliated with Shanghai Jiaotong University School of Medicine, Shanghai, China Medical Centre Dynasty Samara, Russian Federation

Tianjin Medical University Hospital, Tianjin, China

Sahel Teaching Hospital, Sahel, Cairo, Egypt

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Exosomes in rectal cancer

Omics sequencing of exosomes in body fluids of patients with acute lung injury Serum exosomal long noncoding RNAs as potential biomarkers for lung cancer diagnosis

Study title Multicenter clinical research for the early diagnosis of lung cancer using blood-plasma-derived exosome Evaluation of urinary exosome presence from clear cell renal cell carcinoma Study of exosomes in monitoring patients with sarcoma (EXOSARC) Biological: blood samples

Sarcoma

Rectal cancer

Lung cancer

Diagnostic test: blood draw

Diagnostic test: the lung injury, causes and extrapulmonary factors Diagnostic test: collecting samples

Biological: urinary sample

Clear cell renal cell carcinoma

Acute lung injury

Method of intervention Diagnostic test: exosome sampling

Disease Lung cancer

Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hub University of Kansas Medical Center/ Cancer Center, Kansas City, Kansas, USA

CHU de Besançon, Besançon, France, Centre Georges François Leclerc, Dijon, France Nanfang Hospital, Southern Medical University, Guangzhou, China

Chu Saint-Etienne, Saint-Étienne, France

Institution and location of the trial Korea University Guro Hospital, Guro-gu, Seoul, Republic of Korea

Abbreviations: cGVHD chronic graft versus host disease, IBD irritable bowel syndrome, MHs macular holes, MSCs mesenchymal stem cells, PRP platelet-rich plasma, TD1 type I diabetes mellitus

ClinicalTrials. gov identifier: NCT03874559

NCT identifier ClinicalTrials. gov identifier: NCT04529915 ClinicalTrials. gov identifier: NCT04053855 ClinicalTrials. gov identifier: NCT03800121 ClinicalTrials. gov identifier: NCT05058768 ClinicalTrials. gov identifier: NCT03830619

Table 3 (continued)

1026 K. H. Haider and M. Najimi

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identifier: NCT04664738), fistulizing Crohn’s disease, and acute myocardial infarction (AMI). For AMI, a phase Ib/2a entitled “Safety Evaluation of Intracoronary Infusion of Extracellular Vesicles in Patients With AMI” has been initiated (ClinicalTrials.gov identifier: NCT04327635). Led by the principal investigator Dr. Guy Reeder at Mayo Clinic, the interventional study is currently recruiting patients (21–85 years of age) to research the dose-limiting toxicities and maximum tolerated dose of a single dose of PEP™ (during PCI or AMI) at escalating concentrations of EVs with well-defined exclusion and inclusion criteria expected to start from December 2021. The study will involve the evaluation of three different doses of PEP™ (5%, 10%, and 20%) to be administered via intracoronary infusion within 20 min of stent placement during percutaneous coronary intervention (PCI) at least 4 h (but no more than 12 h) after the onset of heart attack symptoms. The patients would be followed up for 1 year for evaluation. Another clinical trial registered on ClinicalTrials.gov involving patients with AMI is for the diagnostic application of exosomes in the peripheral circulation (ClinicalTrials.gov identifier: NCT04127591). The study entitled “Differential Expression and Analysis of Peripheral Plasma Exosome miRNA in Patients with Myocardial Infarction” has been designed to study the possible changes in the miR expression profile in peripheral plasma exosomes during an infarction episode. Led by Dr. He from Xinhua Hospital, Shangai Jiao Tong University, China, the study was expected to be completed by December 2021. Some of the clinical trials for the assessment of the therapeutic efficacy of exosome preparations have been summarized in Table 3 with a summary of the salient results of these studies.

Challenges and Future Perspective The transfer of cell-free therapy based on a stem-cell-derived exosome approach from bench to bedside as a routine therapeutic and diagnostic modality has immense potential. Unlike their mother cells, although exosomes lack proliferation and differentiation potential, they can promote the mobilization and homing of intrinsic/resident stem/progenitor cells to participate in reparative and regenerative processes. As is evident from the published data, the theranostic assessment of exosomes has reached the clinical phase based on safety and efficacy data from the translational studies (Table 3). However, it still requires optimization, and standardization of the protocols that encompass their isolation, characterization and large scale production in accordance with the GMP requirements to the biological and functional characterization, ensure off-the-shelf availability (i.e., storage under optimal conditions to sustain their efficacy), and safe and efficient use in the humans for theranostic applications. Table 4 summarizes some of the challenges that need to be encountered and overcome to streamline their progress to the clinics.

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Table 4 Challenges impeding the progress of exosomes for theranostic applications in the clinics Pharmaceutical challenges • Optimal source for exosome isolation, i.e., stem/progenitor cells, peripheral blood, plant, food sources, etc. • Labor-intensive and time-consuming isolation and purification protocols • Flawed production and quality control of GMP-grade exosome preparations on a large scale • Variation in size, shape, and inherent payload • Maintenance of uniform loading efficiency with the required cargo • Standardized biophysical, biochemical, and biological characterization of exosome preparations • Lack of optimized protocols for long-term storage and stability (i.e., time, temperature, freezing, thawing, reconstitution) to ensure off-the-shelf availability Preadministration challenges • Pharmacodynamical features of exosome preparation • Optimal dosage form for efficient delivery • Mode of delivery • Route of administration • Time of administration • Dose of administration • Single- or multi-dose administration • Site-directed or tissue-targeted administration • Off-targeted distribution of delivered exosomes Postadministration challenges • Lack of optimization of pharmacokinetic features • Preferential uptake and accumulation in the spleen and liver • Biodistribution and bioavailability features • Lack of standardized release profile of the exosome payload • Biological half-life • Long-term safety and efficacy concerns • Toxicological features of exosome preparation • Postdelivery visualization and fate determination of delivered exosomes

Cross-References ▶ Extracellular Vesicles-Based Cell-Free Therapy for Liver Regeneration ▶ Mesenchymal Stem Cells for Cardiac Repair

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Current Trends and Future Outlooks of Dental Stem-Cell-Derived Secretome/ Conditioned Medium in Regenerative Medicine

Israa Ahmed Radwan, Dina Rady, Sara El Moshy, Marwa M. S. Abbass, Khadiga Mostafa Sadek, Aiah A. El-Rashidy, Azza Ezz El-Arab, and Karim M. Fawzy El-Sayed Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dental Stem/Progenitor Cells (DMSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem/Progenitor Cells’ Secretome/Conditioned Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles (EVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosomes (EXs) and Microvesicles (MVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. A. Radwan · D. Rady · S. El Moshy · M. M. S. Abbass Oral Biology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt e-mail: [email protected]; [email protected]; [email protected]; [email protected] K. M. Sadek · A. A. El-Rashidy Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Biomaterials Department, Faculty Dentistry, Cairo University, Cairo, Egypt e-mail: [email protected]; [email protected] A. Ezz El-Arab Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt e-mail: [email protected] K. M. Fawzy El-Sayed (*) Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian Albrechts University, Kiel, Germany © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_47

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Comparison Between Secretome/CM Derived from DMSCs and MSCs from Other Tissue Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretome/Conditioned Medium (SHED-CM) Derived from Stem Cells from Exfoliated Human Deciduous Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Cardiopulmonary Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Hepatic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Immunological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Dental-Pulpal Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHED-CM in Treating Neural Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretome/Conditioned Medium Derived from Dental Pulp Stem Cells . . . . . . . . . . . . . . . . . . The Osteogenic Potential of DPSCs-CM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DPSCs-CM in the Treatment of Liver Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential of DPSCs-CM to Regenerate Dental Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of DPSCs-CM in the Treatment of Neurological Diseases . . . . . . . . . . . . . . . . . . . . . DPSCs-CM in the Treatment of Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretome/Conditioned Medium Derived from Gingival Mesenchymal Stem/ Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GMSCs-CM in Treating Neural Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GMSCs-CM in Treating Skin Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GMSCs-CM Osteogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Periodontal Ligaments Stem-Cell-Derived Secretome/Conditioned Medium . . . . . . . . . . . . . . . . PDLSCs-CM in the Therapy of Neural Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PDLSCs-CM Osteogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PDLSCs-CM in Dental Tissue Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretome/Conditioned Medium Derived from Stem/Progenitor Cells of Apical Papilla (SCAP), Dental Follicle Stem/Progenitor Cells (DFSCs), and Tooth Germ Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Constructing biological substitutes that mimic the structure, architecture, and function of different tissues and organs is the ultimate goal of regenerative medicine. Adult mesenchymal stem/progenitor cells (MSCs) are considered the most widely researched cells in regenerative applications, yet several obstacles that challenge the safe and effective clinical translation of MSC-based therapies still exist. MSCs could partially exert their reparative and regenerative impact through a paracrine effect, mediated by the release of bioactive and trophic factors known as secretome, rather than the actual presence of the engrafted cells in the target site. In addition, MSCs have shown the ability to secrete these various bioactive molecules in their surrounding media (the conditioned media (CM)). MSC-secretome/CM is a set of proteins, lipids, nucleic acids, and trophic factors such as cytokines, chemokines, hormones, growth factors, and extracellular vesicles (EVs). Compared to nondental MSC secretome, dental MSC secretome/CM revealed a higher expression of proliferation-related, metabolic, transcriptional proteins and chemokines, as well as neurotrophins. Dental MSC secretome/CM exhibited experimentally tremendous biological effects, including immunomodulatory, anti-inflammatory, neuroprotective, osteogenic, angiogenic, and antiapoptotic effects, as well as the modulation of

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oxidative stresses. These aforementioned biological effects greatly explain the increasing interest in dental MSC secretome/CM as an acellular regenerative strategy for the treatment of various clinical diseases/injuries while alleviating the limitations and safety concerns associated with MSC-based therapies. Keywords

Cell-free therapy · Conditioned medium · Dental stem cells · Exosomes · Extracellular vesicles Abbreviations

3D ABMSCs AD ALS ASCs bFGF BMMSCs BMP BNDF BSP CCK-8 c-JUN/JNK CM CSF CXCL DFSCs DMSCs DPSCs DSPP EAE ECM EGF EMVs EVs EXs FGF FGF-R FLT-3 GCSF GDNF GFAP GM-CSF GMSCs

Three-dimensional Alveolar bone proper-derived stem/progenitor cells Alzheimer’s disease Amyotrophic lateral sclerosis Adipose stem/progenitor cells Basic fibroblast growth factor Bone-marrow-derived mesenchymal stem/progenitor cells Bone morphogenetic protein Brain-derived neurotrophic factor Bone sialoprotein Cell count kit-8 c-JUN/N-terminal kinase Conditioned medium Colony-stimulating factor CXC motif ligand Dental follicle stem/progenitor cells Dental mesenchymal stem/progenitor cells Dental pulp stem/progenitor cells Dentin sialophosphoprotein Encephalomyelitis Extracellular matrix Epidermal growth factor Exosomes/Matrix vesicles Extracellular vesicles Exosomes Fibroblast growth factor Fibroblast growth factor-receptor Fms-like tyrosine kinase receptor-3 Granulocyte-colony-stimulating factor Glial-cell-line-derived neurotrophic factor Glial fibrillary acidic protein Granulocyte-macrophage colony stimulating factor Gingival mesenchymal stem cells

1038

HEGF HFSCs HGF HLA-DR HUVECs IFNγ IGF IL iNOS KGF LF MAPK MCP-1 MS MSCs MVs NF-Κb NGF NT-3 OCN Oct OSM PCNA PDEGF PDGF PDL PDLSCs PEI PGE2 PLA RUNX2 SCAP SCF SDF SHED SOD SOX-2 sSiglec-9 TFIP11 TGF-β TIMP TLR TNF-α TRAP

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Heparin-binding epidermal growth factor Hair follicle stem cells-CM Hepatocyte growth factor Human leukocyte antigen-DR isotype Human umbilical vein endothelial cell culture Interferon gamma Insulin-like growth factor Interleukins Inducible nitric-oxide synthase Keratinocyte growth factor Liver failure Mitogen-activated protein kinase Monocyte chemoattractant protein-1 Multiple sclerosis Mesenchymal stem/progenitor cells Microvesicles Nuclear factor-kappa B Neural growth factor Neurotrophin-3 Osteocalcin Octamer-binding transcription factor Oncostatin M Proliferating cell nuclear antigen Platelet-derived endothelial cell growth factor Platelet-derived growth factor Periodontal ligament Periodontal ligament stem/progenitor cells Polyethyleneimine Prostaglandin E2 Poly-(lactide) Runt-related transcription factor 2 Stem/progenitor cells from apical dental papilla Stem cell factor Stromal-cell-derived factor Human shed deciduous teeth Superoxide dismutase Sex-determining region Y (SRY)-box 2 Soluble sialic-acid-binding Ig-like lectin-9 Tuftelin-interacting protein Transforming growth factor β Tissue inhibitor of metalloproteinase Toll-like receptor Tumor necrosis factor-α Tartrate-resistant acid phosphatase

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Tuftelin 1 Vascular endothelial growth factor

Introduction The restoration, regeneration, or even repair of damaged tissues and organs is the center of interest of many researchers, aiming to improve the quality of life of many patients in need. Regenerative medicine represents a promising treatment modality that, through employing tissue engineering approaches, can enhance and guide the restoration, maintenance, and improvement of different organs and tissue functions (Atala 2008). Regenerative medicine is a multidisciplinary branch of medicine that aims at constructing biological substitutes that can mimic the patient’s organs and tissues, thus offering a promising therapeutic modality for several disorders and diseases (Berthiaume et al. 2011; Stoltz et al. 2015). The tissue engineering approach combines biocompatible scaffolds, cells, and biologically active molecules into functional tissues (Berthiaume et al. 2011; Gao and Cui 2016; Guan et al. 2017), while regenerative medicine, being a broader field, includes tissue engineering approaches and research on self-healing, where the body can use its endogenous tissue formation ability, which could occur secondary to induction by foreign biological materials, to form new cells and regenerate tissues and organs (Katari et al. 2015). Biocompatible scaffolds employed in tissue engineering are commonly fabricated from bioceramics, polymers, or their composites. Bioceramic and bioactive glass scaffolds and their composites are widely employed (Bose et al. 2012; Pilia et al. 2013; Roseti et al. 2017; Chocholata et al. 2019; Kerativitayanan et al. 2017). In addition, various polymeric scaffolds are fabricated from natural, synthetic, or hybrid polymers in addition to elastic polymer networks such as hydrogels (Rezwan et al. 2006; Pina et al. 2015; Qu et al. 2019). Scaffold biomaterials are often combined with bioactive molecules and/or growth factors that can home, stimulate, and promote the differentiation of tissue-resident stem/progenitor cells (Gomes and Reis 2004; Mallick et al. 2015; Chocholata et al. 2019). Decellularization is an innovative scaffold fabrication technique, where the extracellular matrix (ECM) with its organization, vascular network, and architecture are preserved. Decellularization provides a three-dimensional (3D) cell-free scaffold, ready to be seeded with the desired cell type for specific tissue engineering applications (Paduano et al. 2017; Taylor et al. 2017); such 3D scaffold influences cellular behavior and differentiation potential through its structure and biological signaling (He and Callanan 2013). The decellularization process could be achieved by various methods, which employ enzymes, detergents, and salts combined with specific physical stimuli (Scarritt et al. 2015). Tissue engineering employs several cell types, benefiting from their remarkable properties in each intended application; cells employed include primarily embryonic stem cells, adult stem/progenitor cells, in addition to induced pluripotent stem cells (Egusa et al. 2012, Shah et al. 2016). Adult mesenchymal stem/progenitor cells

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(MSCs) are considered the most widely investigated cell population in tissue engineering applications based on their multipotency and differentiation potential into an array of mesodermal origin, upon proper stimulation, as well as its selfrenewal capabilities. MSCs reside in many locations in adult tissues, including bone marrow, umbilical cord, synovial fluid, and adipose tissues (Pires et al. 2016; Hsiao et al. 2012; Jones et al. 2004). The cellular transplantation of MSCs has been proposed as a treatment modality for functional tissue regeneration and for treating various autoimmune and chronic inflammatory diseases. Unfortunately, the clinical translation of stem-cell-based therapies faces various serious ethical and safety issues (Volarevic et al. 2018). Based on the well-documented paracrine hypothesis (Haider and Aziz 2017), a more promising alternative is the utilization of MSCs-conditioned media (CM), as recently MSCs have been characterized by their ability to secrete various bioactive molecules into their surrounding media (Lei and Haider 2017). Such secreted bioactive molecules, known as secretome, can be easily collected and have been remarkable in the enhancement of mesenchymal tissue regeneration (Caplan 2007; Cicconetti et al. 2007). This book chapter aims to review the efficacy of secretome derived from different populations of dental mesenchymal stem/progenitor cells (DMSCs) in the treatment of various diseases and the regeneration of different tissues, emphasizing and displaying the involved content of bioactive molecules.

Dental Stem/Progenitor Cells (DMSCs) DMSCs are multipotent adult MSCs derived from the ectomesenchymal neural crest cells (Fawzy El-Sayed et al. 2013a; Fawzy El-Sayed et al. 2013b). DMSCs include stem/progenitor cells extracted from pulpal tissues of human shed deciduous teeth (SHED) (Miura et al. 2003; Stanko et al. 2018), dental pulp stem/progenitor cells (DPSCs) isolated from dental pulpal tissues of permanent teeth (Gronthos et al. 2000), stem/progenitor cells isolated from apical papilla (SCAP) at the apices of immature permanent teeth (Jo et al. 2007; Sonoyama et al. 2006), tooth germ progenitor cells isolated from late bell stage third molar tooth germs (Ikeda et al. 2008), dental follicle stem/progenitor cells (DFSCs) isolated from dental follicle surrounding the third molar (Morsczeck et al. 2005), periodontal ligament stem/ progenitor cells (PDLSCs) isolated from the periodontal ligament (Seo et al. 2004; Jo et al. 2007), alveolar bone proper-derived stem/progenitor cells (ABMSCs) (Fawzy El-Sayed et al. 2012a, 2014, 2017), and gingival stem/progenitor cells (GMSCs) isolated from gingival tissues (Palmer and Lubbock 1995; Fawzy-ElSayed et al. 2016, 2019b; Fawzy El-Sayed and Dorfer 2016; El-Sayed et al. 2015). Stem/progenitor cells were also isolated from inflamed pulp (Alongi et al. 2010; Malekfar et al. 2016) and periapical cysts (Marrelli et al. 2013; Tatullo et al. 2017). DMSCs are characterized by their multidifferentiation potential into multiple cell lineages, self-renewal ability, potent regenerative potentials, and immunomodulatory properties (Cordeiro et al. 2008; Dominici et al. 2006; Huang et al. 2010; Leucht et al. 2008; Liu et al. 2015; Nakashima and Iohara 2014; Park et al. 2010).

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In addition to being easily acquired with minimally invasive procedures (Egusa et al. 2012), they were reported to have improved regenerative potential when compared to MSCs derived from other sources. SHED (Isobe et al. 2016), DPSCs (Murakami et al. 2015; Kumar et al. 2017a, b, 2018; Mead et al. 2014; Davies et al. 2015; Isobe et al. 2016), SCAP (Kumar et al. 2017a, b, 2018), and DFSCs (Kumar et al. 2017a, b, 2018) were shown to exhibit higher neurogenic (Isobe et al. 2016; Kumar et al. 2017a), angiogenic (Murakami et al. 2015; Song et al. 2017), osteogenic (Kumar et al. 2018; Davies et al. 2015), hepatogenic (Kumar et al. 2017b), antiapoptotic (Murakami et al. 2015), and pulpal tissue (Murakami et al. 2015) regenerative potential and higher proliferative rates (Abdullah et al. 2014; Wang et al. 2012) in comparison to adipose stem/progenitor cells (ASCs) (Mead et al. 2014; Davies et al. 2015; Murakami et al. 2015) or bone-marrow-derived mesenchymal stem/progenitor cells (BMMSCs) (Isobe et al. 2016; Murakami et al. 2015; Kumar et al. 2017a, b, 2018; Mead et al. 2014; Davies et al. 2015). DMSCs express common MSC surface markers, including CD105, CD73, and CD90, with a lack of expression of CD45, CD34, CD14, CD11b, CD79a, CD19, and human leukocyte antigen-DR isotype (HLA-DR) (Huang et al. 2009).

Stem/Progenitor Cells’ Secretome/Conditioned Medium Besides the direct cellular activity of stem/progenitor cells following their transplantation, they can exert an indirect paracrine effect to enhance tissue repair and regeneration (Fig. 1) (Ankrum and Karp 2010; Baglio et al. 2012; Maguire 2013; Wollert and Drexler 2010). Such paracrine effect is promoted through the secretion of secretome into their surrounding tissues, which exerts an immunomodulatory effect that supports and enhances tissue regeneration and homeostasis, besides enhancing cell migration and proliferation (Bai et al. 2016; Baraniak and McDevitt 2010; Beer et al. 2017; Katagiri et al. 2013; Madrigal et al. 2014; Li et al. 2014; Ranganath et al. 2012). Based on such effects, cell-free therapy, employing stem/ progenitor cells’ secretome, has emerged as an alternative treatment modality to cellbased therapies (Baglio et al. 2012; Ciapetti et al. 2012; Maguire 2013). As discussed earlier, the secretome is an array of bioactive molecules secreted from living MSCs or shed from their surface into the surrounding environment (Beer et al. 2017). This can be replicated in vitro through the stimulation of stem/progenitor cells to release secretome and trophic factors into the culture media, producing the stem/ progenitor cells’ CM (Baraniak and McDevitt 2010; Phelps et al. 2018). Stem/ progenitor-cell-derived secretome has shown several advantages over cell-based therapy, which involves the ease of secretome preservation, sterilization, packaging, and storage for long time durations without the risk of losing its properties. Secretome dosage can be precisely calculated and the production of large quantities can be readily achieved using cell lines, which save time and cost while avoiding any invasive procedures to the patient (Bermudez et al. 2015, 2016; Osugi et al. 2012; Vizoso et al. 2017).

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Fig. 1 Illustrative diagram showing various bioactive molecules detected in secretome derived from dental mesenchymal stem cells

Stem/progenitor cells’ secretome is composed of proteins, nucleic acid, lipids, extracellular vesicles (EVs), and trophic factors such as chemokines, cytokines, growth factors, and hormones (Ranganath et al. 2012), and its content varies according to the anatomic location of the stem/progenitor cells (Assoni et al. 2017). Stem/progenitor cells’ secretome was demonstrated to harbor an array of growth/differentiation factors, including platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); platelet-derived endothelial cell growth factor (PDEGF); insulin-like growth factors I, II (IGF-I, IGF-II); epidermal growth factor (EGF); keratinocyte growth factor/fibroblast growth factor-7 (KGF/FGF-7); fibroblast growth factor 2/basic fibroblast growth factor (FGF-2/bFGF); hepatocyte growth factor (HGF); heparin-binding epidermal growth factor (HEGF); neural growth factor (NGF); and brain-derived neurotrophic factor (BDNF) (Pawitan 2014). In addition to its content of anti-inflammatory cytokines, including transforming growth factor (TGF)-β1 and interleukins (ILs), including IL-6, IL-10, IL-27,

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IL-17, and IL-13, and proinflammatory cytokines, including IL-8/CXC motif ligand (CXCL)-8, IL-9, and IL-1β. Granulocyte-colony-stimulating factor (GCSF), granulocyte-macrophage CSF (GM-CSF), and prostaglandin E2 (PGE2) (Pawitan 2014).

Extracellular Vesicles (EVs) EVs include exosomes (EXs) (40–100 nm), microvesicles (MVs) (100–1000 nm), and apoptotic bodies (1–5 um), and their content depends on the surrounding environment and may change upon cell stimulation. They are secreted by many cell types, including stem/progenitor cells, and they can be isolated from body fluids, like serum, cerebrospinal fluids, and urine (Beer et al. 2017; Lee et al. 2012; Raposo and Stoorvogel 2013; Skog et al. 2008). Upon reaching their target sites, EVs interact with and attach to the target cell surface, then they either remain attached or become internalized via endocytosis or membrane fusion to the target cell plasma membrane to release their content into the recipient cells or become detached from the cell surface following the completion of their action (Kim et al. 2013; Raposo and Stoorvogel 2013).

Exosomes (EXs) and Microvesicles (MVs) EXs and MVs are membrane-bound particles, secreted for normal homeostasis by many cell types, and their secretion increases upon stimulation (Kim et al. 2013; Valadi et al. 2007). EXs and MVs differ in their origin (biogenesis) and physical characteristics, such as size and surface markers (Lee et al. 2012; Mathivanan et al. 2010; Ratajczak et al. 2006). Their content varies according to the producing cells, comprising proteins and lipids, along with protein-coding messenger ribonucleic acids (mRNAs) and noncoding microRNAs (Gyorgy et al. 2011; Ratajczak et al. 2006; Skog et al. 2008; Valadi et al. 2007; Haider and Aramini 2020). EXs and MVs are essential for intercellular communication, with both exerting paracrine and endocrine actions (Kim et al. 2013). EXs and MVs act as vehicles for the transport of bioactive molecules, such as cytokines and growth factors, from producing cells to adjacent or distant target cells via circulation (Kim et al. 2013; Raposo and Stoorvogel 2013; Valadi et al. 2007). They can also modify target cells’ gene expression or protein synthesis through delivering RNA (Nakamura et al. 2015; Tomasoni et al. 2013). EXs are homogenous and smaller in size than MVs, with a diameter ranging from 40 to 100 nm. They originate in multivesicular bodies, which are discharged through exocytosis via fusion with the cell membrane (Lee et al. 2012; Tkach and Thery 2016). Multivesicular bodies are late endosomes that are formed by the maturation of early endosomes; early endosomes are formed following the fusion of endocytotic vesicles (Pant et al. 2012). EXs are rich in annexins, tetraspanins (CD63, CD81, and CD9), and heat-shock proteins (such as Hsp60, Hsp70, and Hsp90), which are usually used for their identification (Biancone et al. 2012). On the other hand,

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MVs (also termed ectosomes) are heterogeneous and larger in size than EXs, with a diameter ranging between 100 and 1000 nm. MVs are produced through direct budding from the cell plasma membrane, and their surface markers originate from the producing cells (Mathivanan et al. 2010; Vishnubhatla et al. 2014). MVs are rich in proteins, lipids, as well as mRNAs and microRNAs (Collino et al. 2010).

Comparison Between Secretome/CM Derived from DMSCs and MSCs from Other Tissue Sources Proteomic analysis identified a total of 1533 proteins in the CM derived from BMMSCs, ASCs, and DPSCs. Nine hundred ninety-nine proteins were common among all three cell sources, which included 124 proteins identified as secreted extracellular proteins. These secreted extracellular proteins are proposed to modulate MSCs’ regenerative potential, such as inflammatory response, angiogenesis, cell migration, ossification, and organ survival. Proteins isolated from BMMSCs-CM were similar to those isolated from ASCs-CM but did not resemble proteins isolated from DPSCs-CM (Tachida et al. 2015). Proteins responsible for immunomodulation, angiogenesis, neuroprotection, chemotaxis, antiapoptosis, and extracellular matrix formation were expressed in both SCAP-CM and BMMSCs-CM. However, there was a significant difference in the expression levels of 151 proteins between the two cell sources, where SCAP-CM showed higher expression of proteins associated with metabolic processes and transcription, along with chemokines and neurotrophins, while expressing lower levels of proteins related to immunomodulation, angiogenesis, adhesion, and extracellular matrix proteins (Yu et al. 2016). One hundred seventy-four cytokines were commonly expressed in SCAP-CM, DFSCs-CM, and DPSCs-CM. Notably, a significantly higher expression of 23 cytokines related to odontoblast differentiation and proinflammatory and anti-inflammatory cytokines was evident in DPSCs-CM, while three cytokines responsible for cellular proliferation were significantly highly expressed in DFSCs-CM and SCAP-CM (Joo et al. 2018). DPSCs-CM showed higher angiogenic, antiapoptotic, and neurite outgrowth; migration activity (Ishizaka et al. 2013; Murakami et al. 2015); and immunomodulation in vitro when compared to BMMSCs-CM, as well as higher vasculogenesis in vivo (Ishizaka et al. 2013). In addition, DPSCs-CM revealed increased migration and angiogenic activity and antiapoptotic effect on mouse embryonic muscle myoblast cells (C2C12); in vitro, this could be based on its high content of monocyte chemoattractant protein-1 (MCP-1) and CXCL14 (Hayashi et al. 2015). A superior nerve regenerative potential was related to DMSCs-CM derived from DPSCs, SCAP, and DFSCs when compared to BMMSCs-CM, evident by the significantly higher colony formation and neurite extension, indicating an enhanced neural differentiation and maturation associated with DMSCs-CM, in comparison to BMMSCs-CM. Such superior neuroregenerative potential can be explained by the significantly higher expression of BDNF, neurotrophin-3 (NT-3) in DMSCs-CM derived from all three cell sources, and the significantly higher expression of NGF in

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SCAP-CM and DFSCs-CM when compared to BMMSCs-CM. Higher levels of interferon gamma (IFNγ), TGF-β, and GCSF were also expressed in DPSCs-CM when compared to BMMSCs-CM (Kumar et al. 2017a). Similar results were notable upon comparing DPSCs-CM with BMMSCs-CM and ASCs-CM (Mead et al. 2014).

Secretome/Conditioned Medium (SHED-CM) Derived from Stem Cells from Exfoliated Human Deciduous Teeth SHED obtained from the pulpal tissues of deciduous teeth exhibits a higher proliferation rate as compared to DPSCs and BMMSCs. Microarray analysis revealed that SHED had higher expression levels of FGF, TGF, connective tissue growth factor, NGF, and bone morphogenetic protein (BMP)-1 (Nakamura et al. 2009). Genes encoding for extracellular, cell surface molecules; cell proliferation; and embryonic tissue development are highly expressed by SHED. Moreover, SHED neurogenic potential might be related to their neural crest embryonic origin. SHED expressed neural cell lineage markers, including nestin, doublecortin, β-tubulin III, NeuN, glial fibrillary acidic protein (GFAP), S-100, A2B5, and 20 ,30 -Cyclic-nucleotide 30 -phosphodiesterase (Sakai et al. 2012). Moreover, SHED releases an array of secretomes with multiple biological therapeutic activities.

SHED-CM in Treating Cardiopulmonary Injuries In vitro, SHED-CM promoted the differentiation of mouse bone-marrow-derived macrophages into M2 macrophages, which expressed Arginase1, Ym-1, and CD206. These findings were further verified in vivo (Hirata et al. 2016; Matsushita et al. 2017; Wakayama et al. 2015) in a bleomycin-induced acute lung injury mice model; the intravenous administration of SHED-CM reduced lung fibrosis and enhanced survival rates. These therapeutic effects could be a result of the reduced expression of proinflammatory cytokines and fibrotic markers, such as α-smooth muscle actin, thus altering proinflammatory M1 into an anti-inflammatory M2 phenotype (Wakayama et al. 2015; Matsubara et al. 2015; Yamagata et al. 2013). Moreover, SHEDCM revealed cardioprotective benefits in ischemic heart diseases by at least two mechanisms, including the suppression of inflammatory responses in myocardial cells and the reduction of cardiomyocyte death. Interestingly, SHED-CM showed superior effects compared to those of BMMSC-CM and ASC-CM; this may be due to the significantly higher expression of HGF in SHED-CM as compared to the other two cell sources (Yamaguchi et al. 2015).

SHED-CM in Treating Hepatic Disorders Single intravenous administration of SHED-CM in a liver failure (LF) mice model revealed a significant therapeutic effect that was not detected in fibroblast-CM

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(Hirata et al. 2016; Matsushita et al. 2017). In vitro, SHED-CM suppressed chronic inflammation and induced tissue repair, where tumor necrosis factor-α (TNF-α), IL-1β, and inducible nitric oxide synthase (iNOS) were strongly suppressed. In addition, SHED-CM induced hepatic stellate cells’ apoptosis and protected hepatocytes from apoptosis by suppressing carbon-tetrachloride-induced apoptosis in hepatocytes (Hirata et al. 2016). SHED-CM attenuated the LF-induced proinflammatory response and generated an anti-inflammatory environment, where SHED-CM stimulated M2 cell markers (CD206 and Arginase1), anti-inflammatory cytokines (IL-10 and TGF-β1), angiogenic factor (VEGF) and antiapoptotic factor (stem cell factor (SCF) and IGF-1) expression, and hepatocyte proliferation. Furthermore, SHED upregulated the expressions of lysophosphatidylcholine activation genes, including HGF, TNF-related weak inducer of apoptosis (TWEAK), FGF-7, and Wnt3a (Matsushita et al. 2017). These data propose that the active biomolecules within the SHED-CM and the endogenous-tissue-repairing factors activated by SHED-CM administration could function together to markedly improve liver injury and survival rates (Hirata et al. 2016, Matsushita et al. 2017).

SHED-CM in Treating Diabetes Mellitus In a streptozotocin-induced diabetes model in rats, the intravenous administration of human SHED-CM and human BMMSCs-CM resulted in pancreatic β-cell regeneration, with elevated insulin secretion in the SHED-CM group. This antidiabetic effect of SHED-CM was found to be superior as compared to BMMSCs-CM (Izumoto-Akita et al. 2015).

SHED-CM in Treating Immunological Disorders In a rheumatoid arthritis rat model, SHED-CM or BMMSCs-CM intravenous injection markedly improved arthritis symptoms and decreased joint destruction, particularly in the SHED-CM group. This therapeutic efficacy was attributed to the anti-inflammatory capacity of SHED-CM associated with the induction of M2 macrophage polarization and the inhibition of osteoclastogenesis (Ishikawa et al. 2016). Moreover, SHED-EXs were effective in suppressing inflammation and maintaining anabolism homeostasis in temporomandibular joint chondrocytes’ cell culture treated with proinflammatory factors (Luo et al. 2019). Similarly, paracrine factors such as chemokines, cytokines and growth factors secreted by the SHED-CM may play a key role in regulating human hair growth. Human SHED-CM showed promising results in the treatment of alopecia. In an in vivo study, the dorsal area of mice was shaved and was injected subcutaneously with human SHED-CM or human hair follicle stem cells-CM (HFSCs-CM). SHEDCM resulted in a faster stimulation of hair growth compared to HFSCs-CM through upregulating positive hair growth regulatory factors, stromal-cell-derived factor (SDF)-1, hair growth factor, PDGF-BB, and VEGF-A (Gunawardena et al. 2019).

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SHED-CM in Treating Dental-Pulpal Disorders Cytokines commonly expressed by SHED-CM are involved in various mechanisms, including the regeneration of the dentin-pulp complex and vascularization. The angiogenic effect of SHED-CM was studied in vitro on human umbilical vein endothelial cell (HUVEC) culture and in vivo on rats’ dental pulp. An inductive effect was observed in HUVEC cultures, indicating that SHED-CM has a proangiogenic influence, suggesting that these cells can enhance the vascularization of regenerated dental tissues. Endodontic treatment was performed on rats’ first molar tooth, followed by overinstrumentation to allow the blood clot formation to fill the root canal, then SHED-CM was applied on top of the blood clot. Vascular connective tissues were induced inside the root canal (de Cara et al. 2019). SHEDCM contained higher levels of angiogenic factors compared to bone-marrow-derived MSC-CM (Konala et al. 2020).

SHED-CM in Treating Neural Injuries SHED-CM contains multiple cytokines and chemokines that have the ability to improve peripheral nerve regeneration and functional recovery (SugimuraWakayama et al. 2015). SHED-CM contains MCP-1 and secretes ectodomain of sialic-acid-binding Ig-like lectin-9 (sSiglec-9) crucial for SHED-CM-mediated functional recovery following severe peripheral nerve injury. This unique combination of neurotrophic factors enhances the neurite extension of the peripheral nerve and promotes the formation of a Schwann cell bridge and axonal extension (Kano et al. 2017). In vivo studies also showed promising results for SHED-CM in neural regeneration (Kano et al. 2017; Sugimura-Wakayama et al. 2015). SHED-CM administration in a rat nerve gap model was successfully able to induce axon regeneration and remyelination by enhancing the viability and neuritogenesis of neurons of dorsal root ganglia (Kano et al. 2017; Sugimura-Wakayama et al. 2015). MCP-1/sSiglec-9 is considered a set of tissue-repairing M2 macrophage inducers that enhance nerve regeneration by M2 macrophage polarization. This antagonizes the proinflammatory M1 conditions associated with a neural insult (Kano et al. 2017; Matsubara et al. 2015), thereby suppressing inflammatory mediators IL-1β, TNF-α, IL-6, and iNOS and increasing the expression of anti-inflammatory markers arginine-1 and IL-10 (Kano et al. 2017). In a perinatal hypoxia-ischemia-induced brain injury mice model, intracerebral administration of SHED-CM was able to generate an anti-inflammatory microenvironment, which reduced tissue loss and resulted in significant recovery in neurological function, survival rate, and neuropathological score (Yamagata et al. 2013). In a further investigation, SHED-EXs enhanced the recovery of focal cerebral injury in rats (Inoue et al. 2013), improved rat motor functional recovery, and reduced cortical lesion in a traumatic brain injury rat model (Li et al. 2017). SHED-EXs were not only able to inhibit the activity of the M1 phenotype of microglia but also augmented the activity of the M2 phenotype of microglia, thereby suppressing neuroinflammation by anti-inflammatory cytokines

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(Inoue et al. 2013; Li et al. 2017). Moreover, SHED-CM enhanced ischemic brain injury by promoting the migration and differentiation of endogenous neuronal progenitor cells and boosted vasculogenesis (Inoue et al. 2013). Both SHED-CM and DPSCs-CM significantly promoted the regeneration of transected axons via inhibiting multiple axon growth inhibitor signals directly or by paracrine mechanisms, as compared to fibroblast-CM or BMMSCs-CM. In vitro, SHED-CM revealed higher levels of MCP-1 and sSiglec-9 compared with BMMSCs-CM (Sakai et al. 2012). Correspondingly, in vivo, the neuroprotective effects of SHED-CM were confirmed (Asadi-Golshan et al. 2018; Matsubara et al. 2015; Sakai et al. 2012). SHED-CM improved markedly the nerves’ functional recovery as compared with BMMSCs-CM (Matsubara et al. 2015, Sakai et al. 2012). The therapeutic effect of SHED-CM was largely attributed to the immunoregulatory functions that activate anti-inflammatory M2-like macrophages and suppress proinflammatory mediators (Matsubara et al. 2015). Furthermore, SHED-CM converted the proinflammatory brain/spinal cord environment induced by amyloid plaques into an anti-inflammatory state and induced neuroregenerative effects by altering microglial phenotype, as shown in a mouse model of Alzheimer’s disease (AD) (Mita et al. 2015) and a mouse model of multiple sclerosis (MS) (Shimojima et al. 2016). SHED-CM administration showed a cognitive function improvement superior to BMMSC-CM and fibroblast-CM despite the similar suppression of the proinflammatory cytokines and the expression of oxidative-nitrosative stress markers attained by SHED-, BMMSC-, and fibroblastCM. However, SHED-CM uniquely activated M2-type microglia, leading to the expression of the mRNA encoding BDNF, a neurotrophin that plays a key role in synaptic remodeling associated with memory formation. Similar neuropathological recovery was observed in a previous study (Yamagata et al. 2013). SHED-CM improved functional behavior in an in vitro model of Parkinson’s disease. SHED-CM was able to differentiate into dopaminergic neurons. The therapeutic success was attributed to the induction of neurite outgrowth, neuronal survival, together with the repression of 6-hydroxydopamine-induced cell death (Fujii et al. 2015). Similarly, SHED-CM showed a positive outcome in a Parkinson’s disease rat model (Jarmalaviciute et al. 2015; Narbute et al. 2019; Chen et al. 2020). The systemic administration of SHED-CM in treating superior laryngeal nerve lesion rat model protected the swallowing reflex, reduced pharyngeal residue, and promoted functional recovery via two mechanisms (macrophage polarization and vascularization) (Tsuruta et al. 2018). Collectively, SHED-CM has neural regenerative potential ascribed to the release of multiple growth factors, including NGF, BDNF, NT-3, ciliary neurotrophic factor, and glial-cell-line-derived neurotrophic factor (GDNF) (Sugimura-Wakayama et al. 2015). SHED-CM can stimulate angiogenesis by VEGF expression (de Cara et al. 2019) as well as inhibit iNOS generation (Mita et al. 2015). Taken together, the previous results validated the potential of SHED-CM/EXs as a candidate for neural treatment and the notion that SHED-CM may act through several mechanisms to promote neural functional recovery.

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Secretome/Conditioned Medium Derived from Dental Pulp Stem Cells DPSCs possess a remarkable differentiation potential into ectodermal, mesodermal, and endodermal cell lineages (Yamada et al. 2019a). DPSCs express neural-stemcell-like markers, besides MSCs markers, like GFAP and nestin, which contribute to the amplification of their self-renewal abilities as well as multipotency (Geng et al. 2017). Moreover, stemness-related markers, like Nanog, octamer-binding transcription factor (Oct)-3/4, and sex-determining region Y (SRY)-box 2 (SOX-2) were also expressed by DPSCs (Yan et al. 2010). The remarkable immunomodulatory properties of DPSCs could be attributed to their ability to express IL-6, IL-8, and TGF-β, which play a role in T-cell functional inhibition (Bianco et al. 2016; Rajput et al. 2014). DPSCs secrete a variety of neurotrophic factors, such as NGF (Zhang et al. 2017b), GDNF (Chang et al. 2014), and BDNF (Kanafi et al. 2014), as well as different angiogenic factors, like VEGF, FGF, and PDGF, with a stimulating increase in their expression following injury (Tran-Hung et al. 2008). Additionally, DPSCs secrete angiogenin, CSF, angiopoietin-1, IL-8, IGF-binding protein-3, and endothelin-1 (Hilkens et al. 2014; Ratajczak et al. 2016; Bronckaers et al. 2013). Despite the fact that DPSCs and SHED are derived from dental-pulpal tissues and have several similar properties, SHED has a higher proliferation rate but a lower osteogenic potential than DPSCs (Yazid et al. 2018). DPSCs, on the other hand, have a higher proliferative capacity and telomerase activity than PDLSCs (Hakki et al. 2015). The previously mentioned characteristics of DPSCs mark their uniqueness, which is further reflected in their secreted secretome/CM.

The Osteogenic Potential of DPSCs-CM DPSCs osteogenic differentiation may be affected by the surrounding microenvironment (Ma et al. 2009). DPSCs cultivated with DPSCs-CM have enhanced mineralization potential (Paschalidis et al. 2014). The regenerative potential of DPSCs-CM was evaluated in an osteogenic distraction mice model under different culture conditions (Fujio et al. 2017). DPSCs-CM improved the expression of osteogenic and chondrogenic markers, especially under hypoxic conditions. These findings indicate that the paracrine effects of DPSCs upregulate the angiogenic factors (VEGF-A and angiopoietin-2) (Fujio et al. 2017) as well as increase mineralization potential through the expression of TGF-β1 (Paschalidis et al. 2014), which triggers new bone formation and improves osteoblastic/chondrogenic markers (Osterix, SOX-5, factor VIII) (Fujio et al. 2017).

DPSCs-CM in the Treatment of Liver Disorders One of the promising regenerative applications of DPSCs-CM is the treatment of liver disorders. DPSCs-CM interestingly demonstrated the existence of a variety of

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liver lineage proteins, including hepatocyte nuclear factor, oncostatin M (OSM), growth-arrest-specific protein 6, and hepatocyte growth factor receptor in vitro (Kumar et al. 2017b), thereby promoting liver repair and regeneration.

Potential of DPSCs-CM to Regenerate Dental Tissues EXs derived from DPSCs revealed an effective stimulatory effect on odontoblastic differentiation, in vitro and in vivo, where it prompted the regeneration of dentalpulp-like tissue in an ectopic tooth transplantation model (Huang et al. 2016). Moreover, DPSCs-CM improved the proliferation as well as the migration of fibroblasts (Nakayama et al. 2017) and myoblasts (Kawamura et al. 2016) in vitro. This improvement was confirmed in vivo, through injecting DPSCs-CM into a root and transplanting subcutaneously in an immunodeficiency mice model (Hayashi et al. 2015). DPSCs-CM possesses powerful trophic factors, such as CXCL14 and MCP1, which promoted migration and angiogenesis. Furthermore, combining G-CSF with CM from mobilized DPSCs enhanced the proliferation and migration effects of DPSCs-CM (Nakayama et al. 2017). The high concentrations of BMP or NT-3 in DPSCs-CM (Joo et al. 2018) may be the cause of improving the odontoblastic differentiation of DPSCs in vitro (Murakami et al. 2015). Despite this outstanding potential, DPSCs-CM failed to induce odontoblastic differentiation in cells of nondental origin, like myoblast (Kawamura et al. 2016). A wide variety of pulp tissue markers were expressed in the tissues regenerated by DPSCs-CM, such as thyrotropin-releasing hormone-degrading enzyme, syndecan-3, G-CSF, CXCL14, neuropeptide Y, BDNF, IL-1α, IL-6, IL-8, IL-16, MCP1 (Hayashi et al. 2015), BMP2, BMP9, PDGF, TGF-β, dentin sialophosphoprotein (DSPP), and runt-related transcription factor 2 (RUNX2) (Huang et al. 2016), in addition to periodontal tissue markers like periostin and periodontal-ligament-associated protein and enamelysin (Kawamura et al. 2016). Several studies have been conducted to compare the regenerative capacity of DPSCs-CM with other cellular sources. An ectopic tooth model was used to compare BMMSCs-CM, ASCs-CM, and DPSCs-CM in regenerating dental pulp tissues. Compared to other cell-derived CM, DPSC-CM had the highest volume of regenerated pulp tissues. DPSCs-CM has shown angiogenesis in the in vitro dental pulp disease model of HUVEC (Kawamura et al. 2016; Murakami et al. 2015) and embryonic myoblasts (Hayashi et al. 2015) and had antiapoptotic activity in the mouse embryonic fibroblast cell line (NIH3T3) (Ishizaka et al. 2013). Compared to BMMSCs-CM and ASCs-CM, DPSCs-CM promoted the formation of new blood vessels (Hayashi et al. 2015). Although DPSCs-CM had no significant influence on the proliferation of endothelial cells, it enhanced their migration in vitro (Bronckaers et al. 2013). Furthermore, DPSCs-CM inhibited apoptosis in HUVECs (Iohara et al. 2008) as well as fibroblast cell lines by modulating caspase-3 activity (Nakayama et al. 2017). Several angiogenic factors have been found in DPSCs-CM, including VEGF, IL-8, IGF-binding protein-3, endostatin (Bronckaers et al. 2013), chemokine CXCL 14 (Hayashi et al. 2015), and MCP-1 (Bronckaers et al. 2013;

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Hayashi et al. 2015). The aforementioned research highlights DPSCs-CM as a promising new treatment tool for the regeneration of dental tissue through different mechanisms of action, including the promotion of odontoblastic differentiation, antiapoptotic factors, and angiogenesis. Exploring the therapeutic potential of DPSCs-CM in the regeneration of nondental tissues will bring enormous benefits in this era.

The Role of DPSCs-CM in the Treatment of Neurological Diseases DPSCs-CM showed great potential in neuron regeneration, where it demonstrated the ability to induce recruitment and neuronal maturation, as well as the neuritogenesis of human neuroblastoma cells in vitro (Gervois et al. 2017), along with neurite outgrowth (Ishizaka et al. 2013). The regenerative power of DPSCsCM, BMMSCs-CM, and ASCs-CM was compared in an in vitro retinal nerve injury model. DPSCs-CM exhibited neuroprotective effects and neurogenesis due to increased levels of different neurotrophic factors (including NGF, BDNF, and VEGF) (Mead et al. 2014). In addition, in an in vitro model of nerve injury, DPSCs-CM promoted the proliferation, differentiation, and migration of Schwann cells and inhibited their apoptosis and increased angiogenesis (Yamamoto et al. 2016). Additionally, DPSCs-CM displayed a neuroprotective effect in an in vitro model of AD. Its effect was due to the upregulation in the expression of B-cell lymphoma 2 and the downregulation in apoptosis regulator Bax in neuroblastoma cells. In comparison to BMMSCs-CM or ASCs-CM, DPSCs-CM contains a high concentration of fractalkine (antiapoptotic factor), VEGF, and neprilysin, which degrade amyloid peptide (one of the most common misfolded proteins found in AD), besides Fms-like tyrosine kinase receptor-3 (FLT-3), GM-CSF, RANTES, and MCP-1, which makes it a promising candidate for the treatment of AD (Ahmed et al. 2016). DPSCs-CM not only provided a neuroprotective effect but also increased the number and total length of HUVEC tubular structures in an in vitro ischemia model (Song et al. 2017). The systemic administration of DPSCs-CM in a mutant superoxide dismutase mouse model of amyotrophic lateral sclerosis (ALS) showed a promising therapeutic potential (Wang et al. 2019). DPSCs-CM enhanced the neuromuscular junction innervation and survival of motor neurons in treating ALS through various trophic factors and cytokines (Wang et al. 2019). In the same way, when DPSCs-CM was injected into the unilateral hind limb skeletal muscles of diabetic polyneuropathy rats, it demonstrated neuroprotective, anti-inflammatory, and angiogenic effects (Makino et al. 2019). Moreover, in a rat aneurysmal subarachnoid hemorrhage model, the intrathecal administration of DPSCs-CM demonstrated enhancement in cognitive and motor impairments, reduction of neuroinflammation, and microcirculation. Different factors contributed to these significant enhancements, including tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, IGF-1, and TGF-β (Chen et al. 2019). Recently, the effect of various manufacturing features, such as the preconditioning of DPSCs with certain

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factors, the period of conditioning, and the storage of CM on the functional activity of DPSCs-CM on neurite length, was evaluated (Chouaib et al. 2021). The results revealed that conditioning DPSCs for 48 h is optimal for the functional activity of DPSCs-CM, DPSCs-CM significantly improved neurites’ outgrowth of sensory neurons in a concentration-dependent manner, and the frozen storage of DPSCsCM did not affect the experimental results. Furthermore, the authors demonstrated that conditioning DPSCs with B-27 supplement had a significant impact on the influence of their CM in enhancing neurite outgrowth in primary sensory neurons by altering its growth factors’ composition. Taken together, these data indicate that DPSCs-CM has many neuroprotective and angiogenic factors, such as NGF, BDNF, and VEGF (Mead et al. 2014), RANTES, fractalkine, FLT-3, GM-CSF, MCP-1, and neprilysin (Ahmed et al. 2016), in addition to IGF-1, TGF-β, TIMP-1, and TIMP -2 (Chen et al. 2019). They also provide evidence that DPSCs-CM has a promising ability to induce tissue regeneration in many neurological diseases.

DPSCs-CM in the Treatment of Autoimmune Diseases Owing to its immunoregulatory properties and anti-inflammatory effects, DPSCs-CM holds a promising potential in the treatment of autoimmune diseases superior to BMMSCs-CM (Yamada et al. 2019b). DPSCs-CM possessed the ability to inhibit allogeneic peripheral blood mononuclear proliferation at different time points (48 and 72 hours after incubation) (Hossein-Khannazer et al. 2020). Recently, DPSCs-CM demonstrated great potential in treating Sjögren’s syndrome (a chronic inflammatory autoimmune disease associated with hyposalivation) in a mouse model (Ogata et al. 2021). DPSCs-CM contained extra anti-inflammatory cytokines as compared to BMMSCs-CM. This was reflected histologically by the lesser inflammation in the submandibular salivary gland, in addition to increased salivary flow rate. DPSCs-CM modulated the submandibular salivary gland local inflammatory microenvironment by altering the expression of inflammatory cytokines. The expression of IL-10 as well as TGF-β was increased, while the expression of Il-4, Il-6, and Il-17a was decreased. Furthermore, DPSCs-CM increased the percentage of regulatory T cells while decreasing the percentage of T-helper 17 cells (Ogata et al. 2021). The previously mentioned anti-inflammatory combined with antiproliferative properties of DPSCs-CM makes it a novel cell-free therapy for treating different autoimmune diseases.

Secretome/Conditioned Medium Derived from Gingival Mesenchymal Stem/Progenitor Cells GMSCs harvested from the gingival connective tissues are MSC subpopulations that possess remarkable regenerative properties (El-Sayed et al. 2015; Jin et al. 2015; Zhang et al. 2012; Fawzy El-Sayed et al. 2012b, 2015). GMSCs are abundant, homogenous, easily obtainable; preserve normal karyotyping; and maintain stable

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morphology with passaging. GMSCs have a fast proliferation rate, with remarkable multidirectional differentiation potential and immune regulatory properties (Fawzy El-Sayed and Dorfer 2016). GMSCs could release an array of secretomes with various biological therapeutic actions (Fawzy El-Sayed and Dorfer 2016; Zhang et al. 2012; Fawzy El-Sayed et al. 2018, 2019a; Zhang et al. 2017a; Mekhemar et al. 2018). GMSCs were found to express CD13, CD38, CD44, CD54, CD117, CD144, CD146, CD166, Sca-1, STRO-1, SSEA-4, Oct-3/4, Oct-4A, Nanog, nestin, integrin β1, and vimentin, in addition to MSC surface markers (El-Sayed et al. 2015; Jin et al. 2015; Xu et al. 2013).

GMSCs-CM in Treating Neural Disorders Various literature pieces suggested that GMSC-derived EXs, EVs, or CM could be an efficient therapeutic approach (Mao et al. 2019; Rao et al. 2019) in managing motor neuron injury (Rajan et al. 2017a), peripheral nerve injury (Mao et al. 2019, Rao et al. 2019), as well as bone (Diomede et al. 2018c) and skin defects (Shi et al. 2017) by increasing the expression of anti-inflammatory cytokine (IL-10), antiapoptotic cytokine (Bcl2) (Rajan et al. 2017a), and neural-growth-associated markers (BDGF, NT3, neurofilament 200, S100) (Mao et al. 2019) (Rao et al. 2019) (Rajan et al. 2017a) (Zhang et al. 2019), as well as by enhancing the proliferation and regeneration of nerve cells detected by proliferating cell nuclear antigens (PCNAs) (Mao et al. 2019), cell count kit-8 (CCK-8) (Rao et al. 2019), and Shh (Zhang et al. 2019), aside from suppressing proinflammatory cytokines (TNF-α (Rajan et al. 2017a), IL-17, IFN-γ) (Rajan et al. 2016; Giacoppo et al. 2017) and proapoptotic (Bax and cleaved caspase-3) and oxidative stress markers (superoxide dismutase (SOD)-1, iNOS, Cox-2) (Rajan et al. 2017a). Upon uploading GMSCs-EXs on biodegradable chitin conduits, an enhanced in vitro growth of DRG axon and Schwann cell proliferation, besides a significant increase in the thickness of nerve fibers and myelin sheath and recovery of the muscle and neuromuscular function in rats of sciatic nerve defect model, were revealed (Rao et al. 2019). Similar regenerative results were attained upon using GMSCs-derived EVs in treating crush-injured sciatic nerve in mice. Moreover, GMSCs-EVs robustly blocked the activity of c-JUN/N-terminal kinase (c-JUN/ JNK), which abolishes the upregulation of Schwann cell repair genes concomitantly with upregulated the expression of c-JUN, Notch1, GFAP, and SOX-2 genes associated with Schwann cell repair (Mao et al. 2019). The neuroprotective capability of human GMSCs-CM on scratch-injured motor-neuron-like NSC-34 cells was evolved by suppressing apoptotic markers (cleaved caspase-3 and Bax) and oxidative stress markers (SOD-1 and iNOS) while upregulating the expression of anti-inflammatory cytokines IL-10 and neurotrophic factors (BDNF and NT3), in addition to NGF and TGF-β (Rajan et al. 2017a). GMSCs-EXs proved to regenerate tongue taste buds and papillae after being transplanted in critical-sized tongue defects in rats with an increased expression of CK14+; CK8+; and types I, II, and III taste bud cell markers (NTPDase 2, PLC-β2,

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and AADC, respectively), as well as nerve fiber markers (UCH-L1/PGP9.5 and P2X3 receptor) and two trophic factors (BDNF and Shh), which are involved in the proliferation and differentiation of basal epithelial progenitor cells into taste bud cells and the reconstruction of submucosal connective tissues (Zhang et al. 2019). The rapid wound healing rate in the gingiva was primarily attributed to the GMSCs and their unique secretory mechanism through the Fas/Fas-associated phosphatase1/caveolin-1 complex, which triggers SNARE-mediated membrane fusion to secrete a large quantity of IL-1 receptor antagonist (IL-1RA)-expressing EVs, inhibiting the proinflammatory cytokine IL-1β (Kou et al. 2018). This finding represents an auspicious application potential for tongue reconstruction in patients suffering from tongue cancer.

GMSCs-CM in Treating Skin Injuries Isolated EXs derived from GMSCs loaded on chitosan/silk hydrogel sponge effectively promoted the healing of skin defects in diabetic rats. This was evidenced by the formation of neo-epithelium and collagen, an increase in the microvessels’ number in the wound bed, and neuronal ingrowth detected by neurofilament heavy chain (NEFH) two weeks postsurgery (Shi et al. 2017).

GMSCs-CM Osteogenic Potential The osteogenic regenerative potential of a poly-(lactide) (3D-PLA) scaffold supplemented with human GMSCs and human GMSCs-CM was revealed in rat calvaria bone defects after 6 weeks. Moreover, in vitro next-generation sequencing confirmed the increase in the genes involved in ossification (ASF1A, GDF5, HDAC7, ID3, INTU, PDLIM7, PEX7, RHOA, RPL38, SFRP1, SIX2, SMAD1, SNAI1, SOX-9, and TMEM64) in the 3D-PLA loaded with GMSCs-CM (Diomede et al. 2018c). This was attributed to the cytokines and growth factors contained in the CM, which could activate the mobilization and osteogenic differentiation of both endogenous MSCs and GMSCs (Bermudez et al. 2015, 2016; Osugi et al. 2012; Vizoso et al. 2017; Diomede et al. 2018c). In a further study, human GMSCs-EVs that were complexed with polyethyleneimine (PEI) to improve their internalization were loaded on 3D-PLA combined with human GMSCs. The PEI-EVs þ 3D-PLA þ human GMSCs revealed more calcium deposits with the upregulation of adhesion molecules regulating genes and genes involved in ossification processes, 6 weeks later in vitro. Also, in vivo computed tomography revealed the formation of new bone spicules and blood vessels in rats’ calvarial bone defects implanted with 3D-PLA þ PEI-EVs þ human GMSCs and 3D-PLA þ PEI-EVs. It was hypothesized that the osteogenic potential of PEI-EVs-human GMSCs loaded on 3D-PLA was mediated mainly by TGFβR1, SMAD1, BMP2, mitogen-activated protein kinase (MAPK)-1, MAPK14, and RUNX2 through TGF-β signaling (Diomede et al. 2018b).

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Recently, it has been hypothesized that the appropriate preconditioning of MSCs with disease-related stimuli can optimize exosomal proteins or miRNA contents, which would efficiently support tissue regeneration and repair. Concomitantly, it has been found that TNF-α preconditioned-GMSC-derived exosomes utilized in treating periodontitis not only enhanced the amount of exosome secreted from GMSCs but also increased the exosomal expression of CD73, thereby inducing antiinflammatory M2 macrophage polarization. Furthermore, the local injection of GMSC-derived exosomes in a ligature-induced periodontitis mice model markedly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts by exhibiting antiosteoclastogenic activity. Such potentials were significantly enhanced by the preconditioning of GMSCs with TNF-α. The previous findings highlighted TNF-α-preconditioned GMSCderived exosomes as a promising strategy that could alleviate the suffering of patients with periodontitis and other inflammatory osteoimmune diseases (Nakao et al. 2021).

Periodontal Ligaments Stem-Cell-Derived Secretome/ Conditioned Medium The periodontal ligament (PDL) is considered an enriched source of stem/progenitor cells utilized in periodontal tissue regeneration (McCulloch and Bordin 1991; Isaka et al. 2001; Fawzy El-Sayed and Dorfer 2017; Nagata et al. 2017) due to the enhanced expression of scleraxis (responsible for the formation of the cementumPDL complex) (Seo et al. 2004). Human PDLSCs are similar to BMMSCs when it comes to immunomodulatory functions, high proliferative rate, and in vitro differentiation ability into adipogenic, osteogenic, chondrogenic, and neurogenic cell lineages (Rajan et al. 2017c; Sedgley and Botero 2012; Achilleos and Trainor 2012). PDLSCs express proteins that are not present in BMMSCs, including CLPP, NQO1, SCOT1, a new isoform of TBB5, and DDAH1 (Eleuterio et al. 2013; Menicanin et al. 2014; Tsumanuma et al. 2011). The therapeutic effects of human PDLSCs in PDL and alveolar bone homeostasis could be further mediated via secreted paracrine signaling molecules (Rajan et al. 2016). Human PDLSCs were reported to regulate the adipogenic as well as osteogenic differentiation of ABMSCs and inhibit ABMSCs induced osteoclastogenic differentiation of human peripheral blood mononuclear cells (Park et al. 2012). Additionally, PDL cells-CM can regulate the expression of cell proliferation and bone homeostasis genes from MSCs cocultured with BMP-2 (Mizuno et al. 2008). Permanent and deciduous PDL cells’ cytokines profile analysis demonstrated a strong expression of proteins concerned with degradation and immune responses in deciduous PDL-CM, while permanent PDL-CM expressed markedly cytokines related to angiogenesis (EGF and IGF-1) and neurogenesis (NT-3 and NT-4), hence they are a powerful candidate for tissue regeneration (Kim et al. 2016). Moreover, PDL epithelial rests of Malassez demonstrated the expression of significant amounts of chemokines, proteins, and growth factors (IL-1, IL-6, IL-8, and

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IL-10; GM-CSF; MCP-1, 2, and 3; amphiregulin; VEGF; GDNF; and IGF-binding protein-2) (Ohshima et al. 2008).

PDLSCs-CM in the Therapy of Neural Disorders The immunosuppressive effects of human PDLSC secretome in managing MS were proved (Rajan et al. 2016, 2017b) through increased levels of IL-10, TGF-β, and SDF-1α (Rajan et al. 2016). In an autoimmune encephalomyelitis (EAE) model, disease regression and remyelination of the spinal cord were referred to human PDLSC-EX/MV (EMV) fractions. PDLSCs-CM and PDLSCs-EMVs reduced proinflammatory cytokines TNF-α, IL-17, IL-6, IL-1β, and IFN-γ and induced antiinflammatory IL-10 expression, besides attenuated expression of apoptosis-related markers Bax, STAT1, caspase-3, and p53 in the spleen and spinal cord (Rajan et al. 2016). In a more recent study, a downregulated expression of NALP3 inflammasome, cleaved caspase-1, IL-1β, IL-18, Toll-like receptor (TLR)-4, and nuclear factor (NF)-κB was demonstrated in an EAE mouse spinal cord after treatment with human PDLSCs-CM and EMVs. Ultimately, it could be deduced that both human PDLSCs-CM and purified EMVs exerted immunosuppressive effects and may serve as an effective economic approach in treating MS (Rajan et al. 2017b). Similarly, human PLSCs-CM under hypoxic conditions repressed inducedEAE in a murine model after being injected through the mice’s tail vein. The marked expression of antiapoptotic and anti-inflammatory markers (protein Bcl-2 and cytokine IL-37, respectively), as well as the suppression of proapoptotic markers (cleaved caspase-3 and Bax, respectively), was concomitantly associated with the regression of the disease’s clinical and histological features. Moreover, a regenerative potential has been observed upon treating the in vitro scratch injury model–exposed neurons NSC-34 via hypoxic-human PDLSCs-CM (Giacoppo et al. 2017). The aforementioned studies propose PDLSCs-CM as a new pharmacologic tool for managing MS through a remarked expression of antiinflammatory cytokines (IL-10, TGF-β) (Rajan et al. 2016, Giacoppo et al. 2017) and antiapoptotic cytokine (Bcl2) (Rajan et al. 2017a; Giacoppo et al. 2017) and the subsequent suppression of proinflammatory mediators (IL-4, IL-17, IFN-γ, TNF-α, IL-6, IL-1β) (Rajan et al. 2016, Giacoppo et al. 2017), proapoptotic markers (Bax and cleaved caspase-3 (Rajan et al. 2016, 2017a; Giacoppo et al. 2017) and p53 and STAT1 (Rajan et al. 2016)), cleaved caspase-1 (Rajan et al. 2017b), and oxidative stress markers (SOD-1, iNOS, COX-2) (Giacoppo et al. 2017; Rajan et al. 2017a). A reduction in NALP3, IL-1β, IL-18, TLR-4, and NF-κB expression was reported to mediate the nerve regenerative effect of PDLSCs (Rajan et al. 2017b). Moreover, PDLSCs-CM increased the expression of markers associated with neural growth, such as BDNF, IL-37, and NT-3, besides autophagy markers (Beclin-1, LC3) (Giacoppo et al. 2017).

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PDLSCs-CM Osteogenic Potential The in vitro and in vivo results following the culturing of 3D collagen membrane loaded with human PDLSCs and CM or EVs or EVs treated with PEI (PEI-EVs) demonstrated an initially upregulated expression of osteogenic markers (RUNX-2 and BMP-2/4), besides high VEGF, VEGF receptor-2, and collagen type 1 protein levels (Pizzicannella et al. 2019). Likewise, loading Evolution (Evo) (a commercially available collagen membrane) with human PDLSCs enriched with EVs and PEI-EVs revealed high osteogenic properties and biocompatibility in vitro and in rats’ calvarial defects. A quantitative reverse-transcription polymerase chain reaction showed the upregulation of osteogenic genes MMP-8, TGF-β1, TGF-β2, tuftelininteracting protein (TFIP11), tuftelin 1 (TUFT1), RUNX2, SOX-9, and BMP2/4 in the presence of PEI-EVs (Diomede et al. 2018a). Ultimately, these results demonstrated that human PDLSCs might be an effective strategy in bone regenerative medicine, consequent to their potential to increase osteogenic and angiogenic mediators through the TGFβ-BMP signaling pathway.

PDLSCs-CM in Dental Tissue Regeneration In treating periodontal tissue defects in a rat model, transplanted PDLSCs-CM enhanced periodontal tissue regeneration via suppressing the inflammatory response induced by TNF-α, IL-6, IL-1β, and COX-2. PDLSCs-CM was enriched by extracellular matrix proteins, angiogenic factors, enzymes, cytokines, and growth factors, as revealed by proteomic analysis (Nagata et al. 2017).

Secretome/Conditioned Medium Derived from Stem/Progenitor Cells of Apical Papilla (SCAP), Dental Follicle Stem/Progenitor Cells (DFSCs), and Tooth Germ Progenitor Cells DFSCs demonstrated proper osteogenic and cementogenic differentiation capacity mediated through the in vitro and in vivo expression of nestin, Notch-1, bone sialoprotein (BSP), collagen type I, osteocalcin (OCN), and fibroblast growth factor receptor (FGFR)1-IIIC (Kemoun et al. 2007; Morsczeck et al. 2005, 2008). Similarly, SCAP possess odontogenic and adipogenic differentiation ability (Abe et al. 2007; Sonoyama et al. 2006) and express neurogenic markers in vitro (Abe et al. 2007). Being the primary source of odontoblasts at the root region, SCAP can differentiate into dentin-pulp complex (Huang et al. 2008). SCAP and DFSCs revealed comparable hepatogenic differentiation potential and superior neurogenic ability to BMMSCs (Rao et al. 2019; Kumar et al. 2017a). The secretome collected from human DMSCs (DPSCs, DFSCs, and SCAP) stimulated colony formation in preneuroblast cell line IMR-32 and neurite differentiation with a significant increase in neural gene expression (MFI, MAP-2,

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β-tubulin III, nestin, and SOX-1), as well as cytokines and growth factors involved in neural regeneration (CSF, IFNγ, TGFβ, NGF, NT-3, and BDNF), more efficiently than BMMSCs’ secretome. On the contrary, IL-17 expression was higher in BMMSCs-CM as compared to DPSCs-CM (Kumar et al. 2017a). DMSCs-CM could further provide a valuable strategy for liver regeneration. The presence of hepatic lineage protein GAS6 in the secretome of DPSCs, SCAP, and DFSCs and different LDL receptor proteins in the secretome of DPSCs and SCAP reflected their role in regulating the transport and metabolism of lipids, as well as hepatic differentiation. Interestingly, OSM and HGFR, important inducers of hepatic lineage differentiation, were expressed solely in DFSC secretome (Kumar et al. 2017b). The presence of osteogenic lineage proteins was demonstrated in high amounts in human dental MSCs-CM. DPSCs-CM expressed seven proteins, including BMP7 and DSPP, while human DFSCs-CM expressed six proteins, including proteins regulating; endochondral ossification (MINPP1), bone turnover (WISP2) and mineralization (enamelin). SCAP-CM expressed 14 proteins including four of the five proteins expressed by BMMSCs-CM, among them FBN1, DDR2 and Zinc finger protein (ZNF)-423, that play important roles in osteoblastic maturation, activation of BMPs and differentiation of bone osteocytes respectively (Kumar et al. 2018). The ability of DMSCs-CM to express these osteogenic proteins provides a great opportunity for several applications of DMSCs-CM in the regeneration of many bone disorders. The biological effects of dental stem cells conditioned medium are summarized in Fig. 2.

Conclusions DMSC-derived secretome possesses a diversity of capabilities for tissue engineering and regenerative medicine. The usage of stem/progenitor cell secretome in regenerative medicine provides an appropriate alternative to stem-cell-based therapies with their numerous limitations. Concerning stem/progenitor cells’ restrictions, they have a low survival rate after their transplantation (Modo et al. 2002), in addition to a significant risk of malignant transformation following their in vitro expansion, a mandatory step before their clinical use (Baglio et al. 2012; Rubio et al. 2008). A cell-free secretome/CM therapeutic strategy could restore back the function of damaged tissues via the activation of signalling pathways based on the transfer of bioactive molecules, proteins and mRNAs to the affected tissues (Haider and Aslam 2018). Cell-free secretome/CM therapy offers a new perspective in regenerative medicine with minimal or no risks of host rejection, antigenicity, tumorigenicity, and infection, usually present in stem-cell-based therapies. Despite the numerous benefits of stem cell secretome applications in tissue regeneration, many obstacles are still present before its translation into clinical trials. It is highly recommended to develop a manufacturing, easy-to-apply protocol, free from any animal-based products, as well as determine its proper

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Fig. 2 Illustrative diagram showing dental MSCs-CM biological effect

dosage, exact protein composition, and mechanism of action before applying secretome to human beings. Through emerging technologies and frequent research, the full potential of DMSC secretome in regenerative medicine would be shortly unleashed, and soon it would be ready for its clinical translation into the dental and medical fields.

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Biological Activity and Approaches of Large-Scale Production M. O. Gomzikova, V. James, and A. A. Rizvanov

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cell-Derived Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Mesenchymal Stem Cell-Derived EVs in Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . Immunosuppressive Activity of EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EV-Mediated Mitochondria Donation by MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Approaches of Large-Scale Production of Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytochalasin B-Induced Membrane Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vesicle Production by Mechanical Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vesicle Production Using a Hyperosmotic Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The discovery of extracellular vesicles has provided an outstanding breakthrough in stem cells and regenerative medicine. It has been shown that cells can transfer information through the secretion of soluble factors, the formation of direct physical contacts, and the secretion of extracellular vesicles containing a wide range of biologically active factors, including proteins, lipids, and genetic information.

M. O. Gomzikova · A. A. Rizvanov (*) Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia e-mail: [email protected] V. James School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_48

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All these factors are protected from destruction by the extracellular vesicles’ bilayer lipid membrane, allowing the transfer of information from cell to cell over considerable distances. Extracellular vesicles derived from stem cells can reprogram a range of target cells, stimulating their viability and migration. The biological capacity and regenerative potential of stem cell-derived extracellular vesicles are comparable to that of the stem cells in vivo. Due to biosafety concerns over the use of cell-based therapies, the potential of a cell-free therapy based on extracellular vesicles, with equal efficacy to intact stem cells, is highly desirable for future therapeutic purposes. This chapter will discuss current research into the biological activity and therapeutic application of extracellular vesicles derived from mesenchymal stem cells. With a focus on approaches for the large-scale production and isolation of extracellular vesicles will enable the transition of extracellular vesicle research into clinical application. Keywords

Cell-free therapy · Cytochalasin B · Extracellular vesicles · Hyperosmotic vesiculation · Immunosuppression · Mechanical extrusion · Mesenchymal stem cells · Mitochondria donation · Regeneration List of Abbreviations

ADSC AMI BM-MSC CIMVs CKD DC dsDNA EVs GvHD ICG ISEV LPS MHC miRNA MISEV mRNA MSC mtDNA MVB MVs PBMCs PHA SC ssDNA

Adipose-derived mesenchymal stem cells Acute myocardial infarction Bone marrow-derived mesenchymal stem cells Cytochalasin B-induced microvesicles Chronic kidney disease Dendritic cells Double-stranded DNA Extracellular vesicles Graft-versus-host disease Indocyanine green The International Society for Extracellular Vesicles Lipopolysaccharide Major histocompatibility complex MicroRNA Minimal information for studies of extracellular vesicles Messenger RNA Mesenchymal stem cells Mitochondrial DNA Multivesicular body Microvesicles Peripheral blood mononuclear cells Phytohemagglutinin Stem cells Single-stranded DNA

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TNTs UACR UC-MSC WJ-MSC

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Tunneling nanotubes Urinary albumin creatinine ratio Umbilical cord mesenchymal stem cells Wharton’s jelly-derived mesenchymal stem cells

Introduction Mesenchymal stem cells (MSCs) have enormous potential in regenerative medicine (Haider and Ashraf 2005). However, the production of cell-based therapeutics is a time-consuming and high-cost process that requires special equipment and expertise. The discovery of the regenerative activity of culture media conditioned by MSCs revealed the role of the MSC secretome in tissue repair and led to the formulation of the paracrine hypothesis (Haider and Aziz 2017; Gomzikova and Rizvanov 2017). MSCs-derived secretome consists of soluble growth factors, cytokines, and extracellular vesicles that mediate the transfer of complex bioactive molecules and the horizontal transfer of genetic information between cells (Lei and Haider 2017). Given the therapeutic significance of the cell secretome, various strategies have been developed to tailor the composition to desired composition (Haider et al. 2008; Durrani et al. 2010; Elmadbouh et al. 2007). The release of extracellular vesicles (EVs) was first described more than 50 years ago (Wolf 1967). For a long time, it was believed that EVs were inert “garbage” released by cells as a means of off-loading the unwanted waste products. More recently, EVs are being considered as critical components of many physiological and pathological processes and are being considered as essential means of intercellular communication and bioactive material transfer. EVs are membrane-bound, spherical vesicles released by almost all types of cells. The main functions of EVs are information transfer to recipient cells or to the extracellular environment to mediate intercellular communication (Colombo et al. 2014). EVs are found in almost all tissues, organs, and body fluids: saliva (Palanisamy et al. 2010), urine (Pisitkun et al. 2004), blood (Benz Jr. and Moses 1974), bronchoalveolar lavage (Levanen et al. 2013), nasal discharge (Levanen et al. 2013), amniotic fluid (Keller et al. 2007), uterine fluid (Griffiths et al. 2008), breast milk (Lasser et al. 2011), bile (Witek et al. 2009), cerebrospinal fluid (Street et al. 2012), synovial fluid (Gyorgy et al. 2012), and can also be obtained from supernatants of cultured cells, including MSCs. EVs contain proteins, lipids, and genetic information obtained from parental cells (Colombo et al. 2014). It is well-established that EVs derived from stem cells retain the same molecular features and demonstrate similar biological activities as the parental cells, such as the presence of specific surface receptors and bioactive molecules. For example, they demonstrate the same potential to stimulate cell proliferation, viability, and chemotaxis of target cells, and suppression of inflammation and oxidative stress (Seo et al. 2019). Thus, the biological effects of MSCs and their secretomes are comparable; however, the secreted products are devoid of a significant number of risks that are otherwise associated with the application of

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whole MSC preparations (Lei and Haider 2017). In addition, the composition and biological properties of the secretome as well as EVs secreted by MSCs can be changed in line with tailored therapeutic aims, with no risk of their oncotransformation. To date, there is reported evidence of the regenerative effects of EVs derived from MSCs in the treatment of ischemic and ischemic/reperfusion damage of myocardial cells (Zhao et al. 2019), diabetic complications (Grange et al. 2019), bone defects (Otsuru et al. 2018), liver damage (Li et al. 2013), lung, nervous tissue, and skin injuries (Khatri et al. 2018; Galieva et al. 2019; Ren et al. 2019). EVs are natural vehicles within the human body, and as such, constitute a promising vector system for the delivery of bioactive molecules and drug targeting. Due to their extremely small size and lipophilic characteristics, EVs demonstrate better tissue distribution and penetration, and are able to successfully cross the blood-brain barrier (Alvarez-Erviti et al. 2011). The ability of EVs to deliver biologically active molecules to the target cells, the absence of nuclei, lack of proliferation and tumorigenicity, biocompatibility, and safety render them a promising therapeutic tool. However, despite encouraging data emanating from the preclinical experimental studies, and clinical trials, currently there are limitations that need to be overcome for their use in the development of EVs-based biotherapeutics. These include the development and optimization of protocols for large-scale production, purification, and storage of EVs. This chapter focuses on the extensive experience of working with EVs besides the in-depth analysis of the published data from other research groups on the development of new drugs and biopharmaceutics based on the secretome products and EVs of human MSCs in the clinical perspective.

Extracellular Vesicles EVs are spherical structures ranging in size from 50 to 2000 nm, surrounded by a phospholipid bilayer and containing receptors and biologically active molecules, including lipid mediators (e.g., eicosanoids), proteins (e.g., cytokines, chemokines, growth factors, transcription factors, ferments) and nucleic acids (mRNA, rRNA, siRNA, miRNA, tRNA, Y-RNA, ssDNA, dsDNA, mtDNA). It is believed that nucleic acids (especially mRNA, miRNA, noncoding RNA) determine much of the high biological activity of EVs (Haider and Aramini 2020). The composition of noncoding RNAs and their profile found in MSC-derived EVs often differs in the composition found in the MSCs themselves; this indicates the existence of mechanisms of specific RNA loading as well as sorting into vesicles (Groot and Lee 2020; Haider and Aramini 2020). EVs can also capture and transfer whole organelles such as mitochondria (Islam et al. 2012), ribosomes (Court et al. 2008), and proteasomes (Yu et al. 2014; Gomzikova et al. 2019a). The composition of the EV “cargo” depends on the type of the parent cell and its pathophysiological state, culture conditions in vitro, as well as the signals from their microenvironment (Yamamoto et al. 2019).

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Cells release different types of EVs into the extracellular space acting on neighboring cells and distant tissues. The range of EVs reported may be broadly categorized as exosomes, microvesicles, and apoptotic bodies (Thery et al. 2018). EVs classification is predominantly based on the route of biogenesis and size (Thery et al. 2018). In addition to the three categories, the biofluid origin EVs are also used to categorize the vesicle types; for example, oviductosomes, prostasomes, epididymosomes, and uterosomes are terms used to indicate vesicles isolated from the oviduct, seminal, epididymal, and uterine fluids, respectively (Machtinger et al. 2016; Al-Dossary et al. 2013). Despite these differences, the critical characteristic of all EVs is the presence of a lipid double-membrane within which bioactive surface molecules are embedded. The International Society for Extracellular Vesicles (ISEV) has developed the Minimal information for studies of extracellular vesicles (MISEV 2018), a comprehensive guide on isolation and classification of different types of EVs (Thery et al. 2018). Exosomes are small vesicles of endosomal origin with 40–150 nm diameters, which commonly exhibit the markers CD63, CD9, Alix, and TSG101. Exosomes are formed by the invagination of the endosomal membrane into the multivesicular body (MVB), followed by MVB’s fusion with the cytoplasmic membrane and release of exosomes into extracellular space (Gomzikova and Rizvanov 2017; Willms et al. 2016). Microvesicles (MVs) have a larger range of sizes spanning 100–2000 nm in diameter and are characterized by cell type-specific surface receptors, phosphatidylserine and flotillin-2. MVs are formed by protrusion and budding directly from the plasma membrane and carry the cytoplasmic content of the parent cell (Skotland et al. 2020; Gomzikova and Rizvanov 2017). Thus, MVs derived from MSCs retain their surface mesenchymal markers, such as CD44, CD90, CD105, and CD146 (Bruno et al. 2017; Gomzikova et al. 2020a). Apoptotic bodies are large vesicles with diameters of 1000–5000 nm; they often exhibit annexin V and carry cargos of DNA and histones formed due to programmed cell death and destruction (Yamamoto et al. 2019). These EV subpopulations overlap in size and density, and the cell origin of the EVs can influence the markers they exhibit, making it challenging to isolate pure populations. Therefore, researchers work with a heterogeneous population of EVs more frequently within arbitrary size ranges dictated by the isolation techniques being applied (Gomzikova and Rizvanov 2017).

Mesenchymal Stem Cell-Derived Extracellular Vesicles MSCs are one of the most promising donor cells for EVs production. They are widespread within an organism and are often found within the bone marrow, adipose tissue, muscles, bones, and umbilical cord blood. They demonstrate low-level immunogenicity due to the low major histocompatibility complex (MHC) class I expression and a lack MHC class II expression (Ryan et al. 2005). EVs derived from MSCs also have inherent low immunogenicity and share the high therapeutic

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potential akin to their parental cells, thus rendering EVs a promising therapeutic option in regenerative medicine. The main advantage of EVs in general and exosomes in particular over soluble factors is that they carry a spectrum of biologically active molecules (proteins, lipids, and nucleic acids) (Haider and Aramini 2020). They not only protect their payload, they also transfer this payload to the recipient cells (Fig. 1). In addition, the main advantage of EVs over MSCs is their small size, better tissue penetration, absence of embolism risk, immune acceptance, lack of a nucleus, and the inability to proliferate (Fig. 1) (Gomzikova et al. 2019a). EVs derived from MSCs promote angiogenesis (Zhang et al. 2015), suppress apoptosis and stimulate cell proliferation (Ren et al. 2019), induce cell migration and tissue regeneration mechanisms (Zhang et al. 2020a), and immunomodulatory activity (Gomzikova et al. 2019a). In addition, MSC-derived EVs can also recruit and reprogram cells required for tissue regeneration (Ratajczak et al. 2006) and deliver biological factors and organelles to the target cells (Gomzikova et al. 2021). Autologous or allogeneic MSCs can be used for the production of their secretome including EVs for subsequent use in cell-free therapy (Haider and Aslam 2018). Using EVs derived from autologous MSCs is safer and ethically acceptable. However, the reparability and regenerative potential of MSCs significantly decreases with age and chronic diseases such as coronary heart disease and diabetes mellitus (Shahid et al. 2016; Haider 2018; Efimenko et al. 2015). Therefore, the application of EVs derived from allogeneic MSCs may be required to have the necessary therapeutic efficiency and be available as an off-the-shelf “ready-to-use” preparation for immediate clinical needs.

Fig. 1 Advantages and disadvantages of MSCs, MSCs-derived bioactive molecules, and MSCsderived EVs as therapeutics

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Role of Mesenchymal Stem Cell-Derived EVs in Regeneration An increase in interest in the potential of EVs peaked after the demonstration that EVs derived from MSCs demonstrated biological activity comparable to parental cells. Numerous studies have since shown that MSCs-derived EVs mimic the effects of stem cells in various experimental models of tissue injuries (Table 1). The beneficial effect of EVs on tissue regeneration is mediated by the stimulation of cell proliferation and migration (Zhang et al. 2020a), inhibition of inflammation (Gomzikova et al. 2019a), and reduction of oxidative stress (Zhou et al. 2013). The use of EVs to stimulate regeneration in cardiovascular diseases has been reported for acute myocardial infarction (AMI), stroke, pulmonary hypertension, and septic cardiomyopathy (Yin et al. 2019). EVs have also been used to demonstrate pro-regenerative activity in treating neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and nervous tissue injury (traumatic brain injury, spinal cord injuries, stroke) (Galieva et al. 2019). The regenerative potential of EVs was also confirmed in treating liver damage, lung injury, and skin damage (Table 1). To date, there are three publications on the use of MSC-derived EVs in the clinic. The first evidence of the application of allogeneic EVs in the clinic was obtained in 2014 by Kordelas et al. (2014). The authors administrated MSC-derived EVs to treat steroid-refractory acute graft-versus-host disease (acute GvHD). No side effects were reported and an improvement in GvHD symptoms was seen, including a decrease in IL-1b, TNF-a, and IFN-γ levels (Kordelas et al. 2014). The second was a clinical trial of allogeneic MSC-derived EVs to treat chronic kidney disease (CKD) (Nassar et al. 2016). In 2016, Nassar et al. reported that the administration of MSCs-derived EVs improved the glomerular filtration rate (eGFR), serum creatinine level, blood urea, and urinary albumin creatinine ratio (UACR) in CKD patients (Nassar et al. 2016; Gomzikova et al. 2019a). More recently, the application of MSCs-derived EVs for the treatment of severe pneumonia was observed in COVID19 patients (Sengupta et al. 2020). The study included 24 patients who received a single intravenous infusion of allogeneic MSC-derived EVs. The use of the EVs was deemed safe as no side effects were evident up to 72 h after infusion (Sengupta et al. 2020). Despite these exciting proof-of-principle studies, further research is needed to evaluate the effectiveness of MSCs-derived EVs, including fully resourced clinical trials with larger patient numbers and adequate control groups. Given the challenges of producing natural EVs for clinical use, our research group has focused on the production of induced microvesicles derived from MSCs with cytochalasin B. These cytochalasin B-induced MVs (CIMVs) demonstrate identical angiogenic activity in vivo and possess the molecular content and angiogenic activity similar to the parent MSCs (Gomzikova et al. 2019b). This was also demonstrated that CIMVs retain a similar composition of growth factors, cytokines, and chemokines, and express surface receptors of MSCs (CD90+, CD29+, CD44+, CD73+) as the parental MSCs. Importantly, CIMVs can transfer membrane receptors to the surfaces of target cells that might be the mechanism that mediates the mimicry and reprogramming seen as part of MSC-induced regeneration.

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Table 1 The regenerative potential of extracellular vesicles derived from mesenchymal stem cells Source of EVs Model of disease Cardiovascular diseases Rat ADSC Myocardial infarction in rats

Adiposederived regenerative cells (ADRCs) Human UC-MSCs

Myocardial infarction in mice

Mouse BM-MSCs

Myocardial ischemia/ reperfusion model in mice

Rat BM-MSCs

Myocardial ischemia–reperfusion injury in rats

Myocardial infarction in rats

Liver diseases Human UC-MSCs

CCl4-induced liver fibrosis in mice

Human UC-MSCs

Acute liver failure induced by LPS þ D-GalN in mice

Lung injury Mouse BM-MSCs

LPS-induced acute lung injury in mice

Observed effect

Reference

Significantly improved cardiac function, suppressed MI-induced myocardial fibrosis and apoptosis, downregulated MI-induced inflammatory factor expression, promoted macrophage M2 t antiinflammatory polarization, activated S1P/SK1/S1PR1 signaling Prevented cardiac rupture, promoted cardiomyocyte survival by delivering miR-214

Deng et al. (2019)

Repaired the ischemic myocardium by inhibiting cardiomyocyte apoptosis and promoting angiogenesis and ECM remodeling, partly by activating the prosurvival Akt/Sfrp2 pathway Attenuated myocardial injury via shuttling miR-182, shifted polarization of macrophages to M2 phenotype Inhibited cardiomyocyte apoptosis, downregulated PTEN level, activated the PI3K/AKT signaling pathway, protected injured cardiomyocytes via miR-486-5p

Ni et al. (2019)

Decreased collagen expression and Smad2 activity, anti-fibrotic and antiinflammatory effects Decreased levels of NLRP3, caspase-1 anti-inflammatory cytokines

Li et al. (2013)

Suppression of signaling pathways of inflammation, reduction of SAA3 expression – Reduction of edema

Eguchi et al. (2019)

Zhao et al. (2019)

Sun et al. (2019)

Zhang et al. (2020b) Yi et al. (2019)

(continued)

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Table 1 (continued) Source of EVs Swine BM-MSCs

Model of disease Influenza virus in pigs

Kidney diseases Rat MSCs Renal ischemia reperfusion injury in rats

Human UC-MSCs

Nephrectomy in rats

Neurological diseases Human Autoimmune BM-MSCs encephalomyelitis in mice

Human ADMSCs

Human BM-MSCs

Human WJ-MSC

Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a progressive model of multiple sclerosis in mice Traumatic brain injury in rats

Perinatal brain injury in rats

Observed effect Inhibited the hemagglutination activity of influenza viruses, decreased apoptosis in lung epithelial cells, reduced production of pro-inflammatory cytokines, alleviated lung lesions

Reference Khatri et al. (2018)

Attenuated pathological damage, inhibited the inflammatory response, inhibited NF-κB activation, inhibited the expression levels of cleaved caspase-3, caspase9, and Bax, and upregulated expression level of Bcl-2 Reduced cell apoptosis and enhanced proliferation, renal function was improved and the histological lesion was mitigated, increased capillary vessel density and reduced renal fibrosis

Galieva et al. (2019)

Reduced demyelination, decreased neuroinflammation, upregulated the number of Tregs, reduced levels of pro-inflammatory cytokines, increased levels of antiinflammatory and neuroprotective proteins Improved motor deficits, reduced brain atrophy, increased cell proliferation in the sub-ventricular zone and decreased neuroinflammation Improved cognitive and sensorimotor functional recovery, increased the number of newborn neurons and endothelial cells, reduced neuro-inflammation Reduced neuroinflammation, decreased expression of inflammationrelated genes and secretion of pro-inflammatory cytokines by glial cells

Zou et al. (2016)

Riazifar et al. (2019)

LasoGarcia et al. (2018)

Zhang et al. (2017)

Thomi et al. (2019)

(continued)

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Table 1 (continued) Source of EVs MSCs

Model of disease Spinal cord injury in rats

Human ADMSCs

Sciatic nerve transection and implantation in rats

Rat ADMSCs

Subcortical stroke in rats

Skin damage Human BM-MSCs

Excisional wounds in streptozotocin-induced diabetic rats

Human UC-MSCs

Full-thickness skin defects in mice

Human ADMSCs

Full-thickness skin defects in mice

Observed effect Improved the functional recovery, reduced neuron loss, delivered miR-21 inhibiting the neurons apoptosis Promoted Schwann cells proliferation, migration, myelination, and secretion of neurotrophic factors in vitro, improved axon regeneration, myelination, restoration of denervation muscle atrophy in vivo Improved long-term functional outcome, enhanced axonal repair and brain connectivity, reduced cell death, reduced astrogliosis, decreased GFAP expression

Reference Kang et al. (2019)

Activated the PI3K/AKT signaling pathway, enhanced wound healing, stimulated angiogenesis in vivo Suppressed myofibroblast formation inhibiting the TGF-β2/SMAD2 pathway Promoted proliferation, migration, and angiogenesis, upregulated gene expression of proliferative markers and growth factors in vitro, increased reepithelialization, collagen deposition, neovascularization, and wound closure in vivo

Ding et al. (2019)

Chen et al. (2019)

OteroOrtega et al. (2020)

Fang et al. (2016) Ren et al. (2019)

BM-MSCs bone marrow-derived MSCs, ADMSCs adipose-derived MSCs, UC-MSCs umbilical cord-MSCs, WJ-MSC Wharton’s jelly-derived mesenchymal stem cells

Recently, using an experimental model of skin photoaging, it was observed that induced MVs decreased epidermal thickening and improved the organization of the dermal layer (Syromiatnikova et al. 2020). Furthermore, the authors confirmed that CIMVs demonstrate high biological activity and regenerative potential of parental MSCs. Hence, it was proposed that human MSCs-derived CIMVs might be a promising cell-free option for regenerative medicine.

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Immunosuppressive Activity of EVs The immunomodulatory activity of EVs was first described by Raposo et al. The authors showed that EVs derived from B-cells carry MHC class II molecules and participate in the regulation of T-cell response (Raposo et al. 1996). Thus, MSCs possess profound immunosuppressive activity, inhibiting T- and B-cell proliferation, modulating regulatory T cell function maturation and activation, as well as influencing antigen presentation by dendritic cells, decreasing the secretion of pro-inflammatory cytokines and cytotoxicity (Gomzikova et al. 2019a). It was confirmed that MSC-derived EVs also inhibit the activity and maturation of T cells (van den Akker et al. 2018; Khare et al. 2018) and suppress natural killer (NK) cell activity (Table 2) (Di Trapani et al. 2016). Furthermore, MSC-derived EVs can inhibit B-cell activity and direct monocytic cell polarization towards an immunosuppressive M2 phenotype. They also stimulate the differentiation of T-lymphocytes toward an anti-inflammatory phenotype of regulatory T lymphocytes (Tregs) (Table 2) (Zhang et al. 2014; Morrison et al. 2017). The immunosuppressive activity

Table 2 Immunosuppressive effects of MSCs-derived EVs on immune cells Immune cells T-cells

B-cells

NK-cells Dendritic cells

Macrophages

Effect of MSCs-derived EVs Inhibition of T-cell proliferation Induction of differentiation toward regulatory T cells Upregulation of the immune-modulating factor IL-10 Inhibition of B-cell proliferation Inhibition of maturation Decreasing of immunoglobulin secretion Inhibition of NK-cell proliferation Reduced NK-cell number Suppression of DC activation Upregulation of immunomodulatory factors (TGF-β and PGE2) Inhibition of DC proliferation Inhibition of chemotaxis Shift the M1/M2 balance Inhibition of M1 (mir-147 dependent) Downregulation of pro-inflammatory signaling (CCL5, TNF-α, and IL-6) Upregulation of Arg1 (M2-derived) Stimulation of M2 polarization (by delivery of activated signal transducer and astat3)

Reference Di Trapani et al. (2016), Blazquez et al. (2014) Wen et al. (2016), Del Fattore et al. (2015) Del Fattore et al. (2015) Di Trapani et al. (2016) Budoni et al. (2013) Di Trapani et al. (2016) Koch et al. (2015) Di Trapani et al. (2016)

Shen et al. (2016) Song et al. (2017) Spinosa et al. (2018) Willis et al. (2018)

Zhao et al. (2018)

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of EVs has been also described in depth in other published reviews (Gomzikova et al. 2019a). In numerous preclinical studies, treatment with EVs has been shown to inhibit immune cell proliferation, reduces inflammation, and improves injury symptoms. The immunosuppressive and regenerative effects of EVs derived from BM-MSCs were demonstrated in an experimental model of cardiac ischemia in rats (Teng et al. 2015). The authors showed inhibition of T-cell proliferation in vitro and attenuated infarct size in vivo after the application of EVs (Teng et al. 2015). The use of MSC-derived EVs was also effective in mediating immunosuppression, reducing neuronal degeneration, suppression of microglia activation, and inducing tissue regeneration in experimental animal models of neuronal tissue injury, such as stroke, traumatic brain injury, and acute spinal cord injury (Hu et al. 2016; Drommelschmidt et al. 2017; Ruppert et al. 2018). The immunomodulatory effects of EVs derived from human MSCs in experimental animal models of autoimmune diseases also demonstrated a range of beneficial effects, including a decrease of inflammatory infiltrates and brain atrophy coupled with an increase in cell proliferation in experimental model of multiple sclerosis (Ruppert et al. 2018). In mouse model of type 1 diabetes, EVs caused inhibition of antigen-presenting cell activation and suppression of Th1, Th17 (Shigemoto-Kuroda et al. 2017). For experimental models of rheumatoid arthritis, inhibition of T-lymphocyte proliferation and attenuation of inflammatory response have been reported (Cosenza et al. 2018). In vivo models of atopic dermatitis showed an improvement of the symptoms, with decreased expression of inflammatory cytokines and reduced eosinophils, mast cell infiltration, and CD86+, CD206+ cells in the skin under the area of atopic dermatitis (Cho et al. 2018). Finally, EVs improved symptoms and decreased mortality in recipient mice undergoing acute graft versus host disease (Wang et al. 2016; Fujii et al. 2018). Furthermore, the authors showed that the use of EVs led to a decrease of CD4+ and CD8+ T-lymphocytes, as well as suppression of T-lymphocyte differentiation into the effector phenotype and an overall reduction in organ damage (Wang et al. 2016; Fujii et al. 2018). The immunosuppressive activity of the induced microvesicles (CIMVs) derived from MSCs have also been demonstrated (Gomzikova et al. 2020b). The data showed that CIMVs inhibited phytohemagglutinin (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs), with a more pronounced effect on T-lymphocytes. Moreover, CIMVs significantly suppressed activation of T-helpers (CD4 þ CD25+), B-cells (CD19 þ CD25+), and T-cytotoxic lymphocytes (CD8 þ CD25+) in vitro (Gomzikova et al. 2020b). Using an experimental model of mice immunization with ovine red blood cells, it was demonstrated that MSCs-derived CIMVs suppressed the humoral immune response and antibody production in vivo (Gomzikova et al. 2020a). Interestingly, no immunosuppression was observed in the animals pretreated with MSCs-derived EVs. In contrast, MSCs themselves and CIMVs-derived from them were similarly effective in suppressing antibody production in vivo (Gomzikova et al. 2020a). Therefore, the authors proposed that CIMVs are potentially a more appropriate substitutes for the MSCs-based cell therapy than endogenously produced EVs, combining advantages of safety and ease of production with retaining the parental MSCs immunomodulatory activity (Gomzikova et al. 2020a).

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EV-Mediated Mitochondria Donation by MSCs The regenerative activity of MSCs is accomplished by a complex influence on the injured cells. This includes the aforementioned stimulation of cell migration and proliferation, besides immunosuppression. More recently it is becoming widely accepted that the regenerative effect of MSCs is also in part mediated by the donation of mitochondria to recipient cells. Mitochondria donation from MSCs leads to the rescue of injured cells, improved oxidative phosphorylation, increased ATP production, and restoration of mitochondrial function (Gomzikova et al. 2021). Moreover, MSCs can also regulate the activity of immune cells through mitochondria transfer (Luz-Crawford et al. 2019; Gomzikova et al. 2021). The most recent studies to evidence mitochondrial transfer from MSCs to recipient cells are summarized in Table 3. Mitochondria transfer can be via cell-to-cell-based or cell-free mechanisms. The cell-based mechanism includes the transfer of mitochondria through tunneling nanotubes (TNTs) or cell fusion. The cell-free mechanism is via either the naked transfer of the mitochondria or encapsulation of the mitochondria within EVs (Gomzikova et al. 2021) (Fig. 2). However, due to the risks of cell-based therapy associated with the oncogenic transformation of transplanted cells and their differentiation into undesired cell lineages (Gomzikova et al. 2019a), the cell-free strategies of mitochondrial transfer are more attractive for the development of therapeutic

Table 3 Mitochondria donation from MSCs to recipient cells Donor cells Human marrow stromal cells MSCsderived from iPSCs

MSCs

MSCs and the MSC cell line HS27

BM-MSCs

Recipient cells, injury model Mouse neurons, exposure to hydrogen peroxide

Effect Increased neuronal survival, improved metabolism in vitro

Reference Tseng et al. (2021)

PC12 cells, CoCl2-induced hypoxia

Reduced apoptosis and restored δψm, ameliorated mitochondrial swelling, the disappearance of cristae, and chromatin margination in vitro Increased aerobic capacity and upregulation of mitochondrial genes in vitro Prevents ALL cell apoptosis and death from exogenously administered ROS-inducing agents in vitro

Yang et al. (2020)

Corneal endothelial cells (cecs), 661W cells and ARPE-19 cells REH, SD1, SEM, and TOM1 cell lines, expose to chemotherapy agents in vitro and in vivo on the model of acute lymphoblastic leukemia (ALL) T helper 17 (Th17) cells, rheumatoid arthritis

Oxygen consumption increase by Th17 cells and interconversion into T regulatory cells in vitro

Jiang et al. (2020) Burt et al. (2019)

Luz-Crawford et al. (2019)

(continued)

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Table 3 (continued) Donor cells BM stromal cell line

Recipient cells, injury model Acute lymphoblastic leukemia cells

BM-MSCs

Human umbilical cord vein endothelial cells, cytarabineinduced stress

MSCs

Neonatal mouse cardiomyocytes, hypoxia/ reoxygenation stress In vivo: intra-arterial injection on the model of ischemic stroke

MSCs

Wharton’s jelly MSCs (WJMSCs)

Fibroblasts were isolated from a MELAS patient skin punch biopsy

iPSCsderived MSCs

Injected into the vitreous cavity of one eye

BM-MSCs

Neurons, injection into the spinal cord in vivo, oxygenglucose deprivation injury in vitro, ischemic injury of the spinal cord in vivo

MSCs

Neural stem cells in vitro, intranasal administration in vivo, cisplatin damage in vitro and in vivo

Effect Metabolic support, changes in genes related to energy metabolism and redox status in vitro Reduced apoptosis, promoted proliferation and restored the migration ability and capillary formation in vitro Anti-apoptosis effect in vitro

Improved mitochondrial activity of injured microvasculature, enhanced angiogenesis, reduced infarct volume, and improved functional recovery in vivo Mutation burden of MELAS fibroblasts was reduced to an undetectable level, with longterm retention. Improves mitochondrial functions and cellular performance, including protein translation of respiratory complexes, ROS overexpression, mitochondrial membrane potential, mitochondrial morphology and bioenergetics, cell proliferation, mitochondriondependent viability, and apoptotic resistance in vitro RGC survival was significantly increased with improved retinal function in vivo Improved the bioenergetics profile, decreased apoptosis and promoted cell survival in post-OGD motor neurons in vitro, improved locomotor functional recovery in SCI rats in vivo Decreases cisplatin-induced NSC death, reversed decrease in mitochondrial membrane potential in vitro. Prevented the loss of DCX+ neural progenitor cells in vivo

Reference Usmani et al. (2019)

Feng et al. (2019)

Zhang et al. (2019) Liu et al. (2019)

Lin et al. (2019)

Jiang et al. (2019)

Li et al. (2019)

Boukelmoune et al. (2018)

(continued)

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Table 3 (continued) Donor cells MSCs

Recipient cells, injury model Skin fibroblasts from mitochondrial disease patient in vitro, intravenous injection in vivo

BM-MSCs

Jurkat cells, treatment with chemotherapeutic drugs

Effect Rescues impaired mitochondrial morphology, enhances host metabolic capacity, and induces widespread host gene shifting in vitro and in vivo Increased chemoresistance in vitro

Reference Newell et al. (2018)

Wang et al. (2018)

approaches. Therefore, mitochondrial transfer into the adult cells is in demand to treat of a range of mitochondria-related diseases, such as diabetes, neurodegenerative diseases, aging, and age-related metabolic disorders. EV-mediated mitochondria donation by MSCs was first discovered by Islam et al. (Islam et al. 2012). The authors used an experimental LPS-induced acute lung injury model to demonstrate the transfer of mitochondria encapsulated in EVs from bone marrow stem cells (BMSCs) to injured lung alveolar epithelial cells (Islam et al. 2012). Later, Falchi et al. observed membrane vesicles sized between 1 and 8 μm containing mitochondria in cultures of human fetal astrocytes (Falchi et al. 2013). Hayakawa et al. also detected extracellular particles containing mitochondria in cultures of rat cortical astrocytes (Hayakawa et al. 2016). Finally, Phinney et al. confirmed the EVs-mediated mitochondria donation mechanism and suggested that it might be a rescue mechanism following oxidative stress and clearance of mitochondria (Phinney et al. 2015) (Fig. 2). Recently, it was found that myeloid-derived regulatory cells (Hough et al. 2018), renal scattered tubular cells (Zou et al. 2018), HSCs, B cells, T cells (Zhang et al. 2020c), and neural stem cells (Peruzzotti-Jametti et al. 2020) are also able to release EV-encapsulated mitochondria. Our research group has demonstrated that induced microvesicles (CIMVs) contain mitochondrial DNA (Gomzikova et al. 2020a), as well as functionally active mitochondria (unpublished data). Therapeutic approaches for delivering functional mitochondria in human cells are being developed that might be applied to treat the deficiency of mitochondrial function in disease.

Approaches of Large-Scale Production of Vesicles Due to manufacturers and the pharmaceutical industry becoming aware of the potential of EVs, methods for the large-scale production of vesicles are required. Typically, EVs are harvested from conditioned medium derived from MSCs culture. Considering that during 1 h, one cell releases about 50–150 vesicles, mass production using this protocol would be pretty expensive as well as time-intensive (Wan et al. 2018). Initially, one of the most common protocols used for the isolation of EVs was serial centrifugation. This procedure required the high-speed centrifugation of the conditioned medium/liquids

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Fig. 2 The scheme of EVs encapsulated mitochondria (A) and EVs-mediated mitochondria donation from donor cell to recipient cell (B)

containing EVs (up to 100,000–120,000  g) to precipitate the vesicles in a range of sizes down to 1000 nm or less. The yield of EVs from this method was relatively low and the method itself was labor-intensive and time-consuming. In addition, coprecipitation of protein aggregates, apoptotic bodies, and nucleosomal fragments can reduce the purity of the samples obtained (Momen-Heravi et al. 2013). Fractionation methods such as density gradient centrifugation, ultrafiltration, affinity and gel chromatography, flow field-flow fractionation, and specialized commercial kits have been developed to increase the purity of EVs. However, these methods are often equally time-consuming, expensive, and may require specialist equipment and advanced expertise. Therefore, further development to improve both isolation and large-scale production methods are required before the clinical and commercial benefits of vesicles can be fully developed.

Cytochalasin B-Induced Membrane Vesicles As previously detailed, treatment of cells with cytochalasin B can induce higher rates of vesicle production (Pick et al. 2005). Cytochalasin B acts to inhibit the polymerization of the actin cytoskeleton (Gomzikova and Rizvanov 2017), causing the increased release of vesicles from the cytoplasmic membrane. This mechanism shares similarities with the natural mechanism of microvesicles production from the cytoplasmic membrane. It is known that for the formation of natural microvesicles, molecular changes within cells are required. The critical event in these changes is the local destruction of the actin cytoskeleton by Ca2+-dependent enzyme calpain (Piccin et al. 2007). Obtained induced microvesicles (CIMVs) surrounded by the cytoplasmic membrane contain the functionally active surface receptors of parental cells (Pick et al. 2005) and have a diameter of 100–1000 nm, comparable

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with naturally occurring EVs (Gomzikova et al. 2017). It was found that 17  6% of the cell membrane surface is transformed to induced microvesicles for 1.5 h after treatment with cytochalasin B (Pick et al. 2005; Gomzikova et al. 2020a). These induced microvesicles have been successfully derived from a variety of cell types, including HEK293 (Pick et al. 2005; Mao et al. 2011; Lim et al. 2014), 3T3 fibroblast (Mao et al. 2011), HUVECs (Peng et al. 2015), MDCKII-MDR1 (Eyer et al. 2014), SH-SY5Y (Gomzikova et al. 2017), PC3 cells (Gomzikova et al. 2018), and MSCs (Gomzikova et al. 2020a). Induced microvesicles have already been shown to have a range of beneficial uses; they have already been employed as bioelectronic sensors of non-small cell lung cancer to detect heptanal in patient plasma (Lim et al. 2014). Induced microvesicles are also promising biocompatible vectors of fluorescence dyes, molecular compounds, and other nanoparticles (Mao et al. 2011). Peng et al. demonstrated that induced microvesicles could be used as a vector to deliver antitumor agents. Following the drug delivery via induced microvesicles, they observed inhibition of tumor growth in an experimental mouse model of xeno-grafted tumors while also reporting that the toxicity of the drug was markedly lower when compared to the administration of the free drug (Peng et al. 2015). Induced microvesicles have also been used to encapsulate ICG (indocyanine green) (Sheng et al. 2016; Gomzikova et al. 2018) and methylene blue (Han et al. 2016). Encapsulation of ICG within the induced microvesicles led to a reduction in the clearance of ICG and increased the effectiveness of photothermal antitumor therapy in vivo (Sheng et al. 2016; Gomzikova et al. 2018). Vesicles loaded with methylene blue showed lower cytotoxicity while maintaining the effect of photodynamic anticancer therapy (Han et al. 2016). Oshchepkova et al. demonstrated that CIMV-encapsulated oligonucleotides are protected from the nucleases and transferred into recipient cells (Oshchepkova et al. 2019). These data demonstrate that CIMVs inherit the biological activity of parental cells and can be used as cell-free biotherapeutics. Moreover, CIMVs derived from SH-SY5Y cells contain growth factors of the parental cells and can stimulate capillary tube formation in vitro and angiogenesis in vivo (Gomzikova et al. 2017). Additionally, MSCs-derived CIMVs inherit the angiogenic (Gomzikova et al. 2019b), immunosuppressive (Gomzikova et al. 2020a, b), and regenerative activity (Syromiatnikova et al. 2020) of the parental stem cells. Thus, the effective biological activity of CIMVs and ease of loading with desired cargo components (e.g., drugs), together with the easier procedure of production and isolation of a more homogenous population of vesicles makes them a promising therapeutic tool for clinical use and regenerative medicine.

Vesicle Production by Mechanical Extrusion Membrane vesicles can also be obtained by extruding cells through polycarbonate filters. Wu H.W. et al. proposed to produce membrane vesicles using mechanical extrusion through filters with pore sizes of 1 μm or 2 μm. The authors applied this

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method to obtain membrane vesicles from human retinal pigment epithelium (ARPE-19) cells and demonstrated the resulting population of vesicles had diameters 0.2  0.1 μm and 0.8  0.5 μm (Wu et al. 2012). Xu L.Q. et al. applied the same technique using filters with a pore size 3 μm and produced vesicles from BM-MSCs, containing mitochondria (Xu et al. 2017). Despite the ability to produce vesicles of uniform size, the technique risks the degradation of the nuclei and contamination of the vesicles with nuclei material. These factors need to be thoroughly investigated before transferring this technique for mass production and clinical use.

Vesicle Production Using a Hyperosmotic Solution It was found that osmotic stress induces cells to release vesicles. Del Piccolo et al. developed a protocol based on sequential treatment of cells with hypo- and hypertonic buffers to induce vesiculation (Del Piccolo et al. 2012). Cells are washed twice with 30% PBS in deionized water – a hypotonic buffer that induces cell swelling. Then the cells are placed in a hypertonic solution containing 200 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, and 0.75 mM CaCl2 to stimulate vesicle release (Del Piccolo et al. 2012).

Conclusion and Future Directions The further study of the mechanisms of EV biogenesis, release, and effect on target cells is an important area of research that creates a theoretical basis for their therapeutic application in regenerative medicine. EVs derived from MSCs carry complex biological cargos and have a range of effects on target cells. However, the full range of these EVs’ properties and effects remain to be elucidated. Safe and effective clinical use of EVs requires overcoming several technical difficulties associated with their production. First, it is necessary to develop universal and easily reproducible protocols for the isolation of vesicles, ensuring their high yield with minimal contamination, and improvement in storage methods. Secondly, despite the relative safety of extracellular vesicles administration compared to stem cells, it is necessary to pay close attention to the cultivation conditions of cells (in particular to the presence of animal components in the EV preparations). EVs derived from MSCs are a promising therapeutic tool, which have advantages over cell-based therapy in terms of safety, ease of storage, and clinical use. In addition, the ability to modify the composition of EVs will open up broader prospects for their use in clinical practice. However, for this potential to be achieved, additional improvements to optimize their production, isolation, and storage are critical before transitioning to the development of biotherapeutics and routine clinical use proceeds.

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Cross-References ▶ Sources and Therapeutic Strategies of Mesenchymal Stem Cells in Regenerative Medicine Acknowledgments This chapter has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). This work was funded by RSF according to the research project №21-75-10035.

References Al-Dossary AA, Strehler EE, Martin-Deleon PA (2013) Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm. PLoS One 8:e80181 Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29:341–345 Benz EW Jr, Moses HL (1974) Small, virus-like particles detected in bovine sera by electron microscopy. J Natl Cancer Inst 52:1931–1934 Blazquez R, Sanchez-Margallo FM, de la Rosa O, Dalemans W, Alvarez V, Tarazona R, Casado JG (2014) Immunomodulatory potential of human adipose mesenchymal stem cells derived exosomes on in vitro stimulated T cells. Front Immunol 5:556 Boukelmoune N, Chiu GS, Kavelaars A, Heijnen CJ (2018) Mitochondrial transfer from mesenchymal stem cells to neural stem cells protects against the neurotoxic effects of cisplatin. Acta Neuropathol Commun 6:139 Bruno S, Tapparo M, Collino F, Chiabotto G, Deregibus MC, Soares Lindoso R, Neri F, Kholia S (2017) Renal regenerative potential of different extracellular vesicle populations derived from bone marrow mesenchymal stromal cells. Tissue Eng Part A 23:1262–1273 Budoni M, Fierabracci A, Luciano R, Petrini S, Di Ciommo V, Muraca M (2013) The immunosuppressive effect of mesenchymal stromal cells on B lymphocytes is mediated by membrane vesicles. Cell Transplant 22:369–379 Burt R, Dey A, Aref S, Aguiar M, Akarca A, Bailey K, Day W, Hooper S (2019) Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress. Blood 134:1415–1429 Chen J, Ren S, Duscher D, Kang Y, Liu Y, Wang C, Yuan M, Guo G (2019) Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function. J Cell Physiol 234:23097–23110 Cho BS, Kim JO, Ha DH, Yi YW (2018) Exosomes derived from human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis. Stem Cell Res Ther 9:187 Colombo M, Raposo G, Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289 Cosenza S, Toupet K, Maumus M, Luz-Crawford P, Blanc-Brude O, Jorgensen C, Noel D (2018) Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis. Theranostics 8:1399–1410 Court FA, Hendriks WT, MacGillavry HD, Alvarez J, van Minnen J (2008) Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system. J Neurosci 28:11024–11029 Del Fattore A, Luciano R, Pascucci L, Goffredo BM, Giorda E, Scapaticci M, Fierabracci A, Muraca M (2015) Immunoregulatory effects of mesenchymal stem cell-derived extracellular vesicles on T lymphocytes. Cell Transplant 24:2615–2627

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Facilitators of Repair and Regeneration Laura Yedigaryan and Maurilio Sampaolesi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skeletal Muscle Repair and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Introduction to Skeletal Muscle Regeneration Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of (Other) Residential Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Case for Muscular Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myofiber Intracellular Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins and Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA Cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of Vesicles by Target Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EVs Released by Different Cells in the Injured Muscle Environment . . . . . . . . . . . . . . . . . . . . The Inflammatory Response via EVs and Subsequent Activation of Nearby Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Skeletal muscle may be injured upon physical activity, or due to myofiber frailty caused by degenerative disorders. As a metabolic tissue, skeletal muscle has the innate ability of regeneration. Skeletal muscle regeneration may be endowed to L. Yedigaryan Translational Cardiomyology Laboratory, Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium M. Sampaolesi (*) Translational Cardiomyology Laboratory, Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium Histology and Medical Embryology Unit, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Sapienza University of Rome, Rome, Italy e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_49

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the action of quiescent satellite cells, the resident muscle stem cells, and other interstitial and inflammatory cells that directly and indirectly contribute to adult myogenesis. However, the process of muscle regeneration greatly relies on intercellular communication through signaling factors such as proteins, microRNAs (miRNAs), inflammatory cytokines, and membrane lipids that must be tightly coordinated. It is becoming more evident that the release and transmission of these factors involve extracellular vesicles (EVs) liberated by myofibers and other cells in the milieu of the injured muscle. The cargo of EVs is responsible for altering the state of their target cells by delivering purposeful molecules such as messenger RNAs, miRNAs, lipids, and proteins or by aiming at the alteration of gene expression. These changes activate downstream pathways involved in tissue repair. Due to the heterogeneity of EVs with regard to their cargo, location, size, as well as timing of formation and release, the repair and regeneration of skeletal muscle may subsequently be impacted. This chapter focuses on the impact of EVs as biological cues directing stem cell differentiation and modulating the overall process of skeletal muscle regeneration. Keywords

Epigenetics · Extracellular vesicles · Induced pluripotent stem cells · miRNAs · Mesenchymal stem cells · Muscular dystrophies · Myofiber repair · Satellite cells · Skeletal muscle · Stem cells Abbreviations

ADMSC Ago Akt ASCT2 ASM BMD CCL2 CDK2 circRNA CXCL1 CXCL1 DMD ECM ESCRT EV FAP GJA1 HGF IGF-1 IL-4 ILV

Adipose-derived mesenchymal stem cell Argonaute Protein kinase B Alanine-serine-cysteine transporter 2 Acid sphingomyelinase Becker muscular dystrophy C-C motif chemokine ligand 2 Cyclin-dependent kinase 2 Circular RNA C-X-C motif chemokine ligand 1 Fractalkine Duchenne muscular dystrophy Extracellular matrix Endosomal sorting complexes required for transport Extracellular vesicle Fibro-adipogenic progenitor Connexin 43 Hepatocyte growth factor Insulin-like growth factor-1 Interleukin 4 Intraluminal vesicle

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iPSC lncRNA MAB MDC/CCL22 miRNA MSC MVB myomiRs NO Nox1 NRG1 p120 PDGF-α PIC piRNA PSC RISC rRNA S1P SC scaRNA snoRNA snRNA TGF-β TLR tRNA UTR VEGF

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Induced pluripotent stem cell Long noncoding RNA Mesoangioblast Macrophage-derived chemokine microRNA Mesenchymal stem cell Multivesicular body Muscle microRNA Nitric oxide NADPH oxidase 1 Neuregulin 1 protein Catenin delta-1 Platelet-derived growth factor-α Twist2s and PW1+ interstitial cell PIWI-interacting RNA Pluripotent stem cell RNA-induced silencing complex Ribosomal RNA Sphingosine-1-phosphate Satellite cell Small Cajal body-specific RNA Small nucleolar RNA Small nuclear RNA Transforming growth factor-β Toll-like receptor Transfer RNA Untranslated region Vascular endothelial growth factor

Introduction Skeletal muscle represents one of the largest organ systems of the human body. The overall importance of skeletal muscle is attributed to metabolic homeostasis and movement. Therefore, damage to skeletal muscle, resulting in myofiber injury and possible death, highlights the critical ability of skeletal muscle repair and regeneration. Minor injuries may inflict sarcolemmal disruption, which may be repaired by membrane rectification, thus preventing myofiber death. However, severe injuries caused by resistance training overload, heavy load-bearing, and/or genetic defects may cause serious myofiber injury and death. Such injuries are ameliorated by the complex orchestration of inflammatory events and satellite cell (SC) activation and subsequent fusion, constituting the regenerative cycle of skeletal muscle injury. These measures represent the overall method of the restoration attempt upon disruption of homeostasis. Cellular and molecular events during skeletal muscle repair and regeneration constitute complex intracellular and intercellular transactions,

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critical for success in the process. Therefore, we will discuss how extracellular vesicles (EVs) – membrane-bound cargoes released by cells, enable communication within and between cells, devising the strict mechanism of repair and regeneration of the injured muscle (Yedigaryan and Sampaolesi 2021; Yedigaryan et al. 2022).

Skeletal Muscle Repair and Regeneration General Introduction to Skeletal Muscle Regeneration Events Skeletal muscle regeneration may be classified as a process structured through three distinct yet overlapping phases. Initially, severe injury induces necrosis and significant inflammation. Following the clearance of cellular debris, new fibers expressing embryonic and neonatal myosin heavy chain are formed. Hypertrophy and hyperplasia represent the next step in regeneration, partially regulated by the transforming growth factor-β (TGF-β)/Smad and insulin-like growth factor-1 (IGF-1)/protein kinase B (Akt) pathways. While TGF-β is said to negatively regulate muscle growth, IGF-1 induces muscle hypertrophy by controlling the balance between new protein synthesis and existent protein degradation (Schiaffino et al. 2013). The final step of regeneration is characterized by the restoration of vasculature and innervation patterns. Satellite cells (SCs), adult muscle stem cells, are set-aside between the basal lamina and the plasmalemma of the muscle fibers (Mauro 1961). These cells remain quiescent during homeostasis (Rumman et al. 2015; Schultz et al. 1978); however, upon injury, they proliferate and differentiate into myoblasts that further on fuse to form myotubes (in vitro)/myofibers (in vivo) in order to mature and eventually restore damaged fibers (Fig. 1) (Moss and Leblond 1970; Reznik 1969; Snow 1977; Nakamura et al. 2001). The maintenance of skeletal muscle during the lifetime of an individual is orchestrated through the action of SCs. As a result of growth cues or physical trauma, SCs become poised for activation. Through symmetric division, SCs reconstitute their quiescent pool. The outcome of asymmetric division is defined through both the repopulation of the SC pool, and the differentiation of some of these cells

Fig. 1 Progression of satellite cell activation and myotube/myofiber formation. (Created with BioRender.com)

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into myoblasts, capable of fusing with each other or damaged fibers to rebuild muscle function and integrity (Baghdadi and Tajbakhsh 2018). Quiescent SCs are said to express a multitude of genes to uphold their given state (Musarò 2014). The expression of genes such as Hey1 and Hey2 appear to increase once SCs are activated and proliferative (Scharner and Zammit 2011). Pax3 plays an essential role in coaxing quiescent SCs into committing to the myogenic lineage (Relaix et al. 2005; Buckingham 2007). Proliferating SCs express markers such as desmin, Myf5, MyoD, and PCNA (Scharner and Zammit 2011; Creuzet et al. 1998; Yablonka-Reuveni and Rivera 1994). Depending on MyoD expression, SCs may follow two fates. The downregulation of MyoD commits SCs into self-renewal, guaranteeing the regulation of a steady quiescent pool of Pax7-positive SCs. Alternatively, commitment to differentiation is due to the upregulation of MyoD and the consequent downregulation of Pax7, leading to the activation of myogenin expression (Boldrin et al. 2010; Day et al. 2007; Nagata et al. 2006; Relaix and Zammit 2012). Depending on the cellular context, pathways such as Notch and Wnt may either promote or block cell cycle progression. Notch signaling is prevalent during satellite cell proliferation, while Wnt signaling is predominant during differentiation. In some cases, Notch upregulation promotes the transition of activated SCs to proliferative myogenic precursor cells and myoblasts; however, differentiation to myotube formation is stalled. On the other hand, Notch signaling has been demonstrated to be necessary to maintain muscle stem cell quiescence and homeostasis (Bjornson et al. 2012; Mourikis et al. 2012). Besides muscle injuries, two molecules are said to be responsible for the activation of satellite cells after muscle injury (Relaix and Zammit 2012): hepatocyte growth factor (HGF) and nitric oxide (NO). HGF has been shown to activate quiescent satellite cells into entering the cell cycle both in vitro and in vivo. NO, possibly through the action of metalloproteinases, induces the release of HGF from the extracellular matrix. NO is synthesized through the action of nitric oxide synthase on L-arginine substrates. This component is also said to induce the follistatin expression, a molecule known to antagonize myostatin, thus also possibly contributing to the activation of satellite cells.

The Role of (Other) Residential Cells Other than SCs, it is clear that different cell types have an impact on the regeneration process. Following muscle injury, pericytes give rise to vessel-associated progenitors called mesoangioblasts (MABs). MABs are progenitor cells derived from the embryonic aorta (De Angelis et al. 1999). These cells are said to contribute to postembryonic mesoderm development and be rooted in the origin of vascular development (Minasi et al. 2002). While MABs have a lower myogenic potential than SCs, their potential for expansion, migration, and regeneration should not be undermined. Another subgroup of pericytes, located peripheral to the endothelium of microvessels, are constituents of the SC niche (Armulik et al. 2011). These cells modulate the behavior of SCs through the excretion of molecules such as IGF-1

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(Kostallari et al. 2015). Fibro-adipogenic progenitors (FAPs) that reside in muscle fibers interstitially express markers such as Sca1, CD34, and platelet-derived growth factor-α (PDGFR-α). These cells can differentiate into fibroblasts and/or adipocytes (Joe et al. 2010; Uezumi et al. 2010). In physiological settings, following acute injury, some FAPs are eliminated through apoptosis due to the cues generated by pro-inflammatory cytokines such as interleukin 4 (IL-4) (Joe et al. 2010). However, results have shown that coculture experiments demonstrate the importance of FAPs as sources of pro-differentiation factors. These cues drive the proliferation and differentiation of myoblasts, thus aiding in the positive regenerative outcome. Considering pathological settings, such as muscular dystrophies, FAPs represent the primary source of fibrosis (Lemos et al. 2015). Finally, mesenchymal stem cells (MSCs) are natural precursors of fat, cartilage, and bone (Bianco and Robey 2000). However, MSCs expressing specific transcription factors were reported to act as myogenic progenitors (Liu et al. 2017) and named Twist2s and PW1+ interstitial cells (PICs). However, the origin of these cells, as well as the interrelationship among them, remains uncertain.

The Immune Response Upon muscle injury, neutrophils/monocytes are initially recruited, and their action relies on phagocytosis, oxidation, and proteolysis of necrotic tissue (Sakuma and Yamaguchi 2012; Quattrocelli et al. 2010). Macrophages play a critical role in regeneration, since these cells promote myoblast proliferation. Despite the nature of the injury, the muscle regeneration process is reprised of two phases: degeneration (necrosis) and reconstruction (Kawiak et al. 2006). In all cases of muscle trauma, damage of the myofibers and sarcolemma consequently results in the permeability of the myofibers. This permeability leads to an influx of calcium into damaged myofibers, activating muscle proteases such as the calpains that damage muscle-important proteins. Leukocytes invade the damaged site as a first step, followed by neutrophils that lyse muscle cells in a superoxide-dependent manner.

Macrophages and Lymphocytes Initially, neutrophils are recruited, then macrophages (Sakuma and Yamaguchi 2012). Specifically, two different subpopulations of macrophages subsequently invade the injured muscle tissue. The first population (type I [M1]) consists of “inflammatory” macrophages, secreting pro-inflammatory cytokines such as IL-1β. These macrophages are also responsible for the phagocytosis of necrotic tissue. While the “inflammatory” macrophages reach their peak concentration 24 hours after injury, the second population (type II [M2]) of macrophages, the “antiinflammatory” macrophages, reach their peak at 2–4 days after injury (Mantovani et al. 2007). These macrophages secrete cytokines such as IL-10, known to contribute to the termination of inflammation. This second population of macrophages also has a distinct role in releasing factors that contribute to the proliferation, growth, and differentiation of myogenic precursors. Therefore, it could be elucidated that type I

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macrophages are correlated with muscle necrosis, while type II macrophages are associated with regenerative fibers (Pierre and Tidball 1994). Proper muscle regeneration is permitted through the direct contact of SCs and immune cells (Klimczak et al. 2018). As a result of the secretion of chemotactic factors such as vascular endothelial growth factor (VEGF), macrophage-derived chemokine (MDC/CCL22), and fractalkine (CX3CL1) by SCs, the inflammatory response can be initiated. Eosinophils also play an important role in the innate immune response associated with muscle regeneration (Hoffman et al. 1988; Maeda et al. 2017). Due to the lack of ability of muscle fibers to conduct a T cell response under normal circumstances, lymphocytes are not involved in skeletal muscle regeneration (Karpati et al. 1988; Maffioletti et al. 2014). However, this is not the case when it comes to inflammatory muscle diseases. In this particular case, muscle cells act as antigen-presenting cells and attract T lymphocytes to the site of injury. These cells are able to express human leukocyte class I and class II antigens (Wiendl et al. 2003).

The Case for Muscular Dystrophy Muscular dystrophies are a group of genetically heterogeneous neuromuscular disorders associated with progressive muscle weakness and deterioration (Amato and Griggs 2011). The most well-known form of muscular dystrophy is Duchenne muscular dystrophy (DMD). DMD is characterized by the absence of the protein dystrophin. Dystrophin is crucial in upholding the integrity of the sarcolemma, therefore in the absence of this protein, the sarcolemma is rendered unstable and frail (Forcina et al. 2020). Upon contraction, extensive damage of myofibers is apparent and newly regenerated myotubes are not able to rescue the damaged muscle niche. As a result of continuous degeneration, inflammation and regeneration are persistently stimulated, therefore altering the nonpathological dynamic. Concerning the inflammatory response, IL-6 is said to be involved in mediating the unwanted proliferation of SCs while impairing myoblast differentiation (Pelosi et al. 2015; Pelosi et al. 2014; Kurosaka and Machida 2013). Additionally, the transformed behavior of SCs may be dictated by the absence of dystrophin, since daughter cell fate during asymmetric division in dystrophin-deficient SCs may be altered (Chang et al. 2018). While FAPs assist SCs during nonpathological skeletal muscle regeneration, the role of FAPs in muscular dystrophies turns to mediating fat deposition and fibrosis, promoting the malformed dystrophic microenvironment (Uezumi et al. 2010). The contributions of chronic inflammation, defective myogenesis, and persistent degeneration all lead to the continuous defective regeneration of dystrophic muscles.

Myofiber Intracellular Repair It is becoming evident that the release and conduction of signaling molecules such as microRNAs (miRNAs) and other noncoding RNAs, proteins, and lipids involve the release of extracellular vesicles (EVs) by myofibers and other nearby cells in the

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environment of the injured muscle (Bittel and Jaiswal 2019). Many aspects of EVs are heterogeneous such as the size, location, composition, and time of release. As a result of this, the impact on the repair and regeneration of injured skeletal muscles varies. Vesicular activity is mainly the consequence of the rapid influx of extracellular calcium upon injury and subsequent plasma membrane damage. Specifically, the release of calcium initiates the exocytosis of vesicles such as late endosomes/ multivesicular bodies (MVBs) and lysosomes. The release may occur passively or through endosomal sorting complexes required for transport (ESCRT)-mediated release (Andrews et al. 2014; Jimenez et al. 2014; Scheffer et al. 2014; Demonbreun and Mcnally 2017; Romero et al. 2017). The exocytosis of lysosomes aids the endomembrane in closing the wound and allows the release of lysosomal enzyme acid sphingomyelinase (ASM) (Chakrabarti et al. 2003; Jaiswal et al. 2004; Defour et al. 2014; Sreetama et al. 2015), which eventually assists in ceramide release and subsequent plasma membrane repair by removal of damaged membrane through endocytosis and exocytosis (Babiychuk and Draeger 2000; Tam et al. 2010; Corrotte et al. 2013; Romancino et al. 2013; Draeger and Babiychuk 2013). The endocytic vesicles, containing components of the damaged membrane, fuse together to form late endosomes and MVBs (Murphy et al. 2018). Degradation of the internalized damaged proteins and lipids or the inward budding and the consequent creation of intraluminal vesicles (ILV) are the possible next steps. The MVBs may then exocytose their contents. Upon such a fate, ILVs are released and may then be called “exosomes.” Not only is the injured plasma membrane repaired, but EVs are able to generate a tissue-level repair response that goes beyond the minute myofiber repair phase. A study has revealed that exercise-induced injury in mice lead to an increase in circulating vesicles, both during the initial hours after injury as well as 5–7 days postexercise injury (Coenen-Stass et al. 2016). This discloses that these released vesicles are produced by both injured myofibers and regenerative cells. Myofibers produce three types of EVs – apoptotic bodies (50–5000 nm in diameter), exosomes (50–150 nm), and ectosomes (microvesicles) (100–1000 nm) (Fig. 2). The difference between these vesicles lies in size, cellular origin, composition, and mechanism of release. Exosomes are the smallest EVs. Other than lipids, exosomes contain proteins such as myogenic growth factors and contractile proteins (Choi et al. 2016; Demonbreun and Mcnally 2017). miRNAs are ~22-nucleotides long noncoding RNAs and mainly inhibit the translation of mRNAs (Bartel 2004) (Fig. 3). In mammals, cleavage of the target mRNA is very rare due to the lack of extensive miRNA: target base pairing (Xu et al. 2016). One example of such an occurrence is the cleavage of HOXB8 mRNA by miRNA-196. By binding to an Argonaute (Ago) protein and forming the core of the multicomponent RNA-induced silencing complex (RISC), miRNAs are guided to anneal to the 30 untranslated regions (UTRs) of target mRNAs. In response to muscle damage, miRNAs are detected only in vesicles, thus suggesting selective packaging (Siracusa et al. 2016). Moreover, depending on the context: e.g., muscular dystrophy-related damage or muscle injury, the content of specific muscle miRNAs (myomiRs) differs in the released exosomes (Roberts et al. 2012; Matsuzaka et al. 2016; Fry et al. 2017; D’Souza et al. 2018). Currently, more than 1900 different human miRNA sequences have been reported (as of miRBase 22.1, http://www.mirbase.org).

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Fig. 2 The synthesis and release of extracellular vesicles (EVs). EVs can be divided into apoptotic bodies, exosomes, and microvesicles

Fig. 3 The mechanism of miRNA and mRNA interaction. The binding of miRNAs, through the guidance of the RISC complex, to target mRNAs induces translational repression

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Following muscle damage cycling, exosomal release of miRNA-208a, miRNA-126, and miRNA-16 increases (D’Souza et al. 2018). Similarly, overload-induced injury causes the release of miRNA-206 within SC-derived exosomes (Fry et al. 2017). miRNA-30b and miRNA-181a are said to be involved in muscle regeneration and inflammation as well (Chen et al. 2008; Naguibneva et al. 2006). Lastly, DMD-associated muscle damage leads to the exosomal release of miRNA-1, miRNA-206, and miRNA-133a (Matsuzaka et al. 2016). This is not limited to DMD patients, as increased levels of miRNA-1 were found in limb-girdle muscular dystrophy, Becker muscular dystrophy (BMD), and facioscapulohumeral muscular dystrophy patients (Matsuzaka et al. 2014). Additionally, increased levels of miRNA-133a and miRNA-206 were found in BMD patients. These miRNAs play a vast amount of roles in muscle regeneration and development. The main roles entail the regulation of genes involved in proliferation, myogenesis, and the conversion of muscle fiber type (Cacchiarelli et al. 2010; Cazzella et al. 2012). In order for skeletal muscle repair to forgo issues, interactions between endothelial cells, inflammatory cells, MSCs, and myogenic stem cells are critical (Wosczyna and Rando 2018). This is facilitated through EVs. EVs are also crucial in the inflammatory response. MSCs release exosomes enriched with miRNA-1, miRNA-133, miRNA-206, miRNA-125b, miRNA-494, and miRNA-601 that promote a myriad of pro-regenerative cellular processes. Other than regeneration, EVs aid in remodeling damaged tissue by facilitating deposition and degradation of the new extracellular matrix (ECM), angiogenesis, fibroblast activation, and tissue cell replenishment. Under pathological settings, such as DMD, secreted vesicles are released with increased levels of miRNAs that promote fibrosis in skeletal muscles and the surrounding ECM (Zanotti et al. 2018). The ECM is indispensable when it comes to skeletal muscle development. It plays a large role in support as well as signaling (Ishii et al. 2018). Therefore, tissue engineering is a field that is promptly popularized with regard to the potential of inducing skeletal muscle regeneration. Tissue engineering encircles the concept of supporting muscle regeneration by means of three-dimensional implants with preferably the inclusion of scaffolds with bioactive molecules and/or stem cells (Cezar and Mooney 2015; Pascual-Gil et al. 2015; Quattrocelli and Sampaolesi 2015). Hydrogels may be utilized to generate biomimetic skeletal muscle tissues from human-induced pluripotent stem cell (iPSC)-derived cells. Pluripotent stem cells (PSCs) hold tremendous potential when it comes to cell therapy for degenerative diseases (Judson and Rossi 2020). These cells can essentially give rise to any cell of the adult human body. PSCs are highly proliferative and allow the use of the patients’ own cells to avoid immune challenges. In todays’ context, it would be of high merit to discuss iPSCs. These cells may be derived by the reprogramming of any human somatic cell, utilizing four transcription factors, namely Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka 2006). When considering the use of these cells in the context of skeletal muscle regeneration, it is important to develop suitable techniques for differentiation into tissue-specific progenitors with properties appropriate for transplantation. A key advantage would

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be to derive mesodermal myogenic progenitors from iPSCs as a route for cell therapy for skeletal muscle regeneration. Unfortunately, differentiation of iPSCs into skeletal muscle cells has proven to be challenging. Protocols depicting methods based on transgene mediation as well as soluble factor deployment have been described. Via viral gene delivery, positive results have been obtained in both in vitro and in vivo settings. Ectopic expression of Pax3, Pax7, and MyoD1 produce myogenic cells at high efficiencies (Darabi et al. 2011, 2012; Young et al. 2016; Tedesco et al. 2012; Santoni de Sio et al. 2008). However, much remains to be seen on the effect of intracellular cues as well as the transition through cellular intermediates to mimic natural regeneration. The risk of random viral DNA integration also remains a large concern in the field of cell therapy. Other drawbacks include time, cost, tumorigenicity, and proper quality control.

Extracellular Vesicles Proteins and Lipids Considering the origin of exosomes, their lipid content is enriched in lipids from the MVB lipid raft domains (Janas et al. 2015). Lipid rafts contain cholesterols, sphingomyelins, and phosphatidylserine (Choi et al. 2013). Lipid raft–associated proteins suggest the possibility of influence on EV protein sorting. Alternatively, ectosomes contain a more heterogeneous population of lipids (Meldolesi 2018). In addition to lipid content, the protein content difference between exosomes and ectosomes is approximated to be 65% (Le Bihan et al. 2012). EV lipid composition mainly consists of cholesterol, glycosphingolipid, sphingomyelins, phosphatidylserine, and ganglioside GM3 (Choi et al. 2013). Lipids of EVs may induce biological responses such as cell migration and proliferation (Xiang et al. 2018). Target cell interaction may also be influenced by the lipid composition of EVs (Miyanishi et al. 2007; Barreca et al. 2020). Proteomic analysis of exosomes derived from skeletal muscle revealed the presence of functionally critical proteins such as contractile proteins and myokines (Choi et al. 2016; Demonbreun and Mcnally 2017). On the other hand, ectosomes are enriched in membrane-enraptured proteins. A key mechanism with respect to selective cargo loading during skeletal muscle repair and regeneration may be protein lipid modifications. Protein sorting into exosomes may be affected by intracellular lipid modifications at the MVB membrane, leading to sphingosine-1phosphate (S1P) receptor activation and Rho-family GTPase stimulation (Kajimoto et al. 2013; Kajimoto et al. 2017). A study done by Ieronimakis et al. revealed that increasing levels of S1P improved muscle regeneration in mdx mice (Ieronimakis et al. 2013). A proteomic characterization of MSCs was performed by Lai et al., and Kim et al. (Lai et al. 2012; Kim et al. 2011). These authors identified specific markers of MSCs, such as CD63, CD109, CD81, as well as surface receptors important for cell differentiation such as EGF-R, and signaling molecules, for instance, RHO and

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MAPK1, for cell differentiation and self-renewal. Additionally, proteins implicated in intracellular trafficking, EV biogenesis, fusion, cell adhesion, morphogenesis, and migration were characterized.

RNA Cargo Unlike the abundance of ribosomal RNAs in the parent cell, EVs are mainly enriched in small RNAs such as miRNAs, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) (Crescitelli et al. 2013; Jeppesen et al. 2019). Although miRNAs have been the focus of many studies, a substantial amount of different RNA species are present in EVs (mRNA, ribosomal RNA [rRNA], yRNA, vault RNA, transfer RNA [tRNA], small Cajal body-specific RNA [scaRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA]) that may affect host and/or recipient cells (Kalluri and Lebleu 2016; Li et al. 2014; van Balkom et al. 2015; Zakharova et al. 2007; Vechetti Jr. 2019). Due to the differential RNA sorting mechanism, miRNAs are loaded into exosomes as opposed to ectosomes (Roberts et al. 2012; Matsuzaka et al. 2016; Fry et al. 2017; D’Souza et al. 2018). This is due to EXOmotifs in miRNAs, preferentially sorting them into exosomes (Villarroya-Beltri et al. 2013). Muscle damage due to muscular dystrophy or eccentric exercise evokes the elevation of tissue-enriched miRNAs (Coenen-Stass et al. 2016). In vivo, this situation is evident during regeneration, while in vitro, myoblast differentiation evokes such a response. After muscle damage, miRNAs are detected only in vesicles, while the level of many circulating miRNAs declines (Siracusa et al. 2016). This reveals the mechanism of selective packaging and release as opposed to unwarranted leakage. Furthermore, depending on the context of muscular damage, miRNAs loaded into vesicles differ and promote or inhibit cell proliferation and/or myogenic differentiation (Table 1) (Roberts et al. 2012; Matsuzaka et al. 2016; Fry et al. 2017; D’Souza et al. 2018). Some microRNAs such as miRNA-1 and miRNA206 restrict the proliferative potential of these cells, therefore facilitating differentiation. miRNA-1 upholds myogenesis by targeting a transcriptional repressor of muscle gene expression, HDAC4 (van Rooij et al. 2008; Chen et al. 2005). miRNA-206 acts on repressing the expression of Pax7. More specifically, miRNA206 negatively regulates DNA polymerase α translation, and partakes in the downregulation of connexin 43 (GJA1) as well as the repression of cyclin-dependent kinase 2 (CDK2) (Callis et al. 2008). Treatment of muscle with miRNA-431, miRNA-675, and miRNA-26a has been shown to increase muscle regeneration since miRNA-431 targets the expression of Pax7, therefore increasing the expression of MRFs in SCs (Wu et al. 2015). On the other hand, some miRNAs are crucial for upholding the integrity of the quiescent population. An example of such is being miRNA-489. It has recently become apparent that miRNAs possess sorting sequences that determine whether they will be secreted into EVs or retained by cells, and that different cell types make preferential use of specific sorting sequences (Garcia-Martin et al. 2021).

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Table 1 Muscle-specific miRNAs and possible role(s) in cellular processes miRNAs miRNA208a miRNA-126 miRNA-16 miRNA-30b miRNA181a miRNA-1/ miRNA-206

Muscle tissue specificity Heart

miRNA-1

Heart/Skeletal muscle, skeletal muscle Heart/Skeletal muscle

miRNA-206

Skeletal muscle

miRNA133a

Heart/Skeletal muscle

miRNA133b miRNA125b miRNA-494 miRNA-601 miRNA-720

Skeletal muscle

Possible role(s) Promotes muscle fiber shift, regulator of myostatin Promotes muscle growth Regulates blood vessel formation Regulates blood vessel formation Inflammatory response Promotes myogenic differentiation Activate satellite cells and promote myogenic differentiation Inhibits cell proliferation, promotes myogenic differentiation, regeneration, angiogenesis regulation Inhibits cell proliferation, promotes myogenic differentiation, promotes regeneration of skeletal muscle, promotes regeneration of neuromuscular synapses Promotes cell proliferation, inhibits cell proliferation, promotes myogenic differentiation, promotes fusion, regeneration, muscle fiber shift Promotes myogenic differentiation and fusion, promotes regeneration Inhibits myogenic differentiation Inflammatory response Inflammatory response Promotes cell proliferation

Uptake of Vesicles by Target Cells Another important aspect of EVs is the uptake of these vesicles by target cells. The uptake may be done by phagocytosis, macropinocytosis, or receptor-mediated endocytosis. Via different surface receptors and their ligands, exosomes and ectosomes exhibit cell-specific signaling (Sahoo and Losordo 2014). Specific peptides on the EV membrane may prompt EV cargoes to be targeted by neurons or skeletal muscles (Alvarez-Erviti et al. 2011). EVs target different tissues upon skeletal muscle injury and repair. For instance, muscle damage induced by exercise directs EVs to the liver (Whitham et al. 2018). Evidence suggests that the cross talk between other organs may also involve skeletal muscle (Hamrick 2012; Rondon-Berrios et al. 2014). Exosomes are mostly enriched in tetraspanins. Upon muscle injury, exosomes laden with tetraspanins from injured muscle cells aid myoblast fusion. Myoblast spreading and fusion is facilitated by CD9 and CD81 tetraspanins (Hemler 2003). Considering other means of uptake, Syncytin-1-loaded exosomes bind to their transporters’ alanine-serine-cysteine transporter 2 (ASCT2), found in skeletal muscle,

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to induce internalization (Cocucci and Meldolesi 2015; Kowal et al. 2016). The speed of uptake and the maintenance of elevated amounts of exosomes depends on the circumstance of muscle damage. Depending on the method of EV uptake, different intracellular signaling and sorting fates await vesicle cargoes. (Rejman et al. 2004; Svensson et al. 2013; Mulcahy et al. 2014; Verdera et al. 2017; Schneider et al. 2017).

EVs Released by Different Cells in the Injured Muscle Environment EVs are important mediators of intercellular communication that allow the coordinated orchestration of repair and regeneration facilitated by different cell types. The cargoes of EVs affect the recipient cells’ mRNA composition and translation. Early stages of injured muscle repair may be characterized by acute actin reorganization and membrane transformation through the action of annexin binding, mitochondrial redox signaling, and ESCRT activity (Jaiswal et al. 2004; Jaiswal et al. 2014; Bouter et al. 2011; Scheffer et al. 2014; Boye et al. 2017; Horn et al. 2017). EVs may be directly involved in some stages of repair. As an example, secretion of Annexin-A1 containing vesicles as a result of epithelial cell injury induces signaling via NADPH oxidase 1 (Nox1), which acts on Rac and catenin delta-1 (p120) proteins that aid in the prompt closure of epithelial wounds (Leoni et al. 2012, 2015). Annexins are crucial not only for myofiber repair but also regeneration. Shedding of vesicles by injured myofibers is enabled by ESCRTmediated ectosome formation.

The Inflammatory Response via EVs and Subsequent Activation of Nearby Cells EVs shed by myofibers and other cells also initiate intercellular interactions critical for muscle tissue regeneration. Pro-inflammatory cascades are triggered by EVs through transport of antigens loaded onto major histocompatibility class 1 and 2 complexes to T lymphocytes, as the initial event necessary for triggering the inflammatory response following skeletal muscle injury (Taverna et al. 2017). Neutrophil-derived ectosomes stimulate the release of factors from macrophages for eventual pro-inflammatory macrophage induction (Gasser et al. 2003; Tidball 2017). miRNAs delivered via exosomes reduce the expression of Toll-like receptors (TLR) by macrophages and cause cells to take-up other vesicles without activating the immune response, which has been demonstrated in muscular dystrophy (Manček-Keber et al. 2015; Phinney et al. 2015; Hindi and Kumar 2016). As stated previously, muscle regeneration is characterized through the clearance of cellular debris and subsequent initiation of the regenerative response (Chazaud 2015). C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand 2 (CCL2) enrich the muscle environment with cytokines that activate pro-inflammatory macrophages (Tidball 2017). The released cytokines cause

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myotubes to produce exosomes that are loaded with myostatin, while also decreasing the level of decorin, a myostatin antagonist (Kim et al. 2018). Therefore, at this pro-inflammatory stage, exosomes participate in limiting myogenesis and allowing the clearance of damaged tissue by inflammatory cells. Consequently, macrophages polarize to the pro-regenerative state with a rise in MSCs. MSCs release vesicles that stimulate myogenin and MyoD, which facilitate regeneration in target cells (Phinney et al. 2015). In addition to this, these exosomes also improve capillary density, attenuate fibrosis, and hasten regeneration of the injured muscle (Nakamura et al. 2015). The cargo of these vesicles includes VEGF, IL-6 in addition to miRNA-1, miRNA-133, miRNA-206, miRNA-125b, miRNA-494, and miRNA-601. These miRNAs promote pro-regenerative cellular developments. Additionally, MSCs can package and transfer mitochondria and mitochondrial proteins within ectosomes to incoming macrophages, in order to maximize their activity for regeneration (Phinney et al. 2015; Sansone et al. 2017). Skeletal muscle regeneration was found to be enhanced by EVs derived from amniotic fluid MSCs (Mellows et al. 2017; Tsiapalis and O’Driscoll 2020). The effect of adipose-derived mesenchymal stem cells (ADMSCs) on muscle injury demonstrated that repair was facilitated through factors dispersed both within EVs and the secretomes’ soluble fraction (Mitchell et al. 2019). EVs derived from MSCs have also been tested as a means to prevent torn rotator cuff injuries (Wang et al. 2019). In a rat model, MSC-EVs prevented inflammation, atrophy, infiltration of fat, and vascularization of muscles, therefore increasing myofiber regeneration and the rotator cuff microenvironmental properties. Recently, ADMSC-EVs have been shown to cease muscle damage in a critical hindlimb ischemia mouse model (Figliolini et al. 2020). This was facilitated through neuregulin 1 protein (NRG1)-mediated signals that play a crucial role in angiogenesis, muscle protection, and inflammation prevention. Lastly, urine-derived MSC-EVs have been shown to repair pubococcygeus muscle injury in rat models of stress urinary incontinence (Wu et al. 2019). This was done through EV-mediated activation, proliferation, and differentiation of SCs. Dendritic cellderived exosomes and MSC-derived exosomes are being utilized in ongoing clinical trials to treat different types of cancers and graft versus host diseases, respectively (Zhu et al. 2017; György et al. 2015; Jeske et al. 2020). Exosomes derived from human skeletal myoblasts can induce myogenesis during myotube formation (Choi et al. 2016), reduce collagen deposition, and increase the number of regenerating fibers upon muscle injury (Huang et al. 2016; Campanella et al. 2019). Other types of muscle injury evoke the packaging of alternative components into exosomes. Skeletal muscle denervation shifts exosomal miRNA content from miRNA-133a and miRNA-720 to miRNA-206 which stimulates SC differentiation (Gasperi et al. 2017). SC activation and differentiation increases the secretion of growth factors such as HGF and IGF-1 while simultaneously transferring miRNA cargoes such as miRNA-206 and miRNA-1. SCs also release exosomes that help attenuate fibrosis and enhance myofiber regeneration (Braun and Gautel 2011; Forterre et al. 2013; Choi et al. 2016; Murphy et al. 2018). Muscle injury induced by laceration stimulates the secretion of exosomes enriched in myogenic

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growth factors that stimulate the differentiation of adipose-derived stem cells to the myogenic lineage, therefore aiding muscle regeneration (Choi et al. 2016). Oxidatively injured myotubes promote the secretion of vesicles by SCs with cargoes repressing myogenin expression, resulting in faster closure in an in vitro assay (Guescini et al. 2017). Vesicles are also involved in the remodeling of damaged tissue. miRNA-208a regulates fiber-type determination while miRNA-126 and miRNA-16 regulate blood vessel formation (D’Souza et al. 2018). Depending on induced cellular stresses, EVs accomplish specific goals that enable regeneration and remodeling. This is not strictly limited to skeletal muscle, in the early stages of cardiac muscle hypertrophy, the damage promotes cardiomyocyte secretion of miRNA-378-containing EVs. This miRNA plays a role in impairing hyperplasia and the production of collagen (Yuan et al. 2018). Hyperplasia may lead to an increase in heart weight where as a consequence, chronic heart failure, coronary insufficiency, and dilation are noted frequently (Linzbach 1976). Increased collagen synthesis may lead to deterioration of cardiac function and augmentation of myocardial fibrosis (Querejeta et al. 2004). As a means of therapeutic use, EVs may well be superior to synthetic constructs considering the possibility of bioengineering with the desired factor, as well as the strong biostability and biocompatibility with fewer risks of adverse effects as opposed to stem cell therapy (Riazifar et al. 2017). An interesting consideration for the production of EVs in a scalable mode may be the utilization of PSCs since these cells may be expanded robustly in vitro. From PSCs, more muscle-specific cells such as induced MSC-like cells may be derived and utilized for EV production (Jiang et al. 2019; Steens and Klein 2018; Kim and Kim 2019; Duelen and Sampaolesi 2017).

Conclusion Recently, it has become more evident that skeletal muscle regeneration does not studiously rely on the propagation of resident stem cells in order to restore homeostatic conditions. The role of extracellular signaling, facilitated by EVs, suggests the existence of EV packaging and networking upon muscle injury induction. The cargo of EVs: proteins, lipids, and nucleic acids play a key role in aiding muscle repair and regeneration. It is evident that depending on the context of muscle injury, the cargo of EVs is custom-engineered by interstitial and immune cells in order to fulfill the necessary intercellular communication and modulate target cells. It is oftentimes thought that regeneration is the outcome of factors secreted by cells, acting in a paracrine manner. The mechanism behind the specificity of EV release and targeting remains an enigma. Current explications emphasize the possibility of EVs either acting through signaling gradients or specific ligand and receptor interactions. Therefore, focusing on such aspects of EVs may unravel their potential in targeted improvement of the injured muscle niche. New insights into the mechanism behind intercellular communication upon muscle injury repair and regeneration may allow future therapeutic achievements, possibly by combining paracrine cues with stem cell therapy.

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Even though much work remains to be done in order to establish a proper and standardized means of EV production; by inhibiting their deleterious effects and taking advantage of their regenerative properties, EVs present exciting possibilities in treating muscle-specific disorders.

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The Cell-Free Therapy Approach Alberto Gonza´lez-Gonza´lez, Daniel García-Sa´nchez, Ana Alfonso-Ferna´ndez, Khawaja Husnain Haider, Jose´ C. Rodríguez-Rey, and Flor M. Pe´rez-Campo Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells Based Therapies: A Useful Tool? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs Secretome as an Alternative to Cell-Based Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soluble Factors Secreted by MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composition of EVs Cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of the Secretome from Mesenchymal Stem Cells in the Treatment of Musculoskeletal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs Secretome for Bone Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSCs Secretome in the Treatment of Tendon Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulating Secretome Therapeutic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory Priming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addition of Bioactive Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Cell-Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tuning Cell-Substrate Interaction (ECM and Scaffolds) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. González-González · D. García-Sánchez · J. C. Rodríguez-Rey · F. M. Pérez-Campo (*) Grupo de Ingeniería de Tejidos de Cantabria, Dpto. Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IDIVAL, Santander, Cantabria, Spain e-mail: [email protected]; [email protected]; [email protected]; [email protected] A. Alfonso-Fernández Servicio de Cirugía Ortopédica y Traumatología, Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_50

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Disease-Like Priming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149

Abstract

Mesenchymal stem cells (MSCs) have been the most frequent stem cell used in clinical trials due to their easy isolation from multiple adult tissues, their ability to home into the injury sites, and their potential to differentiate into various cell ® types. Despite this, only two drugs, TEMCELL , for the treatment of acute graft® versus-host disease, and Alofisel (Darvadstrocel), for the treatment of fistulae in Crohn disease, have been approved so far by regulatory agencies. For various reasons, MSCs’ approval as a drug is proving difficult, and from the outside, it might seem that the field is in a period of stagnation. The risk of lung entrapment or the fact that transplanted MSCs might lead to the formation of tumors are causes of serious concern. Besides, the realization that the beneficial effects of MSCs rely mainly on their paracrine action rather than engraftment and differentiation, has paved the way for cell-free therapeutic strategies in regenerative medicine that would lack the unwanted effects linked to the administration of live cells. The use of MSCs secretome has key advantages over the cell-based therapies, such as lower immunogenicity, and easiness of production, handling, and storage. More importantly, MSCs can be modulated to alter their secretome composition to better suit specific therapeutic goals, thus opening a large number of possibilities. Altogether these advantages are making MSCs secretome the focus of many investigations in several clinical contexts, enabling the rapid scientific progress of this field. Keywords

Cell-free therapy · Mesenchymal stem cells · Paracrine · Regenerative · Secretome · Stem cells List of Abbreviations

Agn A-MSCs Ang-1 bFGF COX DCs BM-MSCs ECM ELISA EVs HGF HIF-1α

Angiogenin Adipose tissue-derived MSCs Angiopoietin 1 Basic fibroblast growth factor Cyclooxygenase Dendritic cells Bone marrow MSCs Extracellular matrix Enzyme-linked immunosorbent assay Extracellular vesicles Hepatocyte growth factor Hypoxia-inducible factor 1α

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HO-1 ISCT IDO IL10 MCP-1 MVs MS MSCs NK OA PGE PlGF ROS S-MSCs SATB2 SDF-1 TGF-β TIMP VEGF

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Heme oxygenase 1 International Society for Cellular Therapy Indoleamine 2,3-dioxygenase Interleukin-10 Monocyte chemoattractant protein 1 Microvesicles Mass spectroscopy Mesenchymal stem cells Natural killer cells Osteoarthritis Prostaglandin E Placental growth factor Reactive oxygen species Synovial fluid-derived MSCs Special AT-rich sequence- binding protein 2 Stroma cell-derived factor-1 Transforming growth factor β Tissue inhibitor of metalloprotease 1 Vascular endothelial growth factor

Introduction Mesenchymal Stem Cells Based Therapies: A Useful Tool? Mesenchymal stem cells, also known as mesenchymal stromal cells (MSCs), have received center-stage attention by both industry and academia due to their unique qualities. Thanks to their regenerative potential and other remarkable properties MSCs are considered excellent candidates for cellular therapy. The escalating research reflects this on their therapeutic applications during the last decade. Since their discovery, more than a thousand clinical trials, many of them interventional, have been registered and reported, while others are in the pipeline. However, the conclusion of these studies has been highly variable, even reporting contradictory results (Couto et al. 2019). Whereas some MSCs-based studies have shown apparent significant beneficial effects, the others did not find an effect at all. This extreme divergence has been generally attributed to the small size of cases analyzed in some trials or the lack of appropriate control groups in the MSCs-based treatments. However, the primary factor responsible for the lack of consistency in these data is considered to be the heterogeneity of the MSCs populations and the quality of cell preparation used for cell therapy. The International Society for Cellular Therapy (ISCT) has established the minimal criteria to define MSCs. According to these criteria, MSCs are fibroblasts-like multipotent adult stem cells that should have the following three properties (Dominici et al. 2006):

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1. Adherence to the plastic surface under standard culture conditions. 2. Expression of an array of specific surface markers, that is, CD105, CD73, and CD90, while lacking the expression of hematopoietic specific markers, that is, CD45, CD34, CD79a/CD19, and CD14/CD11b. 3. Multilineage differentiation potential into adipocytes, osteoblasts, and chondrocytes in vitro. Although this is a useful and valuable definition that initially provided a common ground for describing MSCs, it did not wholly reflect the complexity of MSCs populations that could be gathered from the increasing knowledge about these cells. The isolation of MSCs using the ISCT-devised standard has helped to standardize isolation and purification protocols; however, it still produces heterogeneous populations of cells with diverging potencies, and the standardization of methods remains highly challenging. Efforts are now directed to develop and optimize standard protocols to produce homogeneous MSCs populations to achieve consistent outcomes and optimize the therapeutic use of these cells. One of the options currently tested to achieve this goal is the use of MSCs derived from induced pluripotent stem cells (iPSCs-MSCs). Besides the uniformity of the iPSCs-MSCs cell population, these cells engraft into the recipient tissue and survive at higher rates than the adult tissue-derived MSCs, which render them more appealing and efficient for allogenic transplantation (Gao et al. 2017). It is pertinent to mention that as the investigations involving iPSCs-MSCs have just started, there are still many questions to resolve about their biological characterization, safety, and feasibilityrelevant challenges before they could be considered a safe and efficient alternative to adult tissue-derived MSCs. The heterogeneity of the MSCs’ population is not the only drawback hindering the development of MSCs-based therapies. Efficient MSCs-based therapies require MSCs to home, survive, engraft, and integrate with the host tissue at the damaged site. The quality of the cell preparation is an important determinant of cell-based therapy (Shahid et al. 2016). MSCs’ performance on these different steps seems to be greatly dependent upon parameters, such as isolation and purification protocols, culture conditions for in vitro expansion, and donor-relevant factors, such as the age of the donor, disease/health status of the donor, or tissue of origin, etc. (Lretlow et al. 2008; Neri 2019; Haider 2018). Furthermore, given the scarcity of these cells in the donor tissues and thus the limited starting material, most of the therapies necessitate a prolonged ex vivo expansion period to obtain enough cells required for transplantation for improved prognosis. The length of this expansion period significantly influences the subsequent treatment outcome (Bertolo et al. 2016) and seems to directly correlate with the loss of the clonogenicity, MSCs’ engraftment potential, and differentiation capacity (Thery et al. 2018b). These effects have been linked to a decrease in chemokine receptors expression during the in vitro expansion period, thus translating into a low chemotactic response (Son et al. 2006). Other factors directly unrelated to the status of the cells, such as the cell delivery method, route of delivery, etc., may also influence the effectiveness of MSCs-based treatments. The treatment strategies involving local administration give superior

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outcomes as compared to those involving systemic administration. The reason for this superiority is that the latter leads to the donor cells getting entrapped within the microvasculature of various non-targeted organs, particularly the lungs (Zheng et al. 2016). Even in reaching the target tissue, MSCs will often encounter a highly unfavorable inflammatory and cytotoxic microenvironment characterized by poor perfusion, low oxygen and nutrients, and altered pH, which poses a significant challenge for donor cell survival. This hostile microenvironment often leads to apoptosis of the transplanted MSCs. Some studies have reported as low as 0.1–1 percent survival of the transplanted cells during the first 24 h after delivery, leading to a poor outcome of the procedure. Moreover, the extensive cell death can trigger immune reactions that might aggravate the condition further or even lead to the rejection of the transplanted cells. Given the poor cell survival rate, the transplanted cells could fail to efficiently direct angiomyogenic repair, a crucial step for successful tissue regeneration (Rezaie et al. 2018). Put together, all these obstacles highly reduce the percentage of transplanted MSCs that effectively contribute to the regeneration of the damaged tissue. It is also important to mention that despite having an excellent safety profile, some published reports have raised a red flag regarding the biosafety of MSCs’ use in humans. For example, MSCs have been linked to potential embolisms in small blood vessels (Wu et al. 2017), mainly when the treatment involves systemic administration. The extensive ex vivo expansion required to achieve the cell number necessary for some procedures might contribute to the genomic instability of the transplanted cells. In fact, chromosomal alterations have been observed in clinicalgrade MSCs cultures (Nikitina et al. 2018), something worrisome considering that MSCs would maintain their proliferative capacity once in the recipient tissue, increasing the potential risk of tumorogenesis. Besides, although this is rare due to their low immunogenic capacity, it has also been described that the repeated transplantation of MSCs can cause the production of alloantibodies that could limit their clinical applications due to immune rejection possibility (Cho et al. 2008). Despite all the drawbacks mentioned above, and mixed outcomes of MSCs-based treatments, these cells are still contemplated as a highly useful tool and near-ideal cell type from among the cells for cell therapy-based regenerative medicine (Rajab et al. 2019). The paradigm is now shifting from cell-based therapy to cell-free therapy. Although it was once believed that the main therapeutic benefits of MSCs-based cell therapy primarily relied on their capability to create new tissue via differentiation, growing evidence suggests that the wide range of bioactive molecules secreted by MSCs is responsible for many of the positive effects of MSCs-based therapies. This would explain why, although in many cases less than 1% of the transplanted MSCs are retained long-term within the target tissue, there are still clear therapeutic benefits linked to their transplantation (Yeo et al. 2013). As we will discuss in-depth later in the chapter, MSCs secrete a wide variety of molecules as part of their paracrine activity that has several beneficial effects on the damaged tissue, including their immunomodulatory action, which is considered highly relevant to host tissue cytoprotection and regeneration. Thus, the use of these paracrine secretions, rich in bioactive factors, produced by MSCs could hypothetically replace these cells in

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some applications that would also benefit from the lack of biosafety concerns associated with using a cell-free therapy. However, it should be kept in mind that, besides their ability to replace the damaged tissue by differentiating into multiple cell types and their paracrine activity, the therapeutic potential of MSCs could also rely on other mechanisms that would require the physical proximity of those cells. MSCs exert a cytoprotective effect on the surrounding cells in the host tissue postengraftment by modulating reactive oxygen species (ROS) production and modifying the nutrient usage mechanistically through intercellular mitochondria trafficking (Paliwal et al. 2018). MSCs also modify the behavior of immune cells by the secretion of specific factors, and cell-to-cell contact (Andreeva et al. 2017; Liu et al. 2020c). Although there is still some controversy in the field, it has been recently shown that MSCs fuse with cancer cells leading, in some cases, to the cessation of their proliferation highlighting the anti-tumorogenic effect of MSCs and opening the opportunity to use MSC-fusion based strategies for tumor biotherapy (Zhang et al. 2020b). These properties of MSCs require direct physical interaction of the transplanted MSCs with the tissue-resident cells. Even though all the aforementioned problems are linked to MSCs-based therapies, these cell-based approaches might still be helpful in treating specific pathologies where these particular activities are proven beneficial.

MSCs Secretome as an Alternative to Cell-Based Therapy The paracrine activity is a common feature of virtually every cell type. The wide array of bioactive molecules secreted by a particular cell type is designated as the “secretome.” This term, initially coined by Tjalsma et al. (2000) who defined it as “both the components of machineries for protein secretion and the native secreted proteins.” This definition, as well as the one later provided by Hathout (2007), who defined the secretome as “all the factors secreted by a cell or tissue into the extracellular space under a defined time and conditions,” only seem to contemplate the soluble part of this secretome while letting out a key part of it: the extracellular vesicles (EVs). Several studies have now confirmed that the MSCs’ secretome can reduce cell injury and improve tissue repair. Moreover, it has immunomodulatory, antiapoptotic, and pro-angiogenic properties via activation of endogenous signaling pathways in neighboring cells that would lead to an improved microenvironment and host tissue regeneration. This realization has paved the way for the use of secretome as a cell-free alternative to overcome the limitations of MSCs-based therapies. More importantly, MSCs-derived secretome is being developed as a pharmaceutical product for use in regenerative medicine. MSCs-secretome based products are now being tested as substitutes of MSCs in the applications where those cells have already proven useful (Bogatcheva and Coleman 2019). The use of MSCs-derived secretome has additional advantages over MSCs, such as the ease of handling and storage and off-the-shelf availability. On the same note, the secretome components are highly stable and are not affected by processes such as freezing, concentration by ultracentrifugation, or lyophilization (freeze-drying).

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Besides, its production is easily scalable to fit the necessary levels for large-scale production, thus opening the possibility of developing novel MSCs-secretome based pharmacologically active agents. Although both the soluble and insoluble fractions of the secretome may contribute to tissue regeneration, they might have different effects (Giannasi et al. 2020; Mitchell et al. 2019) and are the base of different preparations currently being tested for therapeutic purposes.

Soluble Factors Secreted by MSCs Various research groups have attempted to profile and identify the soluble factors secreted by MSCs from different tissue sources using global approaches, including mass spectrometry (MS)-based techniques. However, these analyses have proven to be technically challenging and more often than not, identified false-positive results due to proteins stemming from dead cells or interference from the culture media used. Recently, the use of a highly stringent quantitative MS approach, allowed the identification of 315 proteins actively secreted by human bone marrow MSCs (BM-MSCs) (Baberg et al. 2019). However, it is important to note that despite the secreted paracrine factors from MSCs of different origins being highly similar, MSCs from different tissue sources diverge in the level of production, as recently shown in an experimental animal model (Villatoro et al. 2019). These biological differences should then be considered when choosing the MSCs’ source for secretome production. Proteins secreted by MSCs include cytokines, chemokines, extracellular matrix (ECM) proteases, and growth factors that are involved in a wide range of biological activities in the cells, such as antioxidant and antimicrobial activities, proliferation, cytoprotection, modulation of inflammation, emigrational activity, promotion of angiogenesis, and transdifferentiation. The main activities driven by MSCs soluble factors will be discussed in the following sections. A schematic representation is shown in Fig. 1.

Immunomodulation Besides other mechanisms, the therapeutic benefits of MSCs are primarily attributed to their immunomodulatory function. MSCs can influence both adaptative and innate immune responses through their secreted factors. These secreted factors exert widespread immunomodulatory effects on virtually all immune cells, including T cells, B cells, dendritic cells (DCs), natural killer cells (NK), neutrophils, monocytes, and macrophages. The main immunosuppressive factors secreted by MSCs are prostaglandin E (PGE), hepatocyte growth factor (HGF), indoleamine-2,3-dioxygenase (IDO), transforming growth factor β (TGF-β), and interleukin-10 (IL10). Interestingly, many of these factors exert their function by interfering with the metabolism of certain amino acids. MSCs’ regulatory effect of macrophage function is mediated mainly through the secretion of PGE2, the synthesis of which is mediated by the constitutively produced

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Fig. 1 Schematic representation of MSCs-secreted soluble factors’ effect on immunomodulation, angiogénesis, and apoptosis. (Created with BioRender.com)

COX1 enzyme. In response to a pro-inflammatory environment, MSCs activate the synthesis of the inducible COX2, directly increasing PGE2 synthesis (Murakami and Kudo 2004). Macrophages are generated from monocytes through two different pathways, depending on the environment, a classical activation pathway (M1) or an alternative pathway (M2). Both these types of macrophages release a very different set of factors. While M1 macrophages mainly release an array of pro-inflammatory cytokines, M2 macrophages release IL-10, which has a significant anti-inflammatory role. PGE2 induces the differentiation of macrophages toward the M2 antiinflammatory phenotype (Deng et al. 2016). PGE2 not only regulates macrophage activation but is also able to suppress the proliferation of NK cells and the maturation of B lymphocytes and DCs (Hegyi et al. 2012). IDO also has important effects on T and B lymphocytes’ activation, proliferation, and differentiation. The two isoforms of IDO, namely IDO1 and IDO2, catalyze the degradation of tryptophan needed for T-cell division through the kynurenine pathway. The depletion of tryptophan leads to the arrest of T-cell metabolism and subsequent apoptosis (Böttcher et al. 2016). Furthermore, like the effect on macrophages polarization, T-cell differentiation is also polarized toward T regulatory cells, one of the CD4+ subclasses of T lymphocytes involved in the cellular immune response (Weiss and Dahlke 2019). Regarding B cells, MSCs also affect the humoral activity of these cells by diminishing the expression of chemokine receptors 4, 5, and 7 (CXCR4, CXCR5, and CXCR7), decreasing their overall activation rate. As mentioned earlier, MSCs can also secrete various pro-inflammatory cytokines. Although this might appear counterintuitive, it is essential to clarify that for proper tissue regeneration, an acute inflammatory response is needed right after the injury,

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to allow the migration of MSCs to the injury site and undergo proliferation at a faster rate. However, the subsequent repair phase requires an immunosuppressive state. Therefore, MSCs can sense their microenvironment and respond appropriately in a cue-dependent manner during the healing process (Renner et al. 2009). Finally, it is necessary to highlight that although the paracrine activity of MSCs is indeed responsible for the immunosuppressive function of MSCs, we also need to clarify that this function is also mediated by cell-to-cell contact and by the mitochondrial transfer to the injured cells via tunneling nanotubes (Cselenyák et al. 2010).

Angiogenesis and Revascularization MSCs extensively release a variety of pro-angiogenic factors, including basic fibroblast growth factor (bFGF), angiopoietin 1 (Ang-1), placental growth factor (PIGF), Interleukin 6 (IL-6), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β1), and monocyte chemoattractant protein 1 (MCP-1) (Kwon et al. 2014). The presence of all the aforementioned factors and their respective concentrations greatly varies depending upon the MSCs source and their culture conditions (Kehl et al. 2019). The amniotic MSCs secretome shows a more robust angiogenic profile (Kim et al. 2012b). In comparison with the adipose tissue-derived MSCs, human amniotic fluid-derived MSCs were rich in pro-angiogenic factors, including IGF-1, EGF, and IL8, as determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA) (Kim et al. 2012b). The amniotic fluid-derived MSCs were characterized by the surface markers expression of CD44, CD73, CD90, CD105, and CD106, while showing a minimal expression of CD34, CD14, and CD45. Also, while some factors such as bFGF are not always found as part of the soluble factors secreted by different MSCs, other factors, such as IL6, are always present in most of the analyses performed and reported (Hsiao et al. 2012). Besides the release of pro-angiogenic factors, MSCs also produce anti-angiogenic molecules under a specific set of culture conditions, such as stimulation with inflammatory cytokines. This specific culture environment leads to increased secretion of tissue inhibitor of metalloprotease 1 (TIMP-1), a protein with strong antiangiogenic activity (Zanotti et al. 2016). A balance between the activity of the metalloproteases and their inhibitors is crucial for the degradation of the vascular basement membrane that prevents endothelial cells from leaving their positions. Together with correct extracellular matrix remodeling, this process is of utmost importance to allow the emigration of endothelial cells toward the surrounding tissue to initiate and participate in the generation of neovasculature. Hypoxia provides a strong stimulus for the release of pro-angiogenic factors via the activation of hypoxia-inducible factor 1α (HIF-1 α) and its downstream signaling. Therefore, hypoxic treatment of the cells has been extensively studied to alter the secretome profile of the cultured cells (Hsiao et al. 2013; Hao et al. 2019). An augmented secretion of pro-angiogenic factors in the cells may be induced by subjecting the cells to hypoxic culture conditions and by the presence of different cytokines, such as TGF-α, in the culture media. TGF-α increases the secretion of

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various angiogenic factors through the activation of PI3K/Akt and MAPK signaling pathways. Conditioned media from MSCs treated with TGF- α can induce blood vessel formation in an in vivo assay (De Luca et al. 2011). Aranha et al. have shown that MSCs cultured under hypoxic conditions showed copious secretion of pro-angiogenic factor VEGF in response to the activation hypoxia-inducible factor 1α (HIF-1 α)(Aranha et al. 2010). Preconditioning of MSCs has also been achieved by exposing the cells to intermittent cycles of ischemia and reperfusion. Repeated treatment with anoxia-reoxygenation activated HIF-1α signaling, activation of HIF-1α hypoxamiRs, downstream activation of survival signaling, and altering the paracrine profile of the preconditioned cells (Kim et al. 2009, 2012a). It has been observed that HIF-1α-dependent microRNAs are mechanistically involved in the preconditioning induced survival signaling (Haider et al. 2009). Given the significant role of HIF-1α in hypoxic and ischemic preconditioning and paracrine behavior of the cells, strategies have also been developed to activate HIF-1α through a non-hypoxic mechanism based on genetic modulation of the cells for concomitant overexpression of Akt and Ang-1 (Lai et al. 2012). Besides hypoxia treatment, stem cells have also been preconditioned to support their rate of survival and modify their paracrine behavior (Haider and Muhammad Ashraf 2010). Some of the effective strategies in this regard include genetic modulation and pharmacological manipulation of the cells (Haider et al. 2008; Suzuki et al. 2010; Ahmed et al. 2010; Afzal et al. 2010). In addition, the production and secretion of pro-angiogenic factors by MSCs can also be enhanced by serum deprivation in the culture medium (Bianco et al. 2008). Furthermore, the conditioned medium from the cells cultured without serum could generate longer neovascular sprouts than the control cells-derived conditioned medium with serum during an ex vivo assay (Bianco et al. 2008). Put together, the secretion of the complex set of bioactive molecules in the secretome is susceptible to modulation through different approaches to promote angiogenesis.

Anti-apoptotic Activity Several studies have demonstrated that MSCs can modulate cell apoptosis to restore the local microenvironment homeostasis in two different ways: direct cell-to-cell contact with the affected cell types and second through the secretion of paracrine mediators, such as IGF, FGF-2, HIF-1α, Heme oxygenase 1 (HO-1), and VEGF. Also, two different interleukins secreted by MSCs have a crucial role in the antiapoptotic activity of MSCs-derived secretome on cells present in their microenvironment. For example, a recent study has shown that the anti-apoptotic activity of MSCs-derived secretome occurs mainly via IL10 activity since the abrogation of IL10 with a specific antibody added to the conditioned media significantly reduced the cytoprotective effects of the conditioned medium (Al-Azzawi et al. 2020). On the other hand, MSCs also inhibit lymphocytes and monocytes’ apoptosis through an IL6 mediated mechanism (Xu et al. 2007; Raffaghello et al. 2008). Overall, the paracrine activity of MSCs is key to avoiding caspase activation and subsequent apoptosis of endothelial, epithelial, and immune cells in a highly inflammatory microenvironment in the injured tissue.

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Extracellular Vesicles A variety of nano- and micro-sized vesicles, extracellular vesicles (EVs) shed from the cell surface as part of the paracrine activity of the cell, constitute the insoluble part of the secretome. According to the International Society for Extracellular vesicles (ISEV), the term EVs comprises the non-replicable small particles delimited by a lipid bilayer that do not contain a functional nuclei (Thery et al. 2018b). Initially, EVs release was thought to be a part of the mechanism in the cells aimed to eliminate unneeded cellular compounds; it is now clear that EVs are key players in intercellular communication. However, the EVs population is heterogeneous in size and shape, which has hindered our understanding of their features, molecular composition, and functionality. EVs are now classified, based on their size and biogenesis, into three major categories. Exosomes, Microvesicles, and Apoptotic bodies (Thery et al. 2018a). Exosomes, generally 30–150 nm in diameter, are formed within the endosomal network and are released by the inward budding of multivesicular bodies (MVBs) (Raposo and Stoorvogel 2013, Yáñez-Mó et al. 2015). Alternatively, MVBs can be trafficked to lysosomes for degradation (Colombo et al. 2014). The fate of MVBs depends upon the cholesterol contents in their membrane, since cholesterol is necessary to avoid lysosomal degradation (Pfrieger and Vitale 2018). Microvesicles (MVs) biogenesis involves a less well-understood mechanism, however, it is generally perceived to involve outward budding of the extracellular membrane. MVs have a larger size, with a diameter ranging from 100 to 1000 nm. Because of their biogenesis, MVs are rich in proteins that are abundant in the plasma membrane, such as tetraspanins or proteins involved in cell-to-cell interaction through the recognizing of glycans and might facilitate the MVs-cell interaction. Finally, ApoBDs, released by apoptotic cells, are the largest EVs reaching up to 5000 nm in diameter. The first step in ApoBDs formation is the emergence of blebs in the plasma membrane that later become a protrusion, ending up in the release of the ApoBDs (Jiang et al. 2017). Interestingly, ApoBDs are considered essential players in the immune-modulated mechanism of implanted MSCs in the injured tissues (Galleu et al. 2017). There are two different mechanisms by which EVs contribute toward the intercellular signaling. Firstly, EVs exert their effect upon uptake by the target cells, which involves the fusion of the vesicular membrane with the recipient cell membrane, thus enabling the release of EVs’ cargo into the recipient cell cytoplasm. Alternatively, these vesicles could bind with the recipient cell membrane-bound receptors and activate specific downstream signaling pathways (Abels and Breakefield 2016).

Composition of EVs Cargo Most of the pro-regenerative factors present in the MSCs’ secretome are carried in EVs, mainly exosomes. This cargo is highly dynamic and reflects the parent cell lineage and its metabolic state. According to Exocarta, a manually curated standard

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database of exosomes and their payload of proteins, RNAs, and lipids, there are currently registered 9769 proteins, 3408 mRNAs, 2838 miRNAs, and 1116 lipids identified in exosomes derived from various cell types. Importantly, different cell culture conditions and manipulations can greatly influence cargo loading in the exosomes, which might also impact their bioactivity. This can also be used for our own advantage by creating particular conditions that improve specific bioactivity of the exosomes produced by MSCs, making them more effective for a specific therapeutic application (Haider and Aramini 2019). Although several analyses have been performed to establish a protein profile of EVs cargo’ in different cell types, the diverging isolation and purification protocols have rendered this a daunting task and did not allow the production of conclusive data. Overall, the proteins present in the EVs cargo are proteins that are instrumental in their biogenesis, transmembrane proteins, different types of tetraspanins (CD9, CD63, and CD81), and proteins involved in antigen presentation (MHCI and MHC II). Although it is not as common, certain transcription factors have also been found as part of EVs cargo (Kalra et al. 2012). As regards the lipid contents of EVs, they reflect their cells of origin, although specific lipids such as diacylglycerol and phosphatidylcholine seem to be less represented than the other membrane lipids. On the contrary, phosphatidylserine is more enriched in EVs compared to the cellular plasma membrane. Interestingly, it has been proposed that higher presence of phosphatidylserine in the membrane of some EVs facilitates their uptake by the recipient cells (Bicalho et al. 2013; Zaborowski et al. 2015). Pertaining to the type of the genetic material found in the EVs cargo, it may include genomic and mitochondrial DNA and small RNAs of different types, that is, rRNA,tRNA, mRNAs, and lncRNAs. With a few exceptions that could correspond to either lncRNAs or intact mRNAs, the majority of RNAs found in the EVs cargo are less than 200 nucleotides in length. Among, the different nucleic acids found in the EVs cargo, miRNAs associated with exosomes are the ones that have been more intensively studied due to their capacity to alter the gene expression pattern of the target cell and their demonstrated role in different pathologies, including cancer (Asgarpour et al. 2020; Aramini et al. 2020). Generally speaking, the RNA cargo of the EVs mainly reflects the cytoplasmic contents of the cells of EVs’ origin. However, some RNAs seem to be enriched in the EVs, thus suggesting the existence of some mechanism for cargo selection, such as the presence of particular sequences, named zip code, in the 3’UTR (Bolukbasi et al. 2012) on specific mRNAs or the “GGAG” motif found of several of the exosomal miRNAs (Villarroya-Beltri et al. 2013). Currently, there is mounting evidence showing that the effect of the MSCsderived EVs cargo on target cells is often mediated by the regulation of mitochondrial activity (Hogan et al. 2019). There is a significant increase in mitochondrial respiration and ATP production after exposure of the cells to MSCs derived EVs (Bland et al. 2018; Russell et al. 2019). In addition, several of the miRNAs, proteins, and mRNAs packed in the EVs have a direct or indirect effect on mitochondrial metabolism and baseline activity (Loussouarn et al. 2021). These organelles are

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essential for cellular homeostasis, and thus, there are many pathologies related to mitochondrial dysfunction. Given the significant role of mitochondrial in cellular homeostasis, protocols are also being developed for mitochondrial preconditioning to support cell survival (Lu et al. 2010).

Applications of the Secretome from Mesenchymal Stem Cells in the Treatment of Musculoskeletal Diseases A recent search on the clinical trials database (https://clinicaltrials.gov) for studies using MSCs secreted factors showed a total of 33 ongoing or completed trials. A quick screening of this list reveals that, although the number of studies involving MSCs secreted factors does not compare to the number of clinical studies involving MSCs, the therapeutic potential of MSCs-derived secretome is being tested in a wide variety of pathologies as a part of the fast-emerging cell-free therapy approach (Haider and Aslam 2018). In contrast to the low number of clinical trials using the cell-free therapy approach, there is a substantial number of preclinical experimental animal studies involving treatment with MSCs’ secreted factors-containing conditioned medium. The encouraging data from the preclinical studies have generated immense interest in MSCs-based cell-free research to treat various injuries and pathologies. Interestingly, in the current pandemic scenario, the immunomodulatory and anti-inflammatory capacity of MSCs-derived exosomes is being tested in different clinical trials to treat the inflammatory symptoms associated with COVID-19 pneumonia. MSCs secretome also shows therapeutic effect on the regeneration of the musculoskeletal system attributed to its multiple effects on the bone microenvironment, facilitating the communication between osteoblasts, osteoclasts, and bone marrowderived MSCs (Liu et al. 2017). In this section, the role of MSCs secreted factors and EVs in bone, cartilage, and tendon regeneration would be discussed in detail.

MSCs Secretome for Bone Regeneration Recent research is mainly focused on using MSCs secretome to treat bone-related pathologies encompassing from bone fractures to age-related changes, such as bone mass loss in osteoporosis, tissue degeneration of the osteonecrosis, etc. Systematic reviews have revealed that in almost all bone regeneration studies performed in various small animal models, treatment with MSCs-derived EVs has produced measurable benefits compared to controls. An outline of the different approaches tested in various animal models is shown in Fig. 2. Overall the therapeutic effects of EVs include a higher bone density, increased bone remodeling in osteoporotic models, less presence of necrotic tissue in osteonecrosis models with an increase in bone density, showing an increased number of proliferative cells and osteocytes, and finally, in rat fractures models, an enhanced formation of the callus (Tan et al. 2020).

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Fig. 2 Isolation, purification, and diverse applications of MSCs-derived EVs and conditioned médium on bone regeneration. (Created with BioRender.com)

Non-union bone defect after a long bone fracture is one of the main challenges in the orthopedic medical field due to the difficulties in achieving successful treatment. Current therapies are based on grafts and other bone substitutes; however, these are less effective for reconstructing bone defects that exceed a critical size (Roddy et al. 2018). These disadvantages underscore the need for searching novel therapeutic approaches that would allow the regeneration of these defects. The importance of MSCs-derived exosomes in critical-sized fracture repair was first assayed in CD9-/mice, characterized by impaired fracture healing (Furuta et al. 2016). In this experimental model, treatment with MSCs-derived exosomes rescued the fracture healing retardation observed in the control animals. The authors suggested that the reduced amount of MSCs-derived exosomes released in the CD9-/- mice were related to the healing deficiency. Osteogenesis and angiogenesis are two highly related and intricate processes. The damage to local blood vessels during bone injury would impair bone regeneration. Therefore, the restoration of appropriate regional flow through neovascularization would ensure osteoinductive factors’ access to the injured tissue, bone repair, and bone regeneration (Saran et al. 2014). Studies performed in an experimental rat model of femoral non-union clearly showed that MSCs-derived exosomes enhance osteogenesis and angiogenesis (Zhang et al. 2020a). Similar beneficial effects were observed in osteoporotic animals, indicating that MSCs-derived exosomes can also restore osteoporotic bone (Qi et al. 2016). Since osteogenic capacity is significantly reduced in osteoporotic MSCs, these effects could reflect an enhanced osteogenic

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capacity of endogenous MSCs after exosomal treatment (Del Real et al. 2017). Mechanistically, MCP-1/-3 and IL-3/IL-6 in the conditioned medium have been shown to recruit bone marrow MSCs, mature endothelial cells, and progenitor cells to participate in angiogenesis and induce osteogenic differentiation (Ando et al. 2014). As previously described, MSCs-derived exosomes can also benefit the age-associated systemic bone loss typical of osteoporosis (Zhao et al. 2018). However, the number of studies on this subject is scarce. MSCs-derived exosomes affect osteoblasts’ activity through a SATB2 (special AT-rich sequence- binding protein 2) mediated mechanism (Yang et al. 2019). MALAT1, a lncRNA present in MSCs-derived exosomes, seems to act as a sponge for miR34c, inhibiting SATB2, a protein that is responsible for osteoblast activity (Yu et al. 2019). A common feature of all the studies involving treatment with MSCs secretome is the critical participation of miRNAs in mediating various pathological processes and regeneration of the bone tissue (Chen et al. 2019; Xu et al. 2020). The combination of MSCs-derived conditioned medium, or EVs with different bioscaffolds, is an approach that has achieved optimal results in vitro and in vivo (Diomede et al. 2018a, b). The combination of exosomes derived from iPS-MSCs with tricalcium phosphate scaffolds has been shown to enhance bone regeneration by activating the PI3k/Akt signaling pathway (Zhang et al. 2016). Leaving aside all the aspects that require in-depth studies, MSCs-derived exosomes have already been tested in humans in combination with specific scaffolds. For example, a case-control study used exosomes for alveolar bone regeneration, describing a denser bone formation, lower inflammation, and no abnormal delayed healing (Katagiri et al. 2016). A preclinical trial in humans has also been performed with promising results (Katagiri et al. 2017). Although much work needs to be done in this field, these data constitute the lead of future studies.

MSCs Secretome in the Treatment of Cartilage Defects and Cartilage Degeneration Articular cartilage defects in weight-bearing joints represent one of the major pathologies of the musculoskeletal system. The etiology of these defects is multifactorial, but the most prevalent pathologies related to articular cartilage are primary osteoarthritis (OA) and posttraumatic cartilage degeneration. OA is a degenerative disease characterized by the destruction of articular cartilage and synovial inflammation (Musumeci et al. 2015){Musumeci, 2015 #773}. Regardless of the etiology, the deficient intrinsic cartilage self-repairing ability is the main issue when dealing with cartilage damaging diseases (D’Arrigo et al. 2019). Classical contemporary treatment for OA is only symptomatic and is aimed to alleviate pain and inflammation without successfully containing the vicious process of cartilage degeneration. When these treatments are no longer effective, joint replacement is the only possible way of action. To avoid surgery that could lead to long-term complications, more treatment options are currently being studied. Current surgical interventions include microfractures, mosaicplasty, perforations and abrasions, or the transplantation of autologous chondrocytes (ACI and MACI techniques) that fail to prevent the

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progression of the disease over the long term or to provide long-lasting symptomatic relief (Lee and Wang 2017). Importantly, OA incidence is rapidly increasing due to the global aging of the population and the obesity epidemic, urging new efficient therapies to promote cartilage regeneration. The intra-articular injection of BM-MSCs or adipose tissue-derived MSCs (A-MSCs) directly into the affected joint has significant anti-inflammatory and anti-catabolic effects besides pain relief and improved functional scores (Yokota et al. 2019). These beneficial effects have been attributed to the MSCs’ paracrine capacity (Mancuso et al. 2019). It has been estimated that paracrine activity is responsible for at least 80% of the MSCs favorable effects on cartilage regeneration (Muhammad et al. 2019). However, this approach shares the limitations of other MSCs-based therapies. Besides, cell-based therapies for the treatment of OA are subjected to the same problems already mentioned in the introduction of this work. Most of the current studies related to the role of the secretome in OA treatment focus on the insoluble fraction of this secretome, particularly in the exosomes. To date, MSCs-derived exosomes attenuate cartilage degeneration in different experimental animal models of OA (Ni et al. 2020). Furthermore, compared with MSCs transplantation, exosomes-based treatments exhibit higher stability and flexibility and a wide range of immunomodulatory activity. MSCs-derived exosomes have demonstrated their ability to promote regeneration and repair of both cartilage and subchondral bone (Asghar et al. 2020). Exosomes can also prevent osteoarthritic chondrocytes from undergoing apoptosis via fusion with chondrocyte mitochondria, a process that alleviates the mitochondrial dysfunction generally associated with degenerative chondrocytes (Qi et al. 2019). Some of the exosomal cargo moieties with a pivotal role in cartilage regeneration have already been identified. Mao et al. (Mao et al. 2017) found that miR-92a-3p overexpressing BM-MSCs-derived exosomes were able to promote chondrogenesis, prevent cartilage matrix degradation, and delay OA progression in an experimental model of OA. As previously discussed, MSCs-derived EVs are responsible for a wide range of immunoregulatory activities during the cartilage regeneration process. The initial stages of OA are linked to an increase in the synthesis of pro-inflammatory cytokines, such as ILβ or TNFα, which stimulates the expression of multiple cytokines and Ptgs2, one of the most potent pro-inflammatory genes (Sandell et al. 2008). A reduction in Ptsg2 expression in OA can be achieved by treatment with MSCs-derived exosomes overexpressing micro-RNA-26a-5p. This leads to an overall reduction in the pro-inflammatory factors (Jin et al. 2020). MSCs have been modified by drug preconditioning or gene modification to produce exosomes with a specific payload that could improve cartilage repair. Preconditioning of BM-MSCs with TGFβ3 increases the expression of anabolic markers and decreased levels of catabolic marker genes in OA chondrocytes (Cosenza et al. 2017) and enhances the biomechanical characteristics of engineered cartilage (Byers et al. 2008). On the other hand, preconditioning of BM-MSCs with small-molecule Kartogening produced exosomes that promoted the formation of a more robust chondral matrix through the regulation of the metabolic activity of chondrocytes (Liu et al. 2020a){Liu, 2020 #787}.

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Besides BM-MSCs, MSCs from other tissue sources have also been studied for their therapeutic potential in OA. Synovial MSCs (S-MSCs) showed chondrogenic differentiation and cartilage promotion and attenuated OA in experimental animal studies (Enomoto et al. 2020; Kondo et al. 2019). Exosomes derived from A-MSCs decreased inflammation, promoted cartilage regeneration, enhanced the proliferation and migration of human OA chondrocytes, and successfully maintained the chondrocyte matrix (Damia et al. 2018; Tofino-Vian et al. 2018). MiR-100-5p, found in A-MSCs derived exosomes, decreased the severity of cartilage lesions and improved gait in the experimental mice model (Wu et al. 2019). On the other hand, human A-MSCs-derived exosomes had down-regulated pro-inflammatory genes, enhancing proliferation and chondrogenic potential of periosteal cells via upregulation of miR145 and miR221 (Zhao et al. 2020). Put together, these data reinforce the essential role of exosomes to prevent and treat OA in the future. However, more preclinical experimental studies are needed, especially to know their role in long-term OA evolution. To our knowledge, there is no clinical study evaluating exosomes’ role in the treatment of human OA patients.

MSCs Secretome in the Treatment of Tendon Injuries Tendon injuries, both chronic and acute, are prevalent debilitating diseases. Tendon has a limited inherent self-healing capacity. Either conservative or surgical treatment results in a far from satisfactory prognosis. The situation is further aggravated due to a high prevalence of early re-rupture and tissue scarring tissues in the lesion field (Lui 2020). Cell-based therapy of injured tendons using MSCs promotes tendon repair (Hevesi et al. 2019), although these therapeutic effects are mainly executed by paracrine factors (Connor et al. 2019). Transplantation of rat BM-MSCs derived exosomes, seeded on fibrin glue, into a patellar tendon wound resulted in superior tendon healing (Shi et al. 2019). At the microscopical level, the treated tendons showed better-aligned collagen fibers and better biomechanical results (Yu et al. 2020). Interestingly, this improvement was dose-dependent, with tendons treated with a high dose of exosomes achieving better histological tendon-fiber alignment and vascularity (Gissi et al. 2020). Besides fibrin glue, other carriers have been used in these studies. For example, EVs derived from A-MSCs loaded onto a collagen sheet showed decreased postoperative gap formation and re-rupture on a rat Achilles tendon model (Shen et al. 2020). In addition, collagen formation at the injury site was also facilitated and early inflammatory response after the lesion was abrogated. The aforementioned studies are based on the direct action of MSCs-derived exosomes on tendon healing; however, indirect applications of these MSC-derived EVs have also been tested. Macrophages pre-treated with MSCs-derived exosomes promoted tendon healing, a reduction in inflammation, and an improvement in the biomechanical properties when applied to an experimental murine Achilles tendon model (Chamberlain et al. 2019).

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Although there are registered clinical trials for therapeutic assessment of EVs, to our knowledge none of these trials is on tendon healing. Most of the research done in this area corresponds to preclinical experimental studies mainly focused on acute lesions. Future studies are therefore warranted for their therapeutic assessment in the long-term and chronic settings. Since the therapeutic benefits are dose-dependent, it is important to improve our knowledge on the exosome concentration in doseescalation studies to determine optimal exosomal dose. On the same note, the timing of injection after injury and the frequency of exosomal treatment need to be optimally defined.

Modulating Secretome Therapeutic Properties One advantage of MSCs’ secretome for therapeutic application is that the composition of the secretome as well as that of exosomal cargo can be modulated to fit the specific treatment requirements of the damaged tissue. Consequently, various approaches have been developed to tailor MSCs-derived secretome and its exosomal payload for specific clinical applications. The following section focuses on the conditions and methods designed to enhance the therapeutic efficacy of MSCs secretome for bone, cartilage, and tendon regeneration therapies (Chang et al. 2021). A summary of these methods is shown in Fig. 3.

Hypoxia Induction While standard cell culture is carried out in normoxia (21% O2), oxygen tensions ranging from 0% to 10% are often used to mimic the conditions found in MSCs niches for cell expansion in vitro. Therefore, it has been generally considered that MSCs cultured under hypoxic conditions simulating the in vivo MSCs niche microenvironment would help the cells sustain their biological characteristics and full therapeutic potential (Ferreira et al. 2018). Indeed, hypoxic preconditioning seems to potentiate MSCs’ regenerative and cytoprotective effects and bioactive factors secretion in general (Ferreira et al. 2018). MSCs’ secretome produced under hypoxic conditions is rich in proangiogenic factors, such as VEGF, TGF-β1, angiogenin (Agn), or granulocyte-macrophage colony-stimulating factor (GM-CSF) (Quade et al. 2020). Moreover, other important therapeutic properties such as more vigorous immunomodulatory activity (Costa et al. 2021), enhanced chemotactic attraction to the chemical cues, that is, SDF-1 and monocyte chemoattractant protein-1 (MCP-1) (Quade et al. 2020), superior anti-apoptotic, anti-oxidative stress, and anti-fibrotic functions, have also been attributed to MSCs secretome generated under low-oxygen culture conditions (Collino et al. 2019; Miceli et al. 2021). In fact, hypoxia is the most commonly used priming method for enhancing the production of a secretome with greater regenerative potential. However, there are still some issues to overcome before these parameters can be standardized.

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Fig. 3 The commonly used MSCs’ secretome modulation methods to enhance their therapeutic properties. Hypoxia, inflammatory factor priming, induction of bioactive factors, spheroid culture, cell-substrate interaction, and genetic edition are the most well-studied stimuli that have been reported to treat different pathologies using primed secretome. (Created with BioRender.com)

For instance, a wide range of O2 concentrations has been used to precondition the cells. Also, secretome composition is substantially modified depending on the duration of the cells exposure to hypoxia (Quade et al. 2020). Efforts are underway to establish an optimal hypoxic priming protocol that maximizes the therapeutic characteristics of MSCs’ secretome (Costa et al. 2021).

Inflammatory Priming Activation of MSCs to repair damaged tissue is triggered by inflammatory cytokines such as IL-1β, IFN-γ, and TNF-α (Miceli et al. 2021). Pro-inflammatory stimulus in the culture medium is one of the many available methods to modulate MSCs’ secretome that has received more attention. The three cytokines mentioned above, together with lipopolysaccharide (LPS), are the main effectors used in vitro for simulating the inflammatory microenvironment that elicits the secretion of immunomodulatory factors by MSCs. This priming protocol produces results that encompass, complement system inhibition, inhibition of NK cells, the guidance of monocyte differentiation to M2

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anti-inflammatory phenotype, suppression of cytotoxic T cell proliferation, inhibition of dendritic cell maturation, and an increase in regulatory T cells (Treg). Increased prostaglandin E2 (PGE2), indoleamine-2,3-dioxygenase (IDO), cyclooxygenase 2 (COX-2), and TGF-β are responsible for these effects (Costa et al. 2021; Ferreira et al. 2018). IL-6, the primary inducer of inflammation, is also overexpressed in inflammatory factor-primed MSCs’ secretome, but its concentration decreases over time after priming (Bundgaard et al. 2020). Although this seems to be contradictory, a correct regeneration process initially requires an acute inflammatory phase to trigger the influx of immune cells and homing of MSCs to the injury site that would later participate in the repair process (Bogatcheva and Coleman 2019). Preconditioned MSCs’ secretome facilitates this process as it maintains primary inflammation long enough to trigger subsequent angiogenesis, and then exerts anti-inflammatory effects. Subsequently, a complex remodeling process sets, which is marked by an increase in matrix metalloproteinase 1 and 3 (MMP1 and MMP3), metalloproteinase inhibitor 1 precursor (TIMP1), and plasminogen activator inhibitor 1 (PAI-1) (Maffioli et al. 2017; Bundgaard et al. 2020). Inflammatory factor-primed MSCs’ secretome also performs an essential pro-trophic action. For example, it has been reported that INF-γ-preconditioned MSCs’ secretome promoted tendon healing and chondroprotection (Shen et al. 2020; Maumus et al. 2016; Ragni et al. 2020) while TNF-α priming exerts bone regenerative function (Lu et al. 2017). Although these inflammatory stimuli are similar, many studies have shown diverse effects of MSCs’ priming with pro-inflammatory cytokines as IFNγ, TNF-α, or IL-1β (Ragni et al. 2020). Therefore, further refinement of the protocols in terms of the concentration of cytokines, time of induction, cytokine combination, etc., is required to obtain the optimal MSCs’ secretome composition for optimal prognosis.

Addition of Bioactive Factors MSCs can also be primed by adding soluble bioactive molecules to the culture media during cell culture and expansion. For example, given the benefits of hypoxia priming, treatment of the cells with dimethyloxaloylglycine (DMOG), a small molecule that stabilizes HIF-1α, or deferoxamine (DFO), an iron-chelating agent, may simulate hypoxic culture conditions without exposure to hypoxia. Both hypoxia mimicking agents increase angiogenic growth factor expression in MSCs. In particular, DMOG preconditioning has been shown to significantly induce bone regeneration (Chang et al. 2021). On the same note, Vadadustat, a pharmacological hypoxia inducer, has been used to precondition MSCs. Preconditioning MSCs with Vadadustat has led to an augmented anti-inflammatory effect with a concomitant increase in angiogenic growth factors expression and secretion (Zielniok et al. 2020). Besides hypoxia mimicking agents, FGF2, resveratrol, omentin-1, or thrombin have been studied to prime MSCs, leading to an enhanced angiogenic potential of the cells (Liu et al. 2020b; Sung et al. 2019). Moreover, priming with thrombin contributes to increased EV production and alleviates inflammation. Furthermore, it

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was revealed that anti-inflammatory MSCs secretome could be obtained by priming the cells with valproic acid, melatonin, PGE2, substance P, or tetrandrine. Interestingly, tetrandrine was selected and validated from a group of 1402 FDA-approved drugs using a high throughput screening protocol and showed potent immunosuppressive effects (Cabezas et al. 2020; Chang et al. 2021; Heo et al. 2020). Besides, it was stated that secretome from MSCs primed with TGFβ3, hyaluronic acid, or curcumin showed chondroprotection and improved osteoarthritis (Ruiz et al. 2020; Zhang et al. 2021). Similarly, Kim et al. reported that pioglitazone-primed MSCs’ secretome induced cell proliferation and secretion of soluble collagen in tenocytes, leading to tendon regeneration. Interestingly, increased VEGF contents in pioglitazone-primed MSCs’ secretome had a role in tendon remodeling despite poor vascularity (Kim et al. 2019).

Modulation of Cell-Cell Interactions Although two-dimensional (2D) culture is a standard technique for culturing MSCs in vitro; this setting is artificial compared to physiological conditions. Furthermore, 2D culture provokes loss of crucial receptors, increases the expression of pro-inflammatory chemokines, and is highly deficient in cell-cell interactions, thus causing reduced MSCs stemness (Costa et al. 2021; Chang et al. 2021). On the contrary, the formation of 3D structures, known as MSCs spheroids, provokes changes in cell morphology, cytoskeletal organization, intracellular signaling pathways, and gene expression (Ferreira et al. 2018). In addition, this type of culture aids cell-to-cell and cell-to-ECM interactions. It also contributes to a microenvironment that simulates the natural habitat of the cells in their niches, wherein the inner layer cells are exposed to low hypoxia and nutrients (Chang et al. 2021). These subtle but influential adjustments are directly responsible for enriching immunomodulatory, angiogenic, and prosurvival factors in 3D spheroid-cultured MSCs’ secretome (Chang et al. 2021). Moreover, the secretome would be rich and in exosomal contents (Kim et al. 2018a). On the one hand, due to hypoxia, MSCs located in the spheroid core overexpress angiogenic factors through a HIF-1α and HIF-2α-mediated mechanism. On the other hand, spheroid conformation reduces mitochondrial membrane potential and ATP production, indicating apoptotic processes in MSCs, which lead to IL-1 release. Then, self-induction by autocrine effects of IL-1 enhanced the anti-inflammatory profile of MSCs’ secretome, which closely relates to cell survival and anti-fibrotic factors (Cesarz and Tamama 2016). As of today, a few studies corroborate the beneficial effects of the secretome derived from spheroids cultured MSCs (Santos et al. 2015; Miranda et al. 2019). Apart from these scarce published reports, the alleged beneficial effects of their secretome are generally based on the presence of special components rather than any direct evidence. Importantly, spheroid-priming due to 3D culturing seems to have a synergistic role when combined with other forms of priming, that is, inflammatory stimuli (Redondo-Castro et al. 2018), MSCs’ encapsulation, or the use of microcarriers (Saldana et al. 2019; Tsai et al. 2020).

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There are currently several technical details, such as spheroid induction protocols, optimal cell size, cell density, etc., that need to be defined to standardize the production for the maximal therapeutic potential of their secretome (Costa et al. 2021).

Tuning Cell-Substrate Interaction (ECM and Scaffolds) Another protocol of 3D priming is based on using scaffolds that support, or rather enhance, cell-substrate interaction. Since extracellular signals are crucial for cell adhesion, organization, migration, and growth, this approach of substrate-culture preconditioning can play an essential role in MSCs secretome composition. Thomas et al. studied the changes in MSCs secretome when those cells were cultivated in collagen microgel and demonstrated that proper cell density, collagen-crosslinker ratio, and collagen concentration could substantially increase proangiogenic and immunomodulatory cytokine concentration in MSCs secretome (Thomas et al. 2014). Furthermore, due to the mechanosensitive nature of MSCs, the increased stiffness of polyacrylamide hydrogels functionalized with fibronectin gets translated into higher cell spreading and a more robust cytoskeleton. These molecular and cell structural changes result in proangiogenic growth factor-rich MSCs secretome with significantly higher tubulogenic potential in vitro (Abdeen et al. 2014). On the same note, exposure of MSCs to direct mechanical load significantly alters their secretome profile favoring chondrogenic and angiogenic induction (Gardner et al. 2016; Kasper et al. 2007). The topography of the material supporting MSCs growth also significantly affect the secretome composition. Leuning et al. produced 76 different algorithm-generated well-plate surfaces and displayed that each one provoked a unique cytokine secretion profile (Leuning et al. 2018). On the same note, substrate porosity is relevant to cell culture characteristics and hence, has a significant bearing on their secretome profile. MSCs grown on macroporous scaffold show a substantial enrichment in cytokines related to tissue regeneration in comparison to nanoporous hydrogel substrate, even though both exhibit same stiffness. These changes in secretome profile have been attributed to the increase of N-cadherin mediated cell-cell interactions in the macroporous scaffold, which correlates well with the aforementioned spheroid culture impact on MSCs’ paracrine secretions (Qazi et al. 2017). Apart from this, it is imperative to highlight the use of decellularized ECMs as a scaffold to show positive effects on the proangiogenic profile of MSCs’ secretome (Sears and Ghosh 2020). Therefore, different substrate properties can be combined to enrich MSCs’ secretome profile toward a certain cluster of factors of interest. Likewise, a combination of cell-substrate tuning with other types of preconditioning, such as inflammatory priming, was reported to result in a synergistic effect (Wong et al. 2020). Deconstructing the physical and biochemical cues implied in cell-substrate primed MSCs’ secretome will help to design optimal platforms for induction of a desired therapeutic effect (Abdeen et al. 2014).

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Genetic Editing A genetic edition is a solid tool that has been used to genetically modulate MSCs’ ability for long-term transgene expression and to modify their paracrine behavior (Haider et al. 2008). However, in the majority of cases, the application of geneediting was aimed at improving cellular characteristics such as survival, proliferation, or differentiation capacity and with the aim of using MSCs as vehicles for the delivery of certain molecules (Jiang et al. 2006; Ren et al. 2008; Fierro et al. 2011; Costa et al. 2021). From the standpoint of cell-free strategies, some studies revealed that RUNX2 and BMP2 overexpression in MSCs lead to MSC-EVs with higher osteogenic commitment (Martins et al. 2016; Huang et al. 2020). Similarly, simulating hypoxia conditions through HIF-1α overexpression in MSCs, led to the production of angiogenic exosomes with enhanced pro-osteogenic and pro-angiogenic activities (Li et al. 2017). The genetic modification approach has also been used to reprogram MSCs to pluripotency for use as induced pluripotent stem cells (Buccini et al. 2012). Although genetic modification has been associated with tumorigenic risk, the use of cell-free therapies allows to open a new set of possibilities since modified cells are not directly applied to patients. In any case, more research is needed to characterize genetically-modified MSCs’ secretome. This is a crucial step before this strategy could be applied in clinical trials.

Disease-Like Priming In vitro mimicking MSCs’ specific natural niche microenvironment in certain pathologies may produce a tailor-made secretome that could be extremely useful in the treatment of these pathologies. An example of this priming method is outlined in Fig. 4. Recent studies have attempted to demonstrate this hypothesis. Cifù et al. have shown that MSCs exposed to osteoarthritic synovial fluid increased the release of immunosuppressive factors (Cifu et al. 2020). Moreover, a test applying healthy, traumatic, and degenerative human intervertebral disc cells (IVDs)-derived conditioned medium to MSCs showed that the profile of the produced secretome was highly dependent on the IVDs cells’ status. Moreover, the protein composition enhanced homeostasis maintenance in the case of healthy IVDs and damage repair in the case of traumatic and degenerative IVDs. Even differences between the traumatic and degenerative priming were also observed (Wangler et al. 2021). Importantly, MSCs primed with serum of the patients that have suffered polytraumatism showed a secretory profile that could not be simulated by induction with a selected cocktail of trauma-relevant factors (Amann et al. 2019). These data showed that the serum of the patients might have other factors that would activate a slightly different set of genes. Although disease-like priming seems to be highly promising, it remains to be seen whether this approach may be applied to any type of disease. A way forward could be

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Fig. 4 Osteoarthritic-like priming based on the work by Cifu et al. (2020). OA-primed MSC secretome demonstrated the capacity to acquire functions that support the resolution of osteoarthritic pathology. (Created with BioRender.com)

the emergence of artificial tissue-engineered organs to model diseases in vitro. Once the standardization issues applicable to all the secretome-based techniques are solved, secretome preparations tailored to specific clinical applications may be developed. According to the current tendency for medical treatments, this strategy will also presumably progress toward the so-called personalized medicine.

Future Perspectives MSCs have a significant and recognized role in tissue regeneration. Hundreds of patients have been treated in controlled clinical settings with promising results. However, translation to the clinic is still experiencing many obstacles. According to the regulatory agencies, convincing evidence of safety, especially MSCs from sources other than bone marrow, is lacking (Marks et al. 2017; Mendicino et al. 2014). Also, many clinical protocols were based on the idea that MSCs will always meet damaged tissue needs in what can be called the “MSCs knowing what to do” way (Pittenger et al. 2019). The published clinical data analysis has shown that around 50% of patients do not respond to treatment (Caplan 2018). It remains to be ascertained whether this high number of non-responders is due to differences in cell donors, cell preparation protocols, the disease stage at which the treatment is applied,

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or even the patients’ genetics. To make MSCs’ cultures suitable for clinical use, an in-depth research is needed to identify and define all these variables. During the last decade, the role of the paracrine activity of MSCs in tissue regeneration has been highlighted to explain the therapeutic benefits of cell therapy. A head-to-head proteomic profile of MSCs’ secretome with various other cells has shown that BM-MSCs produced larger amounts of secretome proteins (Billing et al. 2016). This is consistent with the greater importance of the secretome in MSCs biology and makes MSCs the cells of choice for clinical applications. MSCs’ secretome has some practical advantages over MSCs themselves. First are the logistic advantages that allow ease of storage, handling, and off-the-shelf availability. Second, its administration does not require cell-culture-trained personnel. Third, regarding safety, as it does not have a cellular component, secretome is less immunogenic, and the risk of malignancy is reduced. As with cell cultures, a great effort is needed to standardize secretome contents, and the RNA Communication Consortium (ERCC1) (Das et al. 2019) and the ISEV (Thery et al. 2018b) have released guidelines to assist researchers accordingly. Clinical application of the secretome also presents many of the difficulties that affect cell-based applications. The lack of standardization of MSCs cultures is a significant problem in translating it to the clinic, while the origin of MSCs is an essential determinant of variability. A comprehensive study using both RNAseq and quantitative proteomics comparing commercial BM-MSCs and Embryonic stem cell-derived MSCs (ESC-MSCs) has reported 2500 differentially expressed RNAs and, depending on the stringency, 40–200 differentially expressed proteins in the two cell types. Most interestingly, MSCs from young donors have a profile closer to ESC-MSCS, which indicates that MSCs gene expression changes with age (Billing et al. 2016). Furthermore, depending on their origin, MSCs secrete different profiles of paracrine factors (Hsiao et al. 2012), which correlates well with various therapeutic capacities. As an example, A-MSCs have less osteogenic potential than BM-MSCs (Niemeyer et al. 2010), but in contrast, A-MSCs produce better results than BM-MSCs for ischemic stroke therapy (Ikegame et al. 2011). Likewise, the proteomic profile of MSCs’ secretome isolated from three tissue sources identified a total of 1533 proteins, of which only 124 were shared between the three tissue samples (Tachida et al. 2015). These data were confirmed in subsequent studies comparing MSCs’ secretomes from different tissues (Shin et al. 2021), and they possessed different therapeutic abilities (Daneshmandi et al. 2020). When it comes to the secretome, standardization is hampered by the limited expansion capacity of the primary cultures and the need to constantly replenish cell sources. Immortalization of cells could be a valid alternative to achieve an unlimited supply of cells with similar characteristics. It has been reported that MYC-transformed MSCs yield exosomes in the milligram range. While the cells’ adipocytic differentiation was affected, the exosomes maintain the ability to reduce relative infarct size in a mouse model of myocardial ischemia /reperfusion (Chen et al. 2011). Another immortal line, constructed by hTERT overexpression, maintained most primary cells’ characteristics, including immunosuppression (Wolbank et al. 2009). Compared to the possible specialization resulting from

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adapting the cells’ origin to each of the different pathologies mentioned above, using a single cell line may be counterproductive. Nevertheless, the fact that culture conditions determine the secretome’s properties could compensate for this disadvantage. Changes in culture conditions may be used to adapt secretomes to particular therapeutic needs. For example, hypoxic culturing protocols maintain MSCs’ multipotency and cell proliferation (Hawkins et al. 2013). The secretome of TNF-α-activated MSCs showed improved wound healing properties, while 3D culture MSCs in spheroids had better pro-angiogenic potential (Xu et al. 2016). Many other changes in culture conditions, including hydrogels (Qazi et al. 2017), cytokines, hormones, growth factors, and drugs like atorvastatin, are being used to introduce changes in the cells’ secretome profile (Ferreira et al. 2018). The specific products and the precise mechanisms by which the microenvironment modulates the secretome profile are undefined. Still, the path is now open to understanding the conditions via in-depth research (Kusuma et al. 2017). EVs-related research is receiving the most interest from researchers. Not only because of their biological properties but also due to their inherent cargo of bioactive molecules, including RNAs and proteins, which they deliver to the recipient cells. They provide a basis to build nano-sized vehicles for the delivery of pharmaceutically relevant substances. Historically speaking, tumor cells are the primary source of EVs for pharmaceutical applications. Still, MSCs produce a large amount of secretome (Billing et al. 2016), and coupled with potentially lower tumorigenicity, make MSCs a safer source of EVs for drug development (Villa et al. 2019). Unmodified EVs reach high blood concentrations soon after administration, but their clearance rate is very high (Wiklander et al. 2015). In vitro modification of the particles by conjugates of glycerol-phospholipid-polyethene glycol prevents their elimination by the endothelial reticulum system. This procedure, known as cloaking, also allows specific ligands to be attached to the particle to provide tissue specificity. The cloning and transfection of DNA encoding hybrid proteins that alter the membrane’s outer properties, called surface display, is an alternative that is also receiving much attention (Antes et al. 2018). Cell culture in the presence of drugs is a simple method to produce anti-tumorenriched EVs (Goh et al. 2017), but the disadvantage is that the drug itself can lead to toxicity in the cultures. On the other hand, the genetic modification of cells in culture is more efficient using plasmids encoding for miRNA precursors or directly with miRNA mimics. The resultant EVs, mostly exosomes, are enriched in specific miRNAs (Kim et al. 2018b). EVs can also be modified for site-directed delivery to the target tissue. This is usually achieved by adding either small peptides or antibodies which recognize receptors present on the target cell (Murphy et al. 2019). Because of the ease with which they are incorporated into particles, aptamers, and small nucleic acid molecules functionally similar to antibodies are becoming a valuable alternative for tissue-specific delivery of EVs (Zou et al. 2019). In conclusion, the secretome, a highly complex mixture of proteins, lipids, RNAs, and EVs, mimic the therapeutic properties of MSCs cultures. The secretome

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presents several practical advantages that could make it a valid alternative to some MSCs culture applications. It has sometimes been possible to attribute the benefits of the secretome to specific proteins or RNAs. Furthermore, the application of constituent molecules of the secretome in isolation is also possible. However, in most preclinical studies, the molecules responsible are unknown and the effects observed and reported are the outcome of several constituent molecules present in the secretome. Much more information is still needed to enable us to transfer the knowledge to the clinic with confidence. However, combined with a more profound understanding of the ideal culture conditions, the availability of immortalized cell lines would help to develop homogenous product batches suitable for treating specific pathologies. Finally, it is pertinent to mention that EVs, with minor modifications, are ideal vehicles for targeted drug delivery. The knowledge of MSCs biology combined with various biomaterials and pharmaceutical technology offers endless possibilities for developing innovative and smart therapies based on MSCs’ secretomes.

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Part IV Miscellaneous Aspects of Stem Cell Applications

Common Ethical Considerations of Human-Induced Pluripotent Stem Cell Research

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Adekunle Ebenezer Omole, Adegbenro Omotuyi John Fakoya, Kinglsey Chinonyerem Nnawuba, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patentability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Informed Consent and Donors’ Right . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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In 2006, Shinya Yamanaka generated induced pluripotent stem cells (iPSCs), which has been the major scientific event of the decade that caught the eye of many scientists, politicians, and bioethicists. The use of human embryonic stem cells (hESCs) has previously been limited by ethical issues related to the A. E. Omole Department of Anatomical Sciences, American University of Antigua College of Medicine, St. John’s, Antigua and Barbuda e-mail: [email protected] A. O. J. Fakoya (*) Department of Anatomical Sciences, University of Medicine and Health Sciences, Basseterre, Saint Kitts and Nevis e-mail: [email protected] K. C. Nnawuba Department of Clinical Medicine, School of Medicine, Caribbean Medical University, Willemstad, Curaçao e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_21

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destruction of embryos. However, with iPSCs, scientists can now reprogram virtually any human somatic cells through the expression of a combination of embryonic transcription factors to a pluripotent embryonic stem-cell-like state, thereby avoiding the contentious destruction of human embryos. Although the clinical realities of human-induced pluripotent stem cells (hiPSCs) appear very promising, they are still laden with some ethical concerns that scientists and legal authorities in the field of iPSC research must recognize. This chapter briefly reviews some ethical issues associated with the use of hiPSCs and suggests ways to address these challenges. Keywords

Ethics · Human-induced pluripotent stem cells · Human embryonic stem cells · Moral issue · Patenting · Reproduction · Informed consent Abbreviations

CNV ESCs FACS hESCs HFEA hiPSCs iPSCs IVF MACS SCNT SNV

Copy number variations Embryonic stem cells Fluorescence-activated cell sorting Human embryonic stem cells Human Fertilization and Embryology Authorities Human-induced pluripotent stem cells Induced pluripotent stem cells In vitro fertilization Magnetic-activated cell sorting Somatic cell nuclear transfer Single nucleotide variation

Introduction For decades, ethical debates regarding stem cell technology have focused mainly on human embryonic stem cells (hESCs). These cells are harvested from the inner cell mass of blastocysts (preimplantation embryos) and obtained with consent from couples receiving in vitro fertilization (IVF) treatment, from aborted fetuses, or from donated oocytes (Thomson et al. 1998; Smith 2001; Zhang et al. 2006). The embryonic origin of hESCs raises a mix of serious moral and ethical controversies about the onset of human personhood, treatment, and harm to embryos; concerns about the safety and health risks of women donating eggs, the potential exploitation of their ova, and their informed consent; and concerns about respect for human life, human dignity, and justice toward humankind. These ethical debates reveal deeply rooted individually diverging opinions about the nature and origin of human personhood, leading to differing policies and regulations of hESC research worldwide (De Trizio and Brennan 2004; Solo and Pressberg 2007; Dhar and Hsi-En Ho 2009). Furthermore, due to this diversity of opinions and cultural differences, an

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international consensus regarding the regulation of hESC research does not exist (Dhar and Hsi-En Ho 2009). The resulting restrictions and prohibitions on hESC research have contributed largely to the slowness in the progress on the translation of hESC technology into clinical therapy. Hence, there was an urgent need for another substitute for hESCs with the same pluripotency potential that can bypass these ethical issues. Shinya Yamanaka’s 2006 discovery of induced pluripotent stem cells (iPSCs) was a notable breakthrough in stem cell research, which has given it a new impetus (Takahashi and Yamanaka 2006; Takahashi et al. 2007; Omole and Fakoya 2018). Scientists and bioethicists were excited at the ability to fabricate a surrogate cell with a pluripotent embryonic stem cell (ESC)-like state by the genetic reprogramming of somatic cells through the ectopic expression of a specific combination of transcription factors (Ibrahim et al. 2016). Enchanted by the extraordinary initial work of Takahashi and Yamanaka, many research groups followed their transcription factorbased reprogramming approach and reproduced the results in mice using cells from diverse tissue sources (Yu et al. 2007; Wernig et al. 2007; Maherali et al. 2007; Ahmed et al. 2011a; Buccini et al. 2012) and humans (Lowry et al. 2008; Park et al. 2008). The reprogramming technique provided an unparalleled and distinctive opportunity to researchers in the field of stem cells and regenerative medicine for possible applications, including pediatric applications, to manufacture patientspecific stem cells for human-disease modeling, drug screening and development, and customized cell therapy (Cagavi et al. 2018; Omole and Fakoya 2018; Çetinkaya and Haider 2020). Since iPSCs appear to end the disputes over the destruction of embryos in hESC research, human-induced pluripotent stem cells (hiPSCs) have been touted by scientists and ethicists alike as ethically and morally uncomplicated alternatives to hESCs and are tipped as surrogate ESCs, and the ethics surrounding hiPSCs have been primarily evaluated in comparison with hESCs. However, even if future investigations demonstrate that hiPSCs fulfill the expectation that they could be possibly viable and superior substitutes for hESCs in disease research, regenerative medicine, and drug discovery, further scrutiny of the reprogramming technology and the resulting ethical concerns might potentially reduce some of the hiPSC-associated ethical advantages over hESCs (Zacharias et al. 2011). In the earliest report on iPSC generation, tumor formation was noticed in more than 20% of the iPSCs due to the reactivation and overexpression of c-Myc oncogene (Okita et al. 2007; Ahmed et al. 2011; Buccini et al. 2012). There is also the safety risk of insertional mutagenesis from virus-dependent delivery methods, which can lead to tumor formation (Takahashi and Yamanaka 2006; Takahashi et al. 2007; Yu et al. 2007). Ethical and legal challenges are also associated with the potentiality of using hiPSCs for the development of human-animal chimeras, human reproductive cloning, and the derivation of human gametes (Lo et al. 2010; Ishii et al. 2013; Wu et al. 2016; Zheng 2016; Volarevic et al. 2018; Moradi et al. 2019). Additionally, such concerns as the application of intellectual property rights or hiPSC patents, donor information, and consent pose considerable challenges to the advancement of iPSCs and iPSC-based research

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(Lo and Parham 2009; Zarzeczny et al. 2009; King and Perrin 2014; Orzechowski et al. 2020). While many of these ethical challenges are not unique to iPSCs but are also shared by hESCs, the ease of accessibility and the simplicity of procuring starting cell sources for iPSC development, the rapid progress in iPSC research witnessed in the last decade, and the remarkable expectations placed on iPSC technology make it very timely and crucial to consider the ethical and legal issues associated with it. Notably, hiPSCs may provide a renewable source of cells for theranostic applications with moral and ethical advantages over their counterpart pluripotent stem cells. Indeed, hiPSCs have some serious ethical concerns that scientists and bioethicists must recognize. This chapter summarizes some of the primary ethical issues associated with the use of hiPSCs, such as safety, reproduction, patenting, and informed consent/donor’s right, which generally remain unfamiliar to a common reader in the field.

Safety There remains significant uncertainty regarding the properties of hiPSCs, how they are reprogrammed, and their ability to form teratomas. The early iPSC lines were generated by transducing somatic cells using retroviral-vector-carrying gene encoding for various transcription factors (Takahashi and Yamanaka 2006; Takahashi et al. 2007). However, insertional mutagenesis using an integrative gene delivery system is a substantial safety risk of this approach, which may even result in tumorigenicity (Takahashi and Yamanaka 2006; Takahashi et al. 2007; Yu et al. 2007; Omole and Fakoya 2018). About 20% of the offspring generated in the original report on germline-competent iPSCs subsequently developed tumors, which were attributed to the reactivation of c-Myc transgene (Okita et al. 2007). Such data prompted many research groups to eliminate c-Myc from the classical quartet of transcription factors to enhance their safety profile (Nakagawa et al. 2008; Martinez-Fernandez et al. 2009). These safety risks are unique to iPSCs due to the combined effect of the overexpression of reprogramming factors and the integrative viral-vector-based delivery method used in the protocol for iPSC generation. Furthermore, incorrect or incomplete patterning and genetic instability can increase the risk of tumorigenicity (Yamanaka 2020). Incorrect or incomplete patterning involves the persistence of undifferentiated and immature cells in the end product of the reprogramming (iPSCs) as well as the differentiated cells derived from hiPSCs. These undifferentiated contaminating cell population has been associated with teratoma formation. The risk of genetic mutations altered biology, and the attainment of tumorigenic potential from incomplete patterning and genetic abnormalities is not unique to iPSCs but relatively common to all cells, which require long-term expansion in vitro (Wang et al. 2013; Izadpanah et al. 2008; Røsland et al. 2009). Genetic alterations like chromosomal aberrations, single nucleotide mutations, and copy number variations are common during reprogramming (Turinetto et al. 2017; Yoshihara et al. 2017a; González and Haider 2021). Chromosomal alterations can either exist in the somatic cells prior to their use

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for iPSC generation or originate during the reprogramming process (Yoshihara et al. 2017b; Liu et al. 2020). Indeed, the first hiPSC clinical trial in 2014 was momentarily halted after discovering mutations in the hiPSCs of the second patient, although mutations were absent in the primary somatic cells (Kimbrel and Lanza 2015; Attwood and Edel 2019). Following the transplantation of hiPSCs, the expectation is that the cells should develop normally, maintain average growth, function in the in vivo environment, and adequately replace the injured or lost cells in the diseased patient. Nevertheless, these cells may proliferate and increase uncontrollably, creating a tumor at the implantation site. This risk of tumorigenicity might trigger extensive safety and ethical concerns about the use of hiPSCs, hence slowing the progress of its application in stem-cell-based clinical therapy. Interestingly, stem cell scientists have made some progress in addressing some of these limitations caused by tumorigenicity. The c-Myc transgene has been shown to be dispensable for reprogramming (Nakagawa et al. 2008). Thomson’s group developed their iPSCs using a different set of four reprogramming factors: Oct3/4, Sox2, Nanog, and Lin28 (OSNL), substituting Nanog and Lin28 for c-Myc and Klf4 in Yamanaka’s “OSKM” cocktails (Yu et al. 2007). Nonviral delivery methods (plasmid vectors, transposons), nonintegrative delivery methods (Sendai virus, lentivirus, and adenovirus), and protocols based on small molecular treatment of somatic cells have been employed to eliminate the limitations caused by insertional mutagenesis (Pasha et al. 2011; Chen et al. 2013; Driscoll et al. 2015; Lee et al. 2020; Kim et al. 2020; Yoshimatsu et al. 2021). Further intensive studies are fundamental for refining the reprogramming techniques of somatic cells and discovering how to prevent the tumorigenicity of hiPSCs. Another approach in this regard is to develop protocols for the direct reprogramming of somatic cells to the lineage of interest without passing through a pluripotency state (Ahmed et al. 2012). Reliable safety assays should be developed to evaluate the potential of hiPSCs before their application for cell therapy. The development of more effective protocols for iPSC differentiation must first be ensured to generate purified populations of hiPSCs before they are used clinically. Regarding incorrect patterning, stem cell researchers are developing means to address tumorigenicity to meet the safety standards required for clinical therapy using iPSCs. Some purification methods have been adopted to identify and remove the residual undifferentiated pluripotent stem cells (PSCs). They include techniques such as directed differentiation and positive/negative selection markers using antibody cell sorting systems, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) (Abujarour et al. 2013; Wuputra et al. 2020). The researchers in the clinical study on spinal cord injury are contemplating the use of the suicide gene method as an additional method to prevent tumorigenicity (Kojima et al. 2019). All these methods will assist the investigators in carefully selecting iPSC lines with the highest level of purity that will be safe for the purpose of clinical application. Regarding genomic alterations, traditional methods like chromosomal karyotyping can detect abnormalities like deletion, duplication, and rearrangement,

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and iPSC products with such abnormalities can be discarded. Minimal genetic alterations, like single nucleotide variation (SNV) and copy number variations (CNV), can be detected by next-generation sequencing technology (like wholegenome sequencing) (Yamanaka 2020). However, analyzing such minimal genetic abnormality can be difficult due to the present difficulty experienced; currently, we have to sequence a significant portion of our genome and accurately analyze and interpret the risks from the mutations detected (D’Antonio et al. 2018). The understanding and assessment of the mutational burden of iPSCs are important for their use for therapeutic applications. Indeed, it is challenging to ensure whether a mutation/mutations detected in the iPSC products will significantly increase the risk of tumorigenicity after transplantation (Yamanaka 2020). At present, extensive tests must be carried out on the iPSC products to detect significant mutations, and only stem cells that pass the test should be forwarded for clinical use. Furthermore, after successful transplantation of the iPSCs, patients should be monitored for the possibility of developing a tumor. More clinical research work is needed to accurately predict the tumorigenic possibilities of a detected mutation.

Reproduction Indeed, one of the most distinct and ethically worrisome potential uses of iPSCs is the production of human embryos through human reproductive cloning. The use of iPSCs for human cloning is illegal and is prohibited worldwide. Generating full-term mice (considered the most stringent criterion of pluripotency) has been fulfilled using iPSCs through tetraploid complementation assays (Kang et al. 2009; Zhao et al. 2009, 2010). This assay involves the injection of iPSCs into the blastocysts of tetraploid mice, embryos that cannot develop into a fetus by themselves. The union results in reconstructed embryos that later develop into fetuses, confirming that iPSCs can form new lives. Sir John Gurdon achieved the first example of cloning using a method where somatic cells were reprogrammed to the embryonic pluripotent states with the same genetic makeup, which is termed somatic cell nuclear transfer (SCNT) (Gurdon 1962). This was followed by Sir Ian Wilmut, who used the same SCNT method to generate the first mammalian – Dolly the sheep – by somatic cloning (Wilmut et al. 1997). SCNT involves the transfer of somatic nuclei into enucleated oocytes to reconstruct embryos (Matoba and Zhang 2018). Theoretically, this procedure is applicable to humans. Yes, human cloning from hiPSCs is technically possible despite associated safety risks (Wilmut et al. 2015). In both tetraploid complementation and SCNT, normal human oocytes or embryos will be destroyed. In tetraploid complementation, human tetraploid embryos will be generated by the fusion of human diploid embryos. Thus, the normal diploid embryo will be destroyed in the process. The low viability rate during this process will require generating many reconstructed embryos to ensure an increased birth rate of the cloned offspring. Hence, the destruction of many diploid human embryos in the process remains a limitation. Likewise, in SCNT, the enucleated oocytes are also destroyed. This is tantamount to

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sacrificing many lives for one life, thus raising ethical concerns that are comparable to that of hESCs. These concerns include controversies about the onset of human personhood and the treatment and harm to embryos, concerns about the safety and health risks of women donating eggs and their potential exploitation for their ova and their informed consent, and concerns about harm to respect for human life, human dignity, and justice toward humankind. In addition, people may also choose to use genetically modified hiPSCs in human cloning to develop offspring with unique characteristics, therefore treating the cloned offspring as a tool for genetic modification or diversity. This type of gene customization of offspring will not show respect for human life. Good surveillance and regulatory processes are essential to monitoring research projects involving SCNT and tetraploid complementation. Regulations must be developed to ban human reproductive cloning explicitly. Another ethically fraught potential use of iPSCs is the derivation of human gametes (sperm and eggs) and human-animal chimeras. The “first generation” of iPSCs did not contribute to the germline or produce adult chimeras. Yamanaka and others later modified the induction protocols, leading to the generation of iPSCs that were fully reprogrammed and proficient for adult chimera and germline transmission (Okita et al. 2007; Takahashi et al. 2007; Yu et al. 2007). Much progress has since been made in the differentiation of pluripotent stem cells into human sperms and oocytes. Protocols have now been established to successfully differentiate and develop male and female gametes from iPSCs (Panula et al. 2011; Hayashi et al. 2011, 2012; Irie et al. 2015; Sasaki et al. 2015; Yamashiro et al. 2018). It is pertinent to mention that gamete derivation from iPSCs may serve as a powerful research tool to improve our understanding of human development and assisted reproductive techniques for the management of infertility disorders (Fang et al. 2018; Zhang et al. 2020). Nevertheless, the chance that they may be considered for reproductive intents poses ethical concerns about cloning, safety, donors’ consent, and the right of the unborn child to know the parents (Advena-Regnery et al. 2018). Other ethical concerns include the potential risk of changing the natural reproduction method, the generation of gametes for same-sex reproduction, and asexual reproduction (Mathews et al. 2009). Furthermore, since the induction of the hiPSCs into gamete cells is not presently a highly efficient process, an attempt to make embryos from such will result in the extensive destruction of many poor-quality embryos, thus raising the same ethical concerns as for hESCs (Mathews et al. 2009). Chimeras are single organisms containing cells from two or more organisms – that is, it contains two or more sets of DNAs, with the genetic code to make two or more separate organisms. Human-animal chimeras have been used enormously by scientists to improve our understanding of gene function and regulation and disease mechanisms and for testing experimental drugs and gene therapies (Levine and Grabel 2017). They are excellent models of human tissues than nonchimeric animals because they are improved systems for human disease modeling. They provide the opportunity to research human cells and tissues in vivo without the necessity for human experimentation. The technology of interspecies blastocyst complementation has already been used to develop rat organs in mice and vice

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versa (Kobayashi et al. 2010; Isotani et al. 2011; Yamaguchi et al. 2017), though human-mouse chimera research is the routine. Recent advances in genome-editing and stem-cell technology have led to extending this research to larger animals, such as pigs. The combination of geneediting technology and interspecies blastocyst complementation has made it possible to use hiPSCs to generate individualized human organs, thus raising the opportunity of addressing the dire shortage of organs for transplantation (Wu et al. 2017). However, the growing amounts of human tissues in these chimeras and the potential availabilities of these tissues in morally significant sites, such as the brain, raises strong ethical concerns and questions about the moral status of these animals (Savulescu 2016). How many human cells are considered “too many” in a humananimal chimera’s brain? How many human cells are considered “too many” in the human-animal chimera’s body altogether? How many would human cells make a mouse brain start thinking human thoughts? What would happen if an animal with human nervous tissues become self-aware and start thinking and feeling like a person? How do we know if we have crossed the commonly accepted dividing line of human decency, dignity, and morality regarding human-animal chimera research? No one knows the answers to these questions, at least not yet. Nevertheless, these questions reveal the main ethical dilemmas that bioethicists are worried about – that chimeric animals with humanized organs may develop human-like consciousness, which will be ethically unacceptable (Bourret et al. 2016; Kwisda et al. 2020). For further in-depth analysis and detailed arguments and public debates on these concerns, please refer to the works reported by Marino, Knoepfler, Palacios-Gonzalez, DeGrazia, and Greely (Degrazia 2007; Palacios-González 2015; Knoepfler 2016; Marino et al. 2017; Koplin and Wilkinson 2019; Greely and Farahany 2021). Going forward, further debates and research are essential to tackle this major ethical dilemma connected with human-animal chimerism. Therefore, we strongly recommend the practical recommendations for chimeric research contributed by Hyun and colleagues (2007). Overall, the ethical objections to all the issues raised concerning reproduction include the sanctity of human life, human dignity, safety, manipulation of genetic diversity, violation of the clone’s rights, etc. (Pattinson 2007). Despite these ethical objections, the Human Fertilization and Embryology Authorities (HFEA) in 2007 agreed for a cytoplasmic hybrid research program to proceed in the United Kingdom (Editorial (Lancet) 2007; Mayor 2008). Meanwhile, in the United States (September 2015), the National Institutes of Health (NIH) announced the discontinuation of the research funding of iPSC-derived chimeras due to additional controversial ethical issues, which require the attention of enforced policies (NIH, Human Pluripotent Cells into Non-human Vertebrate Animal 2015a, Web, July 1, 2021; NIH, Staying Ahead of the Curve on Chimeras 2015b, Web, July 1, 2021). The authors agree that all aspects of stem cell research should be covered by legislation and strict licensing procedures to curtail the potential for the abuse of this technology. However, we also believe that a flexible, less restrictive regulation that considers the proper justification for embryo research will eventually benefit all.

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Patentability A patent gives an inventor the monopoly right to commercialize an invention for a limited period. Comparable to other property types, a patent makes the inventor the owner of the invention, while the intellectual property right remains valid. This concept of ownership has sparked ethical debate in relation to the patentability of life, centered on the objectification and commercial exploitation of living creatures (Schrecker et al. 1997). Intellectual property rights, when efficiently applied, can present a stumbling block to the progress of iPSC research. There are many approaches used for the generation of iPSCs. If investors hold several patents for these many iPSC generation methods, this can impede the translation of the technology from bench to bedside. Although European patent law (Fig. 1) is set up to protect a person’s dignity, the development of iPSCs has opened a worrying loophole (Meskus and de Miguel Beriain 2013). The European Union Court of Justice, on October 18, 2011, delivered a crucial judgment in the aspect of human embryo

Fig. 1 UK and European legal framework for stem cell line patenting

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protection in the case C-34/10 Oliver Brüstle vs. Greenpeace eV. By referring to the meaning of Article 6(2)(c) of Directive 98/44/EC, this case law clarified that those inventions, which involved human embryo destruction at any point, could not be patented (Spranger 2012). However, iPSCs were not in the contemplation of lawmakers when the Biotechnology Directive (Council Directive 98/44/EC and Parliament, from July 6, 1998) was drafted in 1999 (https://eur-lex.europa.eu/ legal-contenEN/TXT/?uri=celex%3A31998L0044). Based on the ruling, patents on stem cells generated from excess embryos from IVF or SCNT or through parthenogenesis will be banned. However, since iPSCs were not derived from embryos, the ruling leaves the door open to patents on iPSCs. Subsequently, in the United Kingdom, regulatory guidance has been offered, which opens the door for the patenting of iPSCs, potentially reviving ethical concerns (UKIPO. Inventions involving human embryonic stem cells, 2015, March 25, 2015). The authors recommend a participatory, inclusive, and transparent process in establishing a workable iPSC patent system that considers the different moral values of all stakeholders in the stem cell field. Creating such a system may not be an easy task, considering the different moral values of all stakeholders. However, if accomplished, this will facilitate the bridging of a moral divide and ensure a consensus that benefits all. More debate and research are essential if we are to close the gap between patents and innovations.

Informed Consent and Donors’ Right Like any other research involving humans, consent is vital for hiPSC research, whether humans participate as research subjects or donors. Usual ethical standards require that participants are fully informed about the specific details of the proposed study, and they are expected to provide voluntary and well-informed consent to participate in the study. Informed consent ensures that the rights, interests, and dignity of patients are protected and respected. Individuals donating somatic cells for iPSC generation should have enough information and answers to address their concerns. The UK Stem Cell Toolkit (USCTK) summarizes iPSC applications concerning legislation (NIH, UK Stem Cell Toolkit 2018, July 1, 2021). These regulations can be used to determine what to include in a consent form. Informed consent should state if the donated cells involved research or clinical applications, genetic modification, animal testing, in vitro or in vivo trials, and whether it will be involved in a therapeutic or diagnostic product with any potential licensing and if there will be risks, complications, and uncertainties. Donors should refuse specific applications, and the right to withdraw one’s cell lines should be discussed clearly in the form. If other applications were not mentioned in the initial document, consenting the donor to be recontacted for such an effect could prevent conflict (Zarzeczny et al. 2009; Aalto-Setälä et al. 2009; Orzechowski et al. 2020). Clear explanations and consent will need to be provided as well for patients treated with iPSCs.

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Under what circumstances can the participants withdraw from a study? Should a time limit be considered for patient withdrawal? It can get quite complicated when it comes to withdrawal in stem cell research or cell therapy trials. All the steps involved, from obtaining somatic cells from donors to using them to generate iPSCs, are very expensive and time-consuming. So imagine a worse scenario where several donors request a withdrawal after establishing iPSC lines and, at the point, where the iPSCs are to be employed for a clinical study. Such a withdrawal will be very damaging to the research project, and it will be a complete waste of time, money, and other resources (Sugarman 2008). Although the usual standards of research ethics require that participants withdraw from the study at any time, and thus this right must be recognized, there can be “points of no return” that research participants should be informed about (Zarzeczny et al. 2009; Moradi et al. 2019). Points of no return can be when transplanted cells (in cell therapy trials) cannot be removed from the patient’s body, thus receiving an irreversible treatment. Even cells donated for research (e.g., to a stem cell bank) may be challenging to withdraw if they have already been used to create a cell line. If there are any such points of no return relevant to given research projects, prospective participants should be informed about them. All this vital information, and the time limit for withdrawal, should be well specified in the consent form (Caulfield et al. 2007). It is indeed a challenge to balance the varied interest linked with iPSC research, considering the prospective benefits of the investigation as well as the interest of the donor. Nevertheless, the apparent policy positions should be adopted and followed through consistently to avoid unnecessary impediments to the research while ensuring the respect and protection of donors’ rights. Closely related to informed consent is the donor’s right to control the scope of the research carried out on their cells as well as the scientific and commercial uses of stem cell lines derived from their cells. The stem cell lines will carry the deoxyribonucleic acid (DNA) of the donor, which contains a wealth of information, including the genetic susceptibility of the donor to disease. The disclosure of such information could inappropriately breach the donors’ right to their privacy (Sugarman 2008). In the USA, the federal law termed “Genetic Information Nondiscrimination Act of 2008” is a typical example of a legislative way of addressing such issues (Taylor 2012). Donors’ rights regarding iPSC research may be exercised in various ways. Some donors may not permit their cells to be injected into humans, and they may oppose all animal research or the mixing of human and animal genetic materials. These objections may lead to friction between obtaining the benefit of iPSC research and respecting the donor’s autonomy. One excellent way to address this issue is to preferentially utilize somatic cells only from donors willing to support and allow all forms of basic research into stem cells. However, this strategy has its risks. There is a danger of introducing bias in research if one decides to select only cells from donors who allow all forms of basic research. Additionally, what if those cells do not exhibit the properties needed for the research project? What if the recruitment of this type of subjects who agree to all forms of research takes considerable time and slows down the research project?

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Another approach is for researchers to ensure they give precise and thorough explanations about the nature of stem cell research when obtaining informed consent. Although an informed consent procedure that provides complete and relevant information, which enables autonomous decision-making, should be the goal of every recruitment process, this standard is probably not generally lived up to. Providing comprehensive explanations about the nature of stem cell research to the prospective participant can be a difficult task. Information about stem cell research can be quite complicated, and some details may not be understood if one does not provide some background details. In general, whatever approach is considered, the pros and cons should be thoroughly debated before recruiting patients for the study.

Conclusion The use of stem cells remains a controversial topic despite the advent of hiPSCs. While their generation does not involve the destruction of an embryo, as with ESCs, debates on how they should be used are still relevant (Hug and Hermerén 2011). To address all the issues considered in this chapter and ensure that hiPSCs are not exploited or used unethically, pertinent regulations must be implemented. Perhaps the recent workshop held by the NIH can serve as a model for proactive policy evaluation (NIH VideoCast – Workshop on Animals Containing Human Cells 2015; NOT-OD-15-158: NIH Research Involving Introduction of Human Pluripotent Cells into Non-human Vertebrate Animal Pre-gastrulation Embryos). If stem cell scientists, bioethicists, and policy makers can maintain an open dialogue about the current state of research, then potential ethical issues on the horizon can be tackled in advance. Such an approach would allow hiPSCs for human treatment to be appropriately moderated without blocking vital research progress that will benefit all.

Cross-References ▶ Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform for Modeling Arrhythmias ▶ Induced Pluripotent Stem Cells

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Response of the Bone Marrow Stem Cells and the Microenvironment to Stress

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Marrow Adipose Tissue and Leukemia Duygu Uc¸kan-C¸etinkaya and Bihter Muratoğlu

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone Marrow Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Components of the Bone Marrow Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Different Stem Cell Niches in the BM Microenvironment and Their Spatial Position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dynamic Interactions in the BM Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone Marrow and Stress Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell and Microenvironment Response to Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hematopoietic Stress Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Hematopoietic Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BM Microenvironment Alterations, Dysregulation, and Myeloid Leukemia . . . . . . . . . . . . . . . . . Development of Secondary Myeloid Malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BM Microenvironment as a Contributor/Driver of Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic Dysregulation and the Role of Adipocytes in the Malignant Niche . . . . . . . . . . . . . . . Marrow Adipose Tissue and Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Stressors and Adipose Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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D. Uçkan-Çetinkaya (*) Faculty of Medicine, Department of Pediatrics, Division of Hematology, Hacettepe University, Ankara, Turkey Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Ankara, Turkey Institute of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey e-mail: [email protected] B. Muratoğlu Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Ankara, Turkey Institute of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_22

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Abstract

In recent studies, the critical roles of the bone marrow microenvironment (BMME) and mesenchymal stem cells (MSCs) in the pathophysiology of leukemia have been addressed. Yet, the mechanisms need to be defined. Leukemia results from a clonal transformation of hematopoietic precursors by the acquisition of chromosomal rearrangements, genetic mutations, and epigenetic modifications. It has been shown that the initiating intrinsic event then leads to alteration of the BMME to support the survival of leukemic blasts. There is limited data for a primary/initiator role for the BMME leading to the development of leukemia. In humans, the development of myelodysplastic syndrome/secondary acute myeloblastic leukemia years after chemotherapy use, besides intrinsic factors, may point to a critical role of the altered/dysregulated ME in the development of leukemia. Similarly, in inherited diseases with DNA repair defects (e.g., Fanconi anemia), long-term xenobiotic exposure and accumulation in the BMME may contribute to the development of leukemia. These factors suggest a previously underestimated but essential role for the BMME in hematological malignancies. Recently, the role of marrow adipose tissue (MAT), by providing free fatty acids through lipolysis, is being addressed as an important factor supporting leukemic blast survival. On the other hand, adipose tissue inflammation is another topic of interest reported to be associated with obesity, insulin resistance, metabolic syndrome, cardiovascular defects, and cancer. In this chapter, the metabolic response of BMME to stress will be discussed with particular attention to MSCs, MAT, and clinical implications in leukemia. Keywords

Adipocytes · Bone marrow microenvironment (niche) · Leukemia · Marrow adipose tissue · Mesenchymal stem cells · Metabolic · Stress · Xenobiotic List of Abbreviations

AhR AML ANGPT ARNT ATF4 ATF6 ATGL BM BMAL1 BMME BRCA CAR CCL CCL2

Aryl hydrocarbon receptor Acute myeloblastic leukemia Angiopoietin The AhR nuclear translocator Activating transcription factor 4 Activating transcription factor 6 Adipose triglyceride lipase Bone marrow Brain and muscle ARNT-like 1 Bone marrow microenvironment Breast cancer cxcl12-abundant reticular C-C motif chemokine ligand C-C motif chemokine ligand 2

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CCL3 CFU-F CHOP CLOCK cMAT CRY CXCL12 CXCR4 CYP DAMP DDR ECM ECV eIF2-α EPCs ER FA FABP4 FAO FAT FATP FFA FLT3 TKIs FOXC2 GCSF GMPs GSTs HIF1 HIF1-alpha HIF2-alpha HSCs HSL HSP ICL Id1 IGF1 IL-1 beta IL10 IL6 IL8 IR IRE1-alpha ITD LepR LT-HSCs

C-C motif chemokine ligand 3 Colony-forming unit-fibroblast C/EBP homologous protein Circadian locomotor output cycles kaput Constitutive marrow adipose tissue Cryptochrome C-X-C motif chemokine 12 C-X-C chemokine receptor type 4 Cytochrome P450 Danger-associated molecular patterns DNA damage response Extracellular membrane Extracellular vesicle Eukaryotic translation initiation factor-2α Endothelial progenitor cells Endoplasmic reticulum Fanconi anemia Fatty acid-binding protein 4 Fatty acid oxidation Fatty acid translocase Fatty acid transport protein Free fatty acids FMS-like tyrosine kinase 3 inhibitors Forkhead box protein C2 Granulocyte colony stimulating factor Granulocyte-macrophage progenitors Glutathione S-transferases Hypoxia-inducible factor Hypoxia-inducible factor 1 alpha Hypoxia-inducible factor 2 alpha Hematopoietic stem cells Hormone-sensitive lipase Heat shock protein Interstrand crosslink Inhibitor of DNA binding 1 Insulin-like growth factor 1 Interleukin 1 beta Interleukin 10 Interleukin 6 Interleukin 8 Ionizing radiation Inositol-requiring enzyme 1 alpha Internal tandem duplication Leptin receptor Long-term hematopoietic stem cells

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MDR MDS MEIS1 MGL MK MMP MPL MSCs NADH NE NES NF-kB O2 PAH PAMP PER PERK PGC POPs PPAR PRDM16 PTPN rMAT ROS sAML SCF SCF SDS SMAD SNS t-AML t-MNS TCDD TGF-β Tie2 TLR TNF-alpha TPO UCP-1 UPR UPR UV VCAM

D. Uc¸kan-C¸etinkaya and B. Muratoğlu

Multidrug resistance Myelodysplastic syndrome Myeloid ecotropic viral integration site 1 Monoacylglycerol lipase Megakaryocyte Matrix metalloproteinases Myeloproliferative leukemia protein Mesenchymal stem/stromal cells Nicotinamide adenine dinucleotide Norepinephrine Nestin Nuclear factor kappa B Oxygen Polycyclic aromatic hydrocarbons Pathogen-associated molecular patterns Period Protein kinase RNA-like endoplasmic reticulum kinase Peroxisome proliferator-activated receptor gamma coactivator Persistent organic pollutants Peroxisome proliferator-activated receptor PR domain containing protein 16 Protein tyrosine phosphatase nonreceptor type Regulated marrow adipose tissue Reactive oxygen species Secondary AML Stem cell factor Stem cell factor Shwachman-Diamond syndrome Small MAD family Sympathetic nervous system Therapy-related AML Therapy-related myeloid neoplasms 2,3,7,8-tetrachlorodibenzodioxin Transforming growth factor TIE receptor tyrosine kinase Toll-like receptor Tumor necrosis factor alpha Thrombopoietin Uncoupling protein 1 Unfolded protein response Unfolded protein response Ultraviolet Vascular cell adhesion molecule

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VCAM1 VEGF VLA4 XBP1 XREs

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Vascular cell adhesion protein 1 Vascular endothelial growth factor Very late antigen 4 X-box binding protein 1 Xenobiotic response elements

Introduction Being the main reservoir of stem cells, the bone marrow (BM) is a critical tissue in maintaining homeostasis and coordinating stress response in the organism. In addition to its essential role in the production of hematopoietic stem cells (HSCs) and progenitors, it is also a major depot for mesenchymal stem/stromal cells (MSCs) and endothelial progenitor cells (EPCs), making this tissue an essential player in regenerative medicine. Therefore, BM is a critical tissue in determining the organism’s response to stress. Cells are exposed to several types of stress, including oxidative, inflammatory, genotoxic, hypoxic, replicative, or nutrient stress. Uncovering the stress response of cells and tissues is critical to understand the mechanisms of diseases. The autonomic nervous system and hypothalamic-pituitary-adrenal axis, through sympathetic, parasympathetic mediators, and cortisol release, coordinate the stress response, respectively. The released hormones and mediators act on several organs and tissues (Herman et al. 2003; Ulrich-Lai et al. 2009). Bone marrow has neural innervation by the sympathetic nervous system (SNS) under circadian control, which is critical for cell trafficking, both in homeostasis and in pathological states (Lucas et al. 2008; Méndez-Ferrer et al. 2008). The changing demands of hematopoiesis upon exposure to stressors are met by the spectacular ability of the BM microenvironment in coordinating hematological, immunological functions and metabolism (Zhao and Baltimore 2015). Stress may be overcome through compensatory mechanisms. However, upon chronic exposure to stress, the response mechanisms may fail to deal with the stressful condition and lead to chronic inflammation in the BM that may contribute to hematological abnormalities, such as leukemia. Among several components of the BM microenvironment, adipocytes have been attracting a lot of attention lately in malignant transformation of hematopoiesis and metastasis of solid tumors (Cawthorn et al. 2016; Shafat et al. 2017). Adipocytes adjacent to tumor cells are involved in metabolic cross-talk, providing adipokines and lipids. Recent evidence suggests that adipose tissue plays a role in cancer aggressiveness by promoting tumor growth, metastasis, increasing cellular lipid metabolism, and chemoresistance (Lengyel et al. 2018). In this chapter, we summarize the dynamic interactions and alterations in the BM microenvironment in both steady state and upon exposure to stressors and discuss the role of stromal factors in developing myelodysplastic syndrome (MDS) and acute myeloblastic leukemia (AML).

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Bone Marrow Microenvironment Components of the Bone Marrow Microenvironment The BM microenvironment is a specialized environment since it provides a niche for stem/progenitor cells, including HSCs, MSCs, and EPCs. It is an enriched and dynamic milieu responding to physiological or stress signals coordinated according to the need of the relevant tissue. Therefore, in addition to its primary function in hematopoiesis, it is essential in maintaining homeostasis, making it a significant player in regenerative medicine. The BM microenvironment is composed of cells of hematopoietic and non-hematopoietic origin (MSCs, pericytes, adipocytes, osteoblasts, macrophages, endothelial cells, osteoclasts, nerve cells, chondrocytes, fibroblasts), a specialized extracellular matrix (ECM) rich in proteins (fibronectin, collagens I-XI, laminin, tenascin, thrombospondin, elastin), proteoglycans (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate), heparin, glycoproteins, integrins, adhesion molecules, and a soluble environment composed of cytokines, chemokines, growth factors, hormones, metabolites, and extracellular vesicles (ECV), all of which play a critical role in determining stem cell functions such as dormancy, proliferation, adhesion, differentiation, autophagy, apoptosis, and migration (Klamer and Voermans 2014; Crane et al. 2017). Therefore, the marrow is a tissue where active cell trafficking occurs not only under stress but also in a steady state to keep up with the physiological cell turnover. This dynamic microenvironment is regulated through the circadian clock, influenced by extrinsic and intrinsic factors influencing cell to cell, cell to ECM, and cell to soluble microenvironment interactions in the niche (Haus et al. 1983; Méndez-Ferrer et al. 2008). With an understanding of the critical roles of the BM microenvironment in stem cell biology, many studies have been performed in the last two decades to define the physical, biochemical, and biological characteristics of the BM microenvironment. However, most studies about marrow stem cell niches were hypothetical until the recent technological developments in multi-omics and single-cell study methods, imaging techniques, which led to a better understanding of hematopoiesis and its microenvironment (Liu et al. 2020a). Progress in discovering genetic tools for functionally identifying niche cells in vivo (mice and zebrafish experiments) and new imaging techniques led to better identification of stem cell niche factors (Crane et al. 2017; Wolock et al. 2019). The recent discovery of the continuous differentiation model of hematopoiesis has suggested that HSCs may bypass discrete hierarchy and gradually acquire lineage biases along with multiple directions (Laurenti et al. 2019). These new developments in hematopoiesis research necessitate a new understanding of the stromal compartment as well. An important study in this area is the extensive mapping of the differentiation paths of non-hematopoietic cells and delineating the BM stroma’s differentiation hierarchy (Wolock et al. 2019). Another recent study used combined single-cell and spatially resolved transcriptomics techniques for mapping distinct BM niches by determining their molecular, cellular, and spatial

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composition. They could profile all major BM-resident cell types, determine their localization, and clarify sources of pro-hematopoietic factors (Baccin et al. 2020). These studies confirmed the heterogeneity of stromal niche populations and their close functional interaction with the differentiation stage of the nearby stem/progenitor cells emphasizing the critical roles of spatial positioning and the dynamic interactions in the BM microenvironment.

Different Stem Cell Niches in the BM Microenvironment and Their Spatial Position The spatial position of different specialized niches in the BM with diverse functions has been a well-adopted opinion since the earlier studies (Wilson and Trumpp 2006). However, unlike the intestinal and skin stem cell niches (Clevers 2013; Hsu et al. 2014), it is challenging to identify BM niches due to their semi-fluidic soft connective tissue structure inside the bone cavity. The HSCs are located near the endosteal lining of the BM cavities or are in close contact with the endothelium of the sinusoids. The hypoxic and calcium-rich endosteal/osteoblastic niche creates a protective and glycolytic microenvironment for the long-term HSCs (LT-HSCs), keeping them in a quiescent state with low metabolic activity and away from toxic insults (Wilson and Trumpp 2006). However, it was shown that the perisinusoidal region also hosts HSCs, in fact, more than the endosteal region. It is known that the marrow is a highly vascularized tissue, and the entire inter-bone space is filled with BM that is fully vascularized, leaving no room for vessel-distant-niches (Stegner et al. 2017). Thus, endothelial cells are present both in the perisinusoidal region (venous type) and also nearby of the endosteum (arteriolar type) (Klamer and Voermans 2014). Therefore, the term “central niche” rather than “vascular niche” is preferred when referring to the perisinusoidal region (Kiel et al. 2005; Lo Celso et al. 2009; Ding et al. 2012; Kunisaki et al. 2013; Acar et al. 2015). Transition zone vessels, connecting arterioles with sinusoids, are also described in the BM microenvironment, located close to the bone surface (Méndez-Ferrer et al. 2020). It has been shown that the spatial position of cells in the marrow is closely associated with the stage of differentiation of cells, earlier stage HSCs and B lymphocytic progenitors residing preferentially in the endosteal niche (Zhu et al. 2007; Wu et al. 2009; Ding and Morrison 2013; Gazit et al. 2014; Morrison and Scadden 2014). Two stromal cell populations representing endosteal and sub-endosteal niches of mice play active and complementary roles on HSCs’ recruitment from a quiescent niche into a proliferative niche and/or its engagement into the differentiation cascade (Balduino et al. 2012). In the central/perisinusoidal niche, the metabolic milieu with increased reactive oxygen species (ROS) resulting from mitochondrial oxidative phosphorylation plays a role in directing HSCs towards mature hematopoietic cell lineages. It is stated that the leaky sinusoidal blood vessels with fenestrated basal lamina provide a site of blood cell migration between the BM and the circulation upon exposure of HSCs to blood plasma components and increases in ROS levels (Crane et al. 2017).

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Although HSCs’ quiescence is critical in maintaining the stem cell pool, recent studies have also shown the critical role of long-lived progenitors in maintaining steady-state hematopoiesis (Sun et al. 2014). Thus, the spatial position of progenitors is suggested to be involved in the regulation of proliferation and differentiation during both steady-state and under hematopoietic stress (Hérault et al. 2017a). The frequent turnover of mature neutrophils and macrophages necessitates a unique localization of granulocyte-macrophage progenitors (GMPs) to regulate their proliferation, differentiation, and responding to negative feedback in the niche. Individual GMPs are scattered throughout the BM in a steady state, whereas during regeneration, clusters are formed from expanding GMP patches, locally differentiating into granulocytes. Besides the granulocytic lineage, a revised model of megakaryopoiesis based on spatial location has been described in which a vessel-biased megakaryocyte pool rather than the migration mechanism from a distant endosteal niche is emphasized (Larson and Watson 2006; Stegner et al. 2017). These findings demonstrate the importance of spatial position in the stress response of the BM microenvironment. MSCs, being the source of the connective tissue cells, are critical microenvironment factors in coordinating the highly organized BM microenvironment network for healthy hematopoiesis. There is significant heterogeneity among MSCs, both phenotypically and functionally. The standard stromal markers, including CD105, CD29, CD44, CD90, CD73, do not discriminate against different subsets of MSCs. The recent International Society for Cell Therapy (ISCT) standardized norms for characterization of MSCs has been instrumental to remove the inconsistencies regarding the nomenclature as well as biological characteristics of MSCs (Haider 2018). According to ISCT recommendation, MSCs show expression of CD73, CD90, CD105 and lack CD34, CD45, HLA-DR, CD14 or CD11b, CD79a or CD19 membrane surface molecules. It was shown that the perisinusoidal niche is supported by leptin receptor-positive (LepR+) stromal cells with active roles in HSC homing and mobilization (Ding et al. 2012; Ding and Morrison 2013), whereas nestin-positive (NES+) cells in the periarteriolar niche promote quiescence (Kunisaki et al. 2013). Nestin is a typical protein of neural cells (ZIaee et al. 2017). When present on MSCs shows higher colony-forming fibroblast (CFU-F) activity, self-renewal, and multilineage differentiation ability (Méndez-Ferrer et al. 2010b; Zhou et al. 2014). A significant overlap between LepR+ cells and NES+ cells and with those with high levels of CXCL12 (CXCL12-abundant reticular/CAR cells (Sugiyama et al. 2006) have been described. LepR+/CAR cells, although they represent a very low population in the BM, they have long processes that are present throughout the bone marrow and become closely associated with HSCs. Both adipocytes and osteoblasts are cell types differentiated from their precursors, MSCs. A recent study reported two types of cells based on the preferential expression of LepR (Adipo-CAR) and osterix (Osteo-CAR). Spatial analysis revealed localization of Adipo-CAR cells to sinusoids and Osteo-CARs to arterioles in the BM (Baccin et al. 2020). The pericytes in the arteriolar endothelium in the endosteal region were reported to be more quiescent similar to their HSC neighbors, were LepR- and innervated by sympathetic nerves. In contrast, sinusoidal pericytes were positive for the perivascular marker LepR and hosted cycling HSCs (Klamer and Voermans 2014).

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Despite the sophisticated experimental studies providing evidence about the role of differential localization and different functions of BM stem cell niches, there is a lack of confirmatory studies in humans. There is a need for clinical translation of the significantly accumulated basic knowledge and experimental works to humans. Metabolic profiling is a valuable tool to determine the state of cells in different microenvironment conditions. Studies have shown that cells with a low mitochondrial potential are highly enriched for HSCs and progenitors in both mice and humans. The LT-HSCs utilize glycolysis with low oxygen consumption rates, have low mitochondrial potential, and have better repopulation ability. These studies have pointed out the hypoxic metabolism, hypoxia-inducible factor (HIF1α), MEIS 1, and the unique metabolic profile of the hematopoietic niche (Simsek et al. 2010; Kocabas et al. 2015). To investigate the spatial position of human hematopoietic niches, we analyzed BM samples derived by superficial and deep aspiration from healthy human BM transplant donors by metabolomics and transcriptomics analyses and determined region-specific metabolic profiles. Significant differences in energy metabolism were detected between two anatomically different regions of the marrow. As expected, the superficial/cortical region of the marrow was representative of the endosteal niche and was enriched for the pentose phosphate pathway. In contrast, the citrate cycle pathway was enriched in the deeper aspiration region, likely, the central marrow region. The culture-expanded MSCs from the superficial and deeper marrow aspiration regions showed differences in gene expression profiles (Ayhan et al. 2021).

Dynamic Interactions in the BM Microenvironment Increasing knowledge of the dynamic interactions and the metabolic state of the BM microenvironment is essential to understand stress response and disease pathophysiology. The BM is a site of high cell turnover and heavy cell/stem cell trafficking. Following BM transplantation, donor HSCs home to the hematopoietic niche in the marrow and, through adhesive interactions with the cells and the ECM, reside in the endosteal, then in perisinusoidal niches to provide lifelong hematopoiesis of donor type. Homing and retention in the hematopoietic niches are mediated through receptor-ligand interactions between cells and the ECM involving chemokines and adhesion molecules (Heazlewood et al. 2014; Sánchez-Aguilera and Méndez-Ferrer 2017; Wei and Frenette 2018; Pinho and Frenette 2019). Stem cell niches in the BM microenvironment interact with each other and metabolic factors are critical in determining the fate of cell populations in the marrow. Hypoxic state/HIF1α is an important determinant in spatial positioning of LT-HSCs in the quiescent niche in which the metabolic milieu is compatible with a glycolytic environment with low NADH, low mitochondrial activity, and active pentose phosphate pathways. On the other hand, mitochondrial plasticity is also a crucial factor in cell fate determination. Migration of HSCs towards ROS high central/perisinusoidal region favors differentiation, proliferation, and mobilization of HSCs to peripheral blood (Bahat and Gross 2019).

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Many cell types are involved in the retention or mobilization of stem cells. Osteoblasts, macrophages, NES+ MSCs provide HSCs retention and maintenance signals, whereas activation of perisinusoidal LepR+ MSCs, osteoclasts, endothelial cells are needed for mobilization. Interactions between cells and ECM components determine the adherence characteristics of HSCs to their niche. CXCL12/CXCR4, VCAM1/VLA4, Angpt1/Tie2, SCF/C-kit, TPO/MPL signaling, matrix components consisting of adhesion molecules, integrins, N-cadherins, osteopontin, calcium ions, hypoxic environment, and TGF-β signaling are among the essential mechanisms providing adherence and maintenance in the quiescent hematopoietic niche (Lapidot and Petit 2002; Arai et al. 2004; Lapidot et al. 2005; Dar et al. 2005; Ramirez et al. 2009; Bonig et al. 2009; Li 2011). Under steady-state conditions, the great majority of HSCs and progenitors reside in the BM; only a small number rhythmically leave the BM. Autonomic signals from the sympathetic nervous system (SNS) in response to “in need” signals regulate the circadian rhythm of HSC and progenitor mobilization by affecting cytokine, chemokine, cell, and ECM interactions. The HSCs neighboring the arteriolar blood vessels are surrounded by the SNS fibers. HSCs’ maintenance/retention factors such as SCF and CXCL12 are expressed by NES+/NG2+ (pericyte-specific marker) perivascular MSCs. The non-myelinating Schwann cells also maintain HSC quiescence by activating the TGF-β)/SMAD signaling. Following circadian secretion of norepinephrine (NE) by the SNS, CXCL2 levels oscillate within the BM microenvironment, orchestrating steady-state egress. When activated, HSCs relocate near the NES low LepR+ MSCs in the perisinusoidal area and are rhythmically released to peripheral blood (Méndez-Ferrer et al. 2010a). Physiological or stress signals, including cortisol release, toxic insults, chemotherapy, radiation, hypoxia, infection-associated danger signals, and a variety of systemic stressors, metabolites are involved in mobilization. This process is carried out by mechanisms that interfere with retention and activate mobilizing mechanisms resulting in the migration of HSCs towards the perisinusoidal region. Research on stem cell mobilizing strategies has broad implications in clinics, not only in hematological conditions but also in regenerative applications (Wang et al. 2006a, b; Elmadbouh et al. 2007; Haider et al. 2008; Tano et al. 2011; Jiang et al. 2012). Granulocyte colony-stimulating factor (GCSF) is the best known and widely used HSCs mobilizing agent used in HSCs transplantation, also in oncology clinics to hasten recovery from neutropenia. It is endogenously secreted from the BM microenvironment in response to physiological and stress signals. GCSF induces significant changes in the BM; it directly or indirectly interferes with nearly all components of the hematopoietic niche, including the cellular members, ECM, and signaling molecules inducing significant physical and metabolic changes (Greenbaum and Link 2011). The first step in the mobilization process is detachment from the BM niches by interfering with the anchoring interactions mediated by CXCL12, VCAM-1, and their receptors. Interaction of GCSF with its receptor on the SNS leads to: suppression of the niche support function of macrophages and osteolineage cells (Katayama et al. 2006; Chow et al. 2011), inhibition of stromal cell synthesis of retention factors such as CXCL12 (Petit et al. 2002), and

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interruption of CXCL12/CXCR4 signaling leading to increased proliferation and mobilization to blood associated with reduced BM but increased blood CXCL12 levels and upregulated CXCR4 expression. GCSF induced neutrophil activation, degranulation, and release of granulocyte proteases, such as neutrophil elastase and cathepsin, play a role in the proteolytic inactivation of these factors and creation of the chemokine gradient for egress (Ratajczak 2015; Hatfield et al. 2017; Itkin et al. 2017; Wei and Frenette 2018). Proteolytic activation of several factors in BM microenvironment is necessary during HSPCs mobilization. Significant changes occur in all ECM components for mobilization, heparan sulfate proteoglycans being a major one. Heparan sulfate proteoglycans are key regulators of the hematopoietic niche, and their functions are dynamically modified depending on the stage of cell maturity and according to physiological or stress signals. It was shown that heparan sulfate chain structures are continuously remodeled in a spatiotemporal fashion while the stem and progenitor cells progress through various differentiation pathways (Papy-Garcia and Albanese 2017). On the other hand, activation of matrix metalloproteinases (MMP-9, MMP-1) also leads to significant alterations in the ECM. Mobilization from the marrow induces substantial changes in the coagulation and the complement systems as well. Plasminogen plays an essential role particularly in the GCSF-induced mobilization by binding to the BM ECM and, after conversion into plasmin, by degrading various proteins including fibrin, laminin, and activating other proteases, such as MMP-3, MMP-9, MMP-12, and MMP-13, and degrading other matrix components, such as collagen. Similarly, the urokinase plasminogen activator, part of the plasminogen activating system, is also involved in cell traffic (Gong and Hoover-Plow 2012). Moreover, activation of the complement cascade proteins C3 and C5 leads to establishing a highly proteolytic microenvironment that degrades CXCL12. Activation of osteoclasts and secretion of cathepsin K and CD26 (dipeptidyl-peptidase 4) are also involved in the cleavage of CXCL12 and mobilization (Lapidot and Petit 2002; Itkin et al. 2017). Another important function of CD26 in mobilization is its effect on vasculature. The BM endothelial cells are essential players in the regulation of HSC trafficking. CD26 is expressed by endothelial cells and is involved in maintaining vascular barrier function during stress-induced conditions (Itkin et al. 2017). Recently, the critical role of signaling through neuropeptide Y on HSC trafficking has been shown through its effects on BM vasculature, raising the importance of targeting vasculature as a mobilizing strategy. The expression of CD26 by endothelial cells activates NPY-mediated signaling by increasing the bioavailability of the truncated form of NPY. Neuropeptide Y is released from SNS nerves, enhances the activity of MMP-9, and affects HSC function by modulating MSCs, osteoblasts, and macrophages (Singh et al. 2017). Neuropeptide Y-mediated direct regulation of HSCs’ quiescence was also reported by the authors (Ulum et al. 2020). Other groups of molecules involved in the mobilization process are the angiopoietins (Ang) and angiopoietin-like proteins, with important roles in vascular development, angiogenesis, which also affect the BM niche functions (Arai et al. 2004). Angiopoietins are regulators of endothelial cell interactions with supporting perivascular cells.

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Angiopoietin-1 and 2 are ligands of endothelial-specific tyrosine kinase receptor, Tie2. Ang1 and Tie2 interaction is important in quiescent niche function and vascular stabilization. Apart from being an endothelial survival factor, Ang1 protects HSCs from cellular stress, thus contributing to the maintenance of the cells in a quiescent state in the BM niche. On the other hand, Ang2 acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang1/Tie2 signaling promotes apoptosis of endothelial cells (Lei et al. 2007a). It was initially identified as a disruptive agent of the vasculature. It has been shown that Ang2 plays a complex role in angiogenesis, depending on the physiological and biochemical environment, acting either as an agonist or antagonist of Tie2 induced angiogenesis (Lei et al. 2007b). Co-overexpression of Akt and Ang-1 significantly contributes stem cell proliferation via miR-143 which critically regulates Erk5/CyclinD1 signaling (Lai et al. 2012a, b) and also causes stabilization of HIF1alpha and supports endothelial commitment of BM stem cells (Lai et al. 2012b). Ang2 is critical for cytokine-induced vascular leakage and controls the vascular response to inflammation associated with cytokine-induced intracellular calcium influx. With these properties, Ang2 has a role in stress-induced mobilization (Benest et al. 2013). Ang-like 4 (Agptl4) is another factor with critical roles in BM vasculature. Recently, it was shown that Angptl4 inhibition led to increased BM vascular permeability and increased trafficking of HSCs and progenitors into circulation in BM homeostasis and as a stress response (Suzuki et al. 2021). Lipid metabolism also plays a vital role in mobilization. Sphingosine-1 phosphate and ceramide-1 phosphate, the bioactive phosphorylated lipids, are potent chemoattractants involved in HSC, progenitor, and malignant cell trafficking (Shirvaikar et al. 2010; Kim et al. 2010; Ratajczak et al. 2014; Ratajczak 2015; Albakri et al. 2020). Recent studies demonstrated the critical role of lipolysis and the lipolytic enzyme hematopoietic-specific phospholipase C-β2 (PLC-β2) in HSC egress (Adamiak et al. 2016). It was shown to be upregulated in the BM microenvironment during the mobilization process and lead to impairment of membrane lipid raft formation, which is required for the optimal BM retention function of glycolipid glycosylphosphatidylinositol-anchor-associated proteins, CXCR4 and VLA-4. It was also shown that the membrane type 1-MMP upregulation in hematopoietic cells and its enhanced incorporation into membrane lipid rafts contribute to proMMP-2 activation, further facilitates HSCs mobilization upon exposure to GCSF (Shirvaikar et al. 2010). The effect of lipid metabolism in BM trafficking was also demonstrated in the recent study (Suzuki et al. 2021), which reported the important role of dietary fat intake on signaling in BM granulocytes. The BM fat ligand for PPARδ was suggested as a negative regulator of mobilization in fed mice. On the other hand, mice on a fat-free diet showed increased mobilization with PPARδ agonist. The authors point out the clinical relevance of this mechanism in poor mobilizers (Suzuki et al. 2021). Stem cell trafficking has significant clinical implications. Therefore enlightening the mechanisms and revealing therapeutic targets is an interesting subject. The use of antagonists of chemokine receptors, cytokines, chemokines, bioactive lipids, bacterial toxins, proteases, inhibitors of adhesive cell interactions,

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polymeric sugar molecules, and modification of their biological effects are strategies towards optimization of stem cell mobilization in transplantation in preventing cancer metastasis and for organ regeneration (Albakri et al. 2020). Studies on humans consist of investigations performed on GCSF-exposed human blood or BM samples of HSCs transplant donors. Significant alterations in the blood metabolite profile are described in apheresis donors exposed to GCSF consisting of significantly increased levels of several medium and long-chain fatty acids, and polyunsaturated fatty acids and reduced levels of other lipid metabolites, such as phospholipids, lysolipids, and sphingolipids (Hatfield et al. 2017). On the other hand, studies performed by us on BM samples of healthy donors after GCSF exposure revealed alterations in the levels of growth factors, cytokines, and immunomodulatory factors, and upregulation of both hematopoietic colonies and the colony-forming ability of MSCs (fibroblast colony-forming units/CFUFs), indicating the regenerative potential of GCSF (Ok Bozkaya et al. 2015; Aerts-Kaya et al. 2021). The role of GCSF in non-hematopoietic tissue/organ regeneration has gained attention in several clinical disciplines. The multifaceted effects of GCSF, particularly on endothelial cells and angiogenesis, are suggestive of its beneficial roles in injury states. Starting in the 2000s and in recent years, GCSF has been used by some centers for organ injury, such as after neurological insults (Lee et al. 2005; Liu et al. 2020b). Favorable results reported were attributed to activation of several mechanisms, including enhanced angiogenesis associated with increased endothelial proliferation, increased endothelial NO synthase, and Ang-2 expression. In addition, the anti-inflammatory and anti-apoptotic effects of GCSF were also identified as contributing factors to its neuroprotective properties.

Bone Marrow and Stress Response Studies regarding the continuous, dynamic, and organized interactions in the BM niche indicate the critical role of the microenvironment in maintaining homeostasis. Therefore, enlightening the niche regulatory mechanisms under stress is important to delineate the highly active part of the microenvironment in the pathophysiology of cancer and other hematological disturbances.

Cell and Microenvironment Response to Stressors Cells are continuously exposed to internal, external, physiological stimuli or stressors upon which various stress responses are activated to recover cell function and maintain homeostasis within the tissue and the organism. Depending on the intensity and duration of the stimuli, different signals that induce microenvironment and/or systemic responses are generated usually associated with paracrine and endocrine signals. Upon exposure to low doses of stressors, either the former state of the cells is preserved or shows an altered profile and adapt to the new

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environment. In contrast, the repair process may be ineffective in prolonged and/or severe stress leading to cellular senescence or death. The microenvironment of cells is the determinant of the fate of the stressed condition. Persistent cell stress enhances susceptibility to cancer and aging-associated diseases and is usually associated with chronic inflammation. Cells may be exposed to different types of stressors such as genotoxic stress/DNA damage, hypoxia, oxidative stress, nutrient/metabolic stress, and xenobiotics (Ivanusic 2017). Genotoxic stress is caused by ionizing and ultraviolet radiation, exposure to chemotherapy, other toxic agents, and ROS. DNA damage requires complicated molecular mechanisms to maintain genomic stability through DNA repair. Activation of the DNA damage response (DDR) signaling pathway results in either cell cycle arrest and DNA repair or apoptosis (Zambetti et al. 2016; Davalli et al. 2018; Huang and Zhou 2019). Oxidative stress derives from the excess production and accumulation of ROS or a defect in the antioxidant protective mechanism. While low doses of ROS stimulate cell proliferation, higher doses can result in cell cycle arrest, senescence, or cell death (Finkel 2003). Hypoxic stress leads to cell cycle arrest to minimize energy consumption to preserve oxygen for cell metabolism. The cellular response to hypoxia is mediated by both HIF-dependent and HIF-independent pathways, i.e., mTOR signaling. Hypoxia, respiratory poisons, and xenobiotics are stressors that cause mitochondrial stress (Liu et al. 2006; Zhang and Zhang 2018). The availability of nutrients is crucial for metabolic homeostasis and the proper function of the cell. Nutrient stress causes metabolite fluctuations, which are recognized by the lipid membranes of organelles. One of the most essential nutrients is glucose, and both deprivation and excess of glucose can cause cellular stress. Nutrient deprivation activates autophagy in most cells, enabling them to catabolize their components for survival (Sekine et al. 2021). Heat shock causes protein denaturation, unfolding, and aggregation. The molecular chaperones, heat shock proteins (HSPs) are responsible for protein folding, unfolding, and/or refolding in either standard or stressed conditions (Vabulas et al. 2010). Similarly, chemical toxins also cause protein denaturation and activate the unfolded protein response (UPR) in the endoplasmic reticulum and mitochondria. On the other hand, infectious agents can induce several stress responses by activating pattern recognition receptors (Muralidharan and Mandrekar 2013). The microenvironment plays a critical role in activating and performing cellintrinsic or extrinsic stress response mechanisms (Galluzzi et al. 2018). Stressed cells may show altered morphology, phenotype, molecular, metabolic profile, and secretory properties, including the release of ECVs (exosomes) with a variable composition of protein cargo (Abramowicz et al. 2019; Haider and Aramini 2020). Suppression of proliferation and elimination of terminally damaged cells are protective mechanisms against inflammation and cancer. The stress response mechanisms, including the DDR, UPR, mitochondrial stress signaling, may result in temporary adaptation, induce autophagy, or trigger cell death. Autophagy is a significant stress response mechanism important in adaptation to a stressed situation. Potentially harmful or disposable cytoplasmic contents are degraded. Physiological autophagy levels are required for maintaining stable cell homeostasis under stress conditions,

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while exacerbated autophagy induces uncontrolled cell death. Autophagy in one cell releases signals that affect other cells locally and systemically. Thus, autophagy affects the local microenvironment, may elicit a systemic metabolic response, and may contribute to the pathophysiology of diseases, e.g., by providing metabolites such as lactate, ketone bodies, alanine to cancer cells as microenvironment support (Capparelli et al. 2012; Sousa et al. 2016). Hence, depending on the intensity of the aggressive stimuli, autophagy can be beneficial or harmful, mediate antiinflammatory or pro-inflammatory effects, and maybe playing either a cytoprotective role or mediates regulated cell death. Stress-induced regulated cell death, as opposed to programmed cell death, may lead to a release of cellular contents and may trigger pro-inflammatory signals. However, the local microenvironmental factors such as the efficiency of the phagocytic system may affect the outcome of the stress response. Cellular senescence is another stress response of cells wherein DNA damage is irreparable but insufficient to drive regulated cell death, oncogene signaling, or other threats to homeostasis and is associated with permanent growth arrest. However, some metabolic activities of senescent cells, secretion of ROS, and pro-inflammatory cytokines are preserved.

Hematopoietic Stress Response Constant blood cell production and the continuous cell traffic in the BM render the hematopoietic system highly sensitive to toxic agents. Under homeostasis, most of HSCs are quiescent and rarely enter the cell cycle to self-renew or differentiate. There is a fine-tuning balance between the quiescent, proliferating, differentiating, and senescent cells. In response to external stimuli, such as infection, blood loss, or toxic insults, HSCs can leave a quiescent state and become proliferative. Recent evidence suggests that acute and chronic stress impact the number and function of HSCs, including their ability to repopulate and produce mature cells. The BM microenvironment has a significant role in determining the response and maintaining a steady-state and life-long hematopoiesis (Walter et al. 2015; Pratibh et al. 2020). HSCs and BM microenvironment may be exposed to different types of stress, including oxidative, hypoxic stress, inflammation, blood loss, ionizing radiation, cytotoxic chemotherapy, all of which lead to BM injury, disrupt homeostasis, and cause significant alterations in cell composition (Mendelson and Frenette 2015; Pratibh et al. 2020) (Fig. 1). The hematopoietic system can quickly respond to infection and inflammation by increasing myeloid and immune cell production. This is a well-known response presenting as a left shift of myeloid elements in the peripheral blood of patients with sepsis and bacteremia. A similar proliferative response in relevant components of the hematopoietic lineages are observed in other pathological conditions, such as increased megakaryocyte production in patients with immune thrombocytopenia as a BM response to platelet consumption platelet, alternatively, accelerated production of erythroid precursors under conditions of immune destruction or blood loss. These clinical manifestations represent the rapid-acting compensatory function of the BM microenvironment to preserve

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Fig. 1 Summary of the hematopoietic stressors and stress response of the hematopoietic system (Created with BioRender.com)

and reconstitute healthy hematopoiesis. Thus, acute stress of low/medium intensity may be overcome with a limited impact on HSCs. On the other hand, prolonged sustained exposure to stress signals can lead to a shift of HSCs towards HSCs proliferation rather than a quiescent state leading to HSCs exhaustion over time. Chronic inflammation, serial BM transplantation, chemotherapy cycles, genotoxic stress, and aging are conditions leading to disruption of the fine-tuning in the hematopoietic compartment resulting in increased proliferation and HSCs exhaustion. This may lead to the development of cytopenia, aplastic anemia, or upon acquisition of cytogenetic abnormalities in the cycling cells may play a role in the development of hematological malignancies. Recent evidence demonstrates that ablation of inhibitor of DNA binding 1 (Id1) gene can protect HSCs from exhaustion during chronic proliferative stress by promoting HSCs quiescence (Singh et al. 2018). It was shown that Id1 is induced in HSCs by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis and its genetic ablation decreases in HSCs’ proliferation, mitochondrial biogenesis, and ROS production. Considering the role of high ROS levels in the BM microenvironment on increased proliferation and differentiation, targeting molecular pathways that reduce ROS in HSCs is suggested as a protective strategy to promote HSC quiescence and limit HSC loss during chronic stress (Singh et al. 2020).

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Recent studies have described alterations in the replicative status of the stromal cells as well, similar to HSCs. It was reported that almost all niche cells were quiescent during homeostasis (1500 differentially expressed genes in BM HSCs involved in pathways in cancer, transcriptional mis-regulation in cancer, and hematopoietic cell lineage. Investigations in peripheral blood revealed hematopoietic cell lineage and leukocyte transendothelial migration as critical pathways. Evaluation of co-regulated pathways demonstrated neutrophil degranulation, CD93 (involved in the adhesion, migration, and phagocytosis), and 53 genes that pointed out mechanisms in regulating leukemia stem cell self-renewal and quiescence (Sun et al. 2021). Other studies involving modulation of the cytosolic transcription factor aryl hydrocarbon receptor (AhR), the xenobiotic

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response regulator, revealed the important role of AhR in benzene-induced hematotoxicity (Hirabayashi et al. 2004; Scharf et al. 2020). Nanoparticles and particulate matter These are other groups of environmental stressors found in polluted air. The degree of toxic systemic effects is closely linked to particle size. Both nanoparticles and particulate matter PM 2.5 (smaller than 2.5 μ m) (Xing et al. 2016) can reach the bloodstream from alveoli and be distributed into tissues, including the BM. The particles can also contain harmful airborne microorganisms and metals. Chronic exposure leads to local and systemic inflammation, may affect HSC niches, and lead to a release of immature granulocytes into circulation (Tan et al. 2000). Recent studies have described the adverse effects of maternal exposure to PM 2.5 during pregnancy on the offspring hematopoiesis (Bhattarai et al. 2020). Dioxins as Persistent Organic Pollutants (POPs) POPs are released into the atmosphere as undesired byproducts of an anthropogenic and natural origin. These pollutants can originate as byproducts from combustion processes, such as the incineration of solid waste; the chlorine bleaching of paper and wood pulp; the burning of coal in power plants; forest wildfire; and contaminants in pesticides, herbicides, and fungicides. Dioxins are considered the most hazardous persistent organic pollutants to human health, with their significant toxic effects being linked to binding to AhR in several cell types. AhR is the central regulator of responses to environmental factors and xenobiotic metabolism. The most toxic member of the polychlorinated dibenzodioxins family 2,3,7,8tetrachlorodibenzodioxin (TCDD) can bind to AhR, leading to several toxic effects. Many stressors can drive AhR activation and nuclear translocation, leading to increased expression of target genes (e.g., cytochrome P450; CYP1A1, CYP1A2, CYP1B1) (Safe et al. 2018). AhR is also a crucial homeostasis modulator in several tissues and biological processes, including hematopoiesis. Epidemiologic studies have demonstrated associations between TCDD and hematological malignancies such as non-Hodgkin lymphomas, chronic lymphocytic leukemia, and multiple myeloma (Bertazzi et al. 1993; Fracchiolla et al. 2016). It has been reported that the BM of adult mice exposed to acute doses of TCDD becomes hypocellular, with a significant decrease in the total number of HSCs due to AhR modulation (Ahrenhoerster et al. 2014). Polycyclic Aromatic Hydrocarbons (PAH) These are ubiquitous environmental pollutants that include over 200 compounds with two or more fused benzene rings. These compounds are formed due to incomplete combustion of fossil fuels, are created in the car and diesel exhaust, and are formed in smoked or charbroiled food. They are also found in cigarette smoke condensate and tobacco products. Studies have demonstrated an association between exposure to PAHs and cancer initiation and progression. It was shown that PAHs must be metabolically activated to reactivate genotoxins to cause their mutagenic and carcinogenic effects. A recent study described activation of the AhR/CYP1A pathway and epigenetic regulation of cancer stem cells upon PAH

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exposure (Akhtar et al. 2020). Moreover, adverse hematopoietic effects of occupational PAH exposure were shown, and oxidative stress-mediated toxicity was suggested in another recent study (Cao et al. 2020). Heavy Metal Exposure This is also associated with hematopoietic disorders. Industrial and urban growth has increased the risk of exposure of humans to metals. Heavy metals may cause alterations in the BM, causing anemia, immune deficiency, coagulation defects, and may be associated with leukemia development. Among heavy metals, lead, mercury, cadmium, and arsenic have been associated with hematological disorders (dos Vianna et al. 2019). It has been shown that lead exposure even at low levels in humans under occupational exposures is toxic to the BM and leads to reduced colony-forming capacity; arsenic trioxide administered to mice severely damages the BMME and hampers the formation of a healthy matrix to support hematopoietic progenitors; mercury exposure is associated with hematopoietic disturbance characterized by anemia and lymphocytopenia; and cadmium exposure leading to increased myelopoiesis and suppressed lymphopoiesis (Zhang et al. 2016b; Medina et al. 2017; Pereira and Law 2018).

Detoxification Response and Signaling Pathways Drug metabolizing enzymes Cytochrome P450 (CYP) and Glutathione S-Transferases (GSTs) Drug metabolizing enzymes display a high degree of polymorphism in the general population. Functional polymorphisms in the genes encoding xenobioticmetabolizing enzymes result in interindividual differences that determine the effects of toxic exposures (Harris 1989). Cytochrome P450 (CYP) and glutathione S-transferases (GSTs) are the main detoxification pathways. The latter catalyzes the conjugation of the reduced form of glutathione to xenobiotic substrates to detoxify cellular environments. Cytochrome P450 family proteins are the major enzymes involved in drug metabolism and metabolize thousands of endogenous and exogenous chemicals. In addition, they metabolize endogenous and exogenous DNA-reactive chemical compounds and xenobiotics (Zanger et al. 2004). Aryl Hydrocarbon Receptor (AhR) AhR is the central regulator of responses to environmental factors and of xenobiotic metabolism, which was initially described in dioxin toxicity (TCDD; 2,3,7,8-Tetrachlorodibenzo-P-dioxin) (Poland et al. 1976), now is accepted as a crucial regulator of homeostasis in several tissues/systems in the body. There are many endogenous ligands of AhR. The AhR-signaling acts as a xenobiotic sensor regulating drugmetabolizing enzymes of the cytochrome p450 family. Various structurally diverse compounds of the environment, chemicals, toxicants, pollutants, diet, microbiome, and host metabolism can induce AhR activity (Mandal 2005; Vogel et al. 2020). Under homeostatic conditions, AhR remains predominantly in the cytoplasm as part of a protein complex linked to molecular chaperone heat shock protein 90 (HSP90),

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p23, and XAP. Several stressors can drive AhR activation and evoke its conformational transition, resulting in its nuclear translocation. AhR then dissociates from HSP90 and binds to the AhR nuclear translocator (ARNT) The AhR/ARNT complex binds to promoter regions in the DNA known as AhR-responsive DNA elements or xenobiotic response elements (XREs), which leads to an increased expression of target genes (e.g., CYP 1A1, CYP1A2, and CYP1B1). This canonical pathway for AhR activation mediates several toxic responses, including liver damage, chloracne, teratogenesis, cancer, and immunosuppression (Barouki et al. 2007; Fujii-Kuriyama and Kawajiri 2010; Furue and Tsuji 2019). In addition to exogenous triggers, many endogenous ligands have been shown to induce AhR-signaling, with implications on several body systems playing a critical role in balancing physiological functions. For example, the significant effects of AhR signaling on circadian clock genes also pointed out the potential impact of different neurotransmitters and metabolites (Kou and Dai 2021). The AhR modulation has shown significant effects on hematopoiesis. In addition, it has been demonstrated that HSCs from AhR knockout mice leave quiescence and become hyperproliferative, suggesting a role for AhR in negative regulation of excessive proliferation (Singh et al. 2009, 2011). Toll-like receptor (TLR) Signaling Toll-like receptor signaling is needed in responding to inflammatory stressors, microbial agents and is required for innate and adaptive immunity. Pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs) are released in response to injury and stimulate TLR-signaling. In addition to their well-known role in the innate immune response to acute infection or injury, accumulating evidence supports a role for TLRs in the development of hematopoietic and other malignancies. HSCs proliferate in response to PAMPs and DAMPs through TLRs, or in response to pro-inflammatory cytokines through receptors expressed on HSCs (Schuettpelz and Link 2013; Mirantes et al. 2014). Acute stress promotes HSCs’ proliferation and differentiation that is quickly resolved, whereas prolonged or chronic stress can lead to HSC exhaustion (BMT, chronic infection/inflammation, chemotherapy) (Singh et al. 2020). Several hematopoietic disorders, including lymphoproliferative disorders and myelodysplastic syndromes, which possess a high risk of transformation to leukemia, have been linked to aberrant TLR signaling. Furthermore, activation of TLRs leads to the induction of several pro-inflammatory cytokines and chemokines, which can promote tumorigenesis by driving cell proliferation and migration and providing a favorable microenvironment for tumor cells (Monlish et al. 2016).

BM Microenvironment Alterations, Dysregulation, and Myeloid Leukemia Due to close interaction between the hematopoietic compartment and the microenvironment, several nonmalignant and malignant conditions are frequently associated with alterations in the BM microenvironment composition and function (Fig. 2).

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Fig. 2 Summary of the bone marrow niche structure and organization and its role in homeostasis and in hematological malignancies. (Created with BioRender.com)

Leukemia results from a clonal transformation of hematopoietic precursors by acquiring chromosomal rearrangements, genetic mutations, and epigenetic modifications. It has been shown that the initiating intrinsic event leads to alterations in the BM microenvironment to support the survival of leukemic blasts (Blau et al. 2007; Li and Calvi 2017). There is limited data for a driver role for the BM microenvironment, leading to the development of leukemia. It has been suggested that prolonged sustained exposure to stress signals, including chronic inflammation, chemotherapy, environmental toxins induced alterations in the BM microenvironment leading to increased proliferation, and HSCs’ exhaustion over time, resulting in BM dysfunction, failure, or development of hematological malignancy, usually of myeloid lineage (Cho et al. 2020). Significant changes are detected in the BM microenvironment in hematological malignancies, during the course or before the development of leukemia (Dührsen and Hossfeld 1996). Various alterations involving MSCs, osteoprogenitors, adipocytes,

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endothelial cells, ECM, and soluble factors have been reported in the leukemic niche playing significant roles in initiation, progression, and chemoresistance. The majority of studies performed on MSCs described alterations in their adhesive, proliferative, and secretory characteristics. Reduced HSCs’ maintenance factors including CXCL12, KİT ligand, Ang1 were described in leukemic MSCs (Chen et al. 2016). Further studies have shown increased expression of pro-inflammatory molecules such as TNF, IL6, C-C motif chemokine ligand 3 (CCL3), alterations in SNS signaling due to damaged tyrosine hydroxylase sympathetic nerve fibers, altered stem cell trafficking, and dysregulated vascularization in the leukemic BM microenvironment (Maryanovich et al. 2018; Méndez-Ferrer et al. 2020). In humans, the development of myelodysplastic syndrome (MDS)/secondary acute myeloblastic leukemia (sAML) years after chemotherapy use, besides intrinsic factors, emphasizes the active role of the altered and dysregulated microenvironment in leukemogenesis (Sperling et al. 2017). It may be speculated that, in both situations, the BM microenvironment alterations lead to dysregulation in time, playing a critical role in the progression towards malignancy and evolution to AML after a myelodysplastic stage (e.g., 5–7 years after alkylating agent exposure) or without such dysplasia (e.g., 2–3 years after topoisomerase II inhibitor exposure) (Raaijmakers et al. 2010; Duarte et al. 2018; Forte et al. 2019). Apart from those toxic exposures or cancer-predisposing disease, various alterations have been reported in the BM microenvironment that may be closely associated with the leukemogenic process (Ciciarello et al. 2019; Kazianka and Staber 2020). Understanding the mechanisms leading to the development of sAML may help identify the microenvironmental factors in the BM.

Development of Secondary Myeloid Malignancy Unlike de-novo AML, in which there is a lack of a typical clinical history, sAML occurs following environmental or toxic exposures and/or develops in patients with an antecedent hematological disorder; therefore, it represents a model to study alterations in the BM microenvironment that may participate in leukemogenesis. It has been shown that inherited diseases (e.g., Fanconi anemia (FA), ShwachmanDiamond syndrome (SDS)) (Horwitz 1997) and/or environmental exposures (e.g., high-level ionizing radiation, cigarette smoking, long-term occupational exposure to benzene, exposure to certain chemotherapy drugs such as alkylating and platinum agents, epipodophyllotoxins, immunosuppressives, and radiation therapy) may lead to leukemia development in some cases of AML (Daniels et al. 2013; Radivoyevitch et al. 2016; Matsuo et al. 2016). The incidence of sAML is rising in parallel with the increasing number of cancer survivors. Its prognosis is poor compared to de novo AML, which occurs without previous therapy or without the antecedent disease.

Therapy-Related Myeloid Malignancy Secondary AML is further divided into therapy-related AML (t-AML) with previous exposure to chemotherapy and radiotherapy, or AML evolving from antecedent

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hematological disorder including MDS, aplastic anemia, or chronic myeloproliferative disorders. Therapy-related myeloid neoplasms (t-MNs), including AML, occur as late complications of cytotoxic chemotherapy and/or radiation therapy and patients show high-risk features. Lymphoma and breast cancer are the most common primary malignancies associated with t-AML. Patients with an inherited predisposition such as mutations in DNA damage sensing or repair genes or polymorphisms in genes that affect drug metabolism or transport are at higher risk for developing t-MNs (Vardiman et al. 2002; Seedhouse et al. 2004; Larson 2009; Bhatia 2013). A wide variety of agents with different mechanisms of action are associated with the development of t-MNs. The interval between treatment and the outcome of the disease varies according to the type of therapy. In patients exposed to alkylating agents, t-AML is usually preceded by myelodysplastic changes and the latency period is long (5–7 years). Contrarily, in patients previously treated with topoisomerase II inhibitors, the latency period is shorter (2–3 years) than alkylating agent exposure, is not associated with prior MDS, and exhibits rapidly progressive leukemia. These patients usually present with balanced translocations involving 11q23 and 21q22 abnormalities. These agents stabilize the enzyme-DNA covalent intermediate, decrease the religation rate, and cause chromosomal breakage. Repair of chromosomal damage results in chromosomal translocations, leading to leukemogenesis (Bhatia 2013; Tiruneh et al. 2020). Increasing knowledge on the pathophysiology of t-AML may help identification of the contributing microenvironment factors. Among other mechanisms involved in developing t-AML, abnormal p53 activity leads to increased susceptibility to leukemogenesis manifesting with reduced ability to repair DNA damage and genomic instability. Telomere shortening also contributes to t-MDS/AML by limiting hematopoietic proliferation and regenerative capacity and inducing genetic instability. Following genotoxic exposure, the increased replicative demand on hematopoietic cells associated with hematopoietic regeneration can lead to accelerated telomere shortening. On the other hand, some polymorphisms in the MDR1 gene are suggested as risk factors for t-MDS/AML (Bhatia 2013; Tiruneh et al. 2020). An important mechanism implicated in t-AML development includes defects of the individual DNA repair machinery. Double-strand breaks in DNA following ionizing irradiation and chemotherapy exposure may lead to cell death or loss of genetic material, resulting in chromosome aberrations. Unsatisfactory repair results in the acquisition and persistence of mutations. On the other hand, better repair mechanism will inhibit apoptosis, enabling the survival of a cell with damaged or poorly repaired DNA (Leone et al. 2007). Evidence suggests a role for mismatch repair in susceptibility to t-AML reflected as microsatellite instability (Seedhouse et al. 2004). Other mechanisms involved include the base excision repair pathway, which corrects individually damaged bases, occurring as the result of endogenous processes, ionizing irradiation, and exogenous xenobiotic exposure; and nucleotide excision repair that removes structurally unrelated bulky damage induced by ultraviolet radiation, environmental factors, and endogenous processes, and repairs a significant amount of DNA damage caused by chemotherapeutic agents.

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Detoxification pathways play a critical role in the pathophysiology of malignancy. Polymorphisms are frequent in drug-metabolizing enzymes. It is suggested that polymorphisms in detoxification enzyme and DNA repair genes synergistically may affect the risk of AML, such as in patients with deletion of the GSTM1 gene and with increased DNA damage (Seedhouse et al. 2004). Glutathione S-transferases are involved in the detoxification of potentially mutagenic and DNA-toxic metabolites of several chemotherapeutic agents. Accumulation of reactive species that escape detoxification mechanisms or are produced in excess due to drug-metabolizing enzyme polymorphisms, together with impaired DNA damage repair, are predisposing factors to t-AML (Davies et al. 2001; Hatagima 2002; Seedhouse et al. 2004). Polymorphisms in cytochrome P450 family compounds are also implicated in leukemia development and prognosis. AhR activation by external and endogenous stressors and regulation of the CYP family of drug-metabolizing enzymes is essential to maintain homeostasis in the hematopoietic system. A recent study has shown increased quiescence in AML stem cells upon suppression of AhR signaling (Ly et al. 2019). In another study, the AHR pathway was activated in human and murine AML and impaired natural killer cell development and function through regulation of miR-29b expression. This effect was reversed with the AHR antagonist pointing out the clinical implications of AhR modulation as a therapeutic strategy in cancer (Scoville et al. 2018). AhR-mediated response has been reported to have a critical role in benzene-induced AML as well; however, the involvement of CYP1A1 and other CYP compounds is not apparent in benzene-induced leukemia (Yoon et al. 2002). On the other hand, CYP3A was shown to participate in the metabolism of various chemotherapeutics, including topoisomerase II inhibitors and alkylating agents and polymorphisms associated with t-AML. A recent report suggested the favorable role of CYP3A inhibitors in patients with AML carrying FLT3/internal tandem duplication (ITD) mutations through the reversal of the protective effect mediated by the BM microenvironment against FLT3 tyrosine kinase inhibitors (Chang et al. 2019). It was reported that BM stromal cells express CYPs, including CYP3A4, and inhibit the activity of tyrosine kinase inhibitors. Clarithromycin, a clinically active CYP3A4 inhibitor, antibiotic was effective in overcoming BM microenvironment-mediated drug resistance. Toll-like receptor signaling has also been implicated in leukemia. In addition to mediating innate immune system response to acute infection or injury, the TLR signaling pathway was also shown to play a role in developing hematological malignancies by inducing the establishment of a pro-inflammatory tumorigenic environment (Monlish et al. 2016; Singh et al. 2020).

Leukemia-Predisposing Inherited BM Failure Syndromes Fanconi anemia Fanconi anemia (FA) is discussed here as a model disease to evaluate myeloid leukemia development both intrinsic and extrinsically due to the genetic defect in DNA damage repair mechanisms and extreme sensitivity to toxic and environmental agents.

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It is an inherited disease characterized by congenital abnormalities, BM failure, and cancer predisposition manifesting as myeloid malignancy (AML, MDS) and solid tumors. The condition is caused by mutations of the Fanconi anemia/breast cancer (FA/BRCA) pathway, critical in cellular processes and DNA repair interstrand crosslink (ICL). Genomic instability, cell death, alterations in cell cycle, extreme sensitivity to DNA cross-linking agents (cisplatin, mitomycin C, diepoxybutane, endogenous aldehydes), and oxidative damage are the typical cellular, molecular events. Two DNA strands are covalently joined, DNA replication and transcription are impaired, resulting in the accumulation of toxic DNA doublestrand breaks. Two ICL repair mechanisms are present. One mechanism, replication dependent, requires the FANC proteins and the other one operates outside the S phase, involves nucleotide excision repair (Houghtaling et al. 2005). FA proteins have roles in the sensing, recognition, and processing of ICLs. To repair this type of DNA damage, alternative error-prone pathways of DNA ICL repair become activated, leading to the formation of gross structural chromosome aberrations, breakage, translocations, dicentrics, and acentric fragments, and the development of hematological abnormalities and leukemia (Garaycoechea et al. 2018). An essential feature of the disease is sensitivity to aldehydes, which cause various DNA lesions, including ICLs and DNA protein cross-links. Humans are exposed to endogenous and exogenous sources of ICL-inducing agents, and most of this damage is successfully repaired by the FA/BRCA pathway. Reactive aldehydes, the byproducts of normal cellular metabolism, are important genotoxins neutralized by the FA/BRCA pathway. Therefore, the exposure of FA deficient cells to aldehydes results in the accumulation of chromosomal aberrations, including the stem and progenitor cell populations with aldehyde susceptibility. Aldehyde dehydrogenase enzymes are required for aldehyde detoxification, and polymorphisms may affect the phenotype of FA patients (Kim and D’Andrea 2012; Garaycoechea et al. 2012, 2018; Ceccaldi et al. 2016). Several mechanisms lie under hematological abnormalities of FA. p53 hyperactivation is an important mechanism responsible for BM failure. BM cells exposed to replicative stress during prenatal HSC expansion trigger an apoptotic p53/p21mediated response leading to a prenatally reduced fraction of CD34+ cells and a compromised HSC pool. In the postnatal period, DNA damage accumulation contributes to BM failure (Ceccaldi et al. 2012). In addition to p53, there is hyperactivation of the potent growth inhibitory TGFβ pathway (Zhang et al. 2016a). Compensatory overexpression of the MYC oncogene in a subset of HSCs may further increase their replicative stress (Rodríguez et al. 2021). Another mechanism of hematological defects is overexpression of cell cycle regulating ATM and CHK1 kinases in FA cells leading to cell cycle arrest in basal conditions (García-De-teresa et al. 2020). The risk of AML, MDS is significantly increased, necessitating periodic BM investigations. Clonal evolution in the BM occurs in 40% of children and young adults with FA (Bagby and Fleischman 2011). The presence of clonal and non-clonal chromosomal alterations both are valuable biomarkers for detecting progression to cancer.

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Recent studies have focused on identifying of the defects in the BM microenvironment of patients with FA that may contribute to hematological disturbances and malignancy. MSCs, giving rise to cells of stromal members including osteoblasts, adipocytes, and chondrocytes (Caplan 1991), are the essential cellular components of the BM microenvironment; hence, they have been investigated in single-cell, co-hepa studies or in advanced engineered systems to understand the BM niches in healthy and diseased states. Other investigators and we studied the characteristics of BM-MSCs in patients with FA. Although the standard phenotypic and functional characteristics showed similarities to healthy controls, several alterations were detected, including decreased CFU-F ability, proliferative capacity, early senescence, and spontaneous chromosomal fragility (Mantelli et al. 2015). Our studies also showed decreased proliferation, increased ROS levels, and an arrest in G2 upon mutagen stimulation, a lack of TGF-beta synthesis, and early senescence in FANCD2-deficient BM-MSCs (Cagnan et al. 2018). In addition, significant downregulation of the TALE class member PKNOX2 was detected in FA MSSCs. TGF-β1 stimulation increased PKNOX2 expression suggesting an association with disease pathophysiology (Cagnan et al. 2019). In another study, the functional defects in HSC differentiation were attributed to the effects of MSC-glycerolipids on TLR signaling (Amarachintha et al. 2015). The results of these studies suggest the presence of a dysregulated BM microenvironment in FA contributing to the hematological defects presenting as cytopenia, BM failure, and/or leukemia. These findings observed in FA BM-MSCs necessitates further investigations on whether the BM microenvironment has a primary or a significant role in the leukemogenesis process. Shwachman-Diamond syndrome Shwachman-Diamond syndrome is another inherited BM failure caused by mutations in the Sbds gene, which encodes a protein involved in ribosomal maturation. The disease is characterized by skeletal defects, pancreatic insufficiency, neutropenia, and a high probability of developing into MDS and AML. Experimental studies showing the leukemia-driver role of Sbds modifications in the stromal compartments of the BM have been significant achievements towards understanding the development of leukemia. Conditional deletion of the RNA-processing endonuclease enzyme dicer1 in primitive osterix-expressing osteolineage cells was shown to lead the development of MDS/AML in mice demonstrating the role of a nichedriven mechanism mice (Raaijmakers et al. 2010). The loss of dicer1 resulted decreased ribosome maturation factor Sbds expression in mesenchymal/ osteoprogenitor cells. In further experiments, niche alteration led to increased p53 levels and secretion of the pro-inflammatory factors (DAMP genes S100A8 and S100A9) that resulted in mitochondrial dysfunction in mouse and human HSC, and progenitors through binding to TLR-4. The authors suggested that activation of the p53-S100A8/9 axis in BM-MSCs has a predictive value in MDS progression (Zambetti et al. 2016). This study showed that increased inflammatory signals in the microenvironment caused genotoxic stress in experimental mouse model and SDS patients. The recent description of increased inflammatory findings in patients

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with SDS (Furutani et al. 2020) further emphasizes the necessity for close follow-up of patients with BM examinations. It brings out the issue of BM microenvironment evaluations to the clinics. However, this area is new, and clinical translation needs further investigations on this topic.

BM Microenvironment as a Contributor/Driver of Leukemia Investigation of human BM samples obtained after diagnosis of leukemia is not a suitable tool to study niche-driven mechanisms since microenvironmental changes that precede the diagnosis of the disease cannot be detected. Development of donorderived leukemia after allogeneic BM transplantation with a different clone than the patient’s one suggests that the BMME alterations induced by the effects of the conditioning regimen and the transplant process might have played an essential role in leukemic transformation (Wiseman 2011). In vivo experimental niche modifications towards leukemia induction have provided valuable data and provided evidence for a leukemia-driver role for the stroma. These investigations on knockout and conditional deletion models have shown the direct contribution of stromal defects to myeloid leukemia development pointed out the critical roles of different mechanisms involving retinoic acid receptor gamma, the RNA-processing endonuclease enzyme dicer1, the ribosome maturation factor Sbds, and protein tyrosine phosphatase non-receptor type11 and 21 (PTPN11, PTPN21) (Walkley et al. 2007; Garcia and Chen 2017; Alter 2017; Kokkaliaris and Scadden 2020). The stromal cell type plays a role in the leukemogenic process. It was shown that conditional deletion of primitive osterix (Osx)-expressing osteolineage cells, but not mature osteoblasts, led to MDS/AML in mice (Raaijmakers et al. 2010). Our investigations on human BM-MSCs, the precursors of osteolineage cells, supported these findings showing differential expression of dicer1 and differences in microRNA profiles among patients with MDS, AML, and healthy controls (Ozdogan et al. 2017). Moreover, activating mutations of PTPN11 in mice MSCs and osteoprogenitors lead to the development of a myeloproliferative, juvenile myelomonocytic leukemialike disease as observed in patients with Noonan syndrome carrying this mutation. Similarly, overexpression of PTPN21 in BM-MSCs of acute lymphoblastic leukemia (ALL) patients was associated with alterations in MSCs differentiation and immunomodulatory characteristics (Wang et al. 2019). PTPN21 was shown to have a role in regulating cytoskeleton-associated cellular processes (Carlucci et al. 2008), and overexpression in MSCs leads to an acceleration of leukemic and endothelial cell recruitment leukemic cell proliferation and drug resistance (Wang et al. 2019). However, when knocked-down on HSCs (Ptpn21-/-), the cell stiffness was lost, cells gained deformability and mobility (Ni et al. 2019). Another study also provides evidence for a highly active/initiator role for osteoblasts in the leukemogenic process. When a constitutively active form of b-catenin was expressed on osteoblasts, the differentiation program of HSCs was shifted towards myeloid lineage through upregulation of the Notch pathway leading to AML development (Garcia and Chen 2017).

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The majority of studies on BM niche interactions consist of studies on MSCs and osteoblastic lineage cells. The highly active roles of adipocytes and endothelial cells are better defined in recent studies. Marrow adipose tissue has an essential role in developing leukemia and in BM metastasis of solid tumors (Shafat et al. 2017; Kumar et al. 2020; Liu et al. 2020a). However, these studies describe the remodeling of adipocytes by malignant cells, which then significantly affect cancer cell behavior. Endothelial cell modifications are also implicated in leukemogenesis. It has been shown that loss of canonical Notch signaling in endothelial cells leads to constitutive activation of mir-155 and NF-kB signaling, increased levels of pro-inflammatory cytokines (GCSF, TNFα) resulting in expansion of immature myeloid cells and a myeloproliferative-like disease. Another example of endothelial cell involvement in the direct leukemogenesis process is which also deletion of signal-induced proliferation-associated gene 1 (Sipa1) in endothelial cells also resulted in myeloproliferative neoplasms (Fernandez et al. 2008; Wang et al. 2014; Xiao et al. 2018). Expansion of the myeloid compartment of the BM, clonal hematopoiesis, and chronic immune stimulation are risk factors for the development of MDS/AML, which are findings observed in aging hematopoiesis (Kristinsson et al. 2011; Yoshizato et al. 2015). Aging is associated with lymphoid suppression and myeloid skewing attributed to high inflammatory cytokine rantes (CCL5) in the BM microenvironment. In a heterochronic setting, it was shown that aged HSCs placed in a young environment generate fewer myeloid cells providing (Ergen et al. 2012) evidence for a critical role for environmental factors in establishing age-associated hematological defects, including malignancy. The spatial distribution of HSCs in aging BM niches has shown differences by localizing away from the endosteum, potentially hampering the ability of HSCs to remain quiescent and leading to a decreased pool of primitive HSCs (Ho et al. 2019). Considering the increased risk of myeloproliferative diseases, MDS, and AML in the elderly, the myeloproliferative, immunosuppressed, adipogenic, and chronic inflammatory state of the dysregulated BM microenvironment points out its essential role in malignant transformation. Exposure of the BM microenvironment to exogenous and endogenous stressors is associated with the induction of a pro-inflammatory state. Among these, GCSF will be addressed due to its clinical implications and frequent use. Patients with BM failure suffering from chronic cytopenia may be exposed to chronic use of GCSF. Spatial positioning in the BM niches is important in determining stem cell fate. GCSF has gross effects on the BM microenvironment, leading to increased cell trafficking towards the sinusoidal niche. It was shown that upon transmigration of HSCs from the endosteal to the sinusoidal niche, they proliferate and become sensitive to genotoxic stress induced by irradiation or myeloablation. GCSF removes HSCs from the endosteal niche where they are protected from toxic insults, thus increasing their susceptibility towards genotoxicity. Therefore, chronic administration of GCSF in congenital neutropenias has been a concern raising the risk of MDS/AML, particularly in patients with SDS with and in those with Ras or GCSF receptor mutations necessitating special scheduling and dosing recommendations for patients on long-term use of GCSF (Freedman and Alter 2002). Alternatively, GCSF is mainly used in some chemotherapy protocols as an adjunct treatment strategy to

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induce mobilization of leukemic cells from their quiescent niches (Lapidot et al. 2007) and render them vulnerable to cytotoxic or pro-apoptotic chemotherapy (de la Rubia et al. 2002). Spatial positioning of HSCs and leukemic stem cells is critical in disease pathophysiology. In addition to inducing HSC mobilization, SNS signals are essential for the regeneration of hematopoiesis following genotoxic stress (Lucas et al. 2013). Exposure to neurotoxic agents such as chemotherapy or irradiation was shown to damage the BMME leading to sympathetic neuropathy associated with MSC and endothelial cell proliferation, further sensitization towards genotoxic insults leading to a reduced niche size and failure to support hematopoietic recovery. Studies revealed that AML disrupts the SNS nerves and the quiescence of Nestin+ niche cells. Interestingly, SNS neuropathy is also involved in the promoting leukemic BM infiltration (Hanoun et al. 2014). In the leukemic stroma, adrenergic signaling maintaining niche quiescence was shown to be transduced by the β3 adrenergic receptor as opposed to β2 receptors in the healthy niche regulating osteoblasts. These findings demonstrate the important role of sympathetic neuropathy in niche dysregulation. GCSF use is also associated with involvement of both β2 and β3 adrenergic receptors, pointing out the contributory role of GCSF in the development of a dysregulated BM microenvironment (Takeda et al. 2002; Elefteriou et al. 2005; Méndez-Ferrer et al. 2010a; Arranz et al. 2014; Hanoun et al. 2014; Man et al. 2021). Among several molecules implicated in BM microenvironment cell trafficking and spatial location, the transcription factor Twist1 has been shown to play a newly described role that may have implications in AML pathophysiology. Its deficiency induces cycling and mobilization of HSCs by inhibition of retention factors, stimulation of GCSF expression on stromal cells leading to myeloid proliferation (Niu and Cancelas 2018). Another issue regarding the role of BM microenvironment in leukemia is the development of drug resistance executed through several mechanisms, including the establishment of multidrug resistance (MDR) phenotype by increased expression of ABC transporters, MSC release of soluble factors, ECVs, the establishment of tunneling nanotubes and mitochondria transfer (Griessinger et al. 2017). The BM-MSCs are highly involved in regulating immune interactions, and MSCmediated immune modulation/suppression could contribute to tumor progression and drug resistance within the BM niche. Additionally, MSCs may give nutritional support to leukemic cells by synthesizing enzymes (e.g., asparagine synthetase) to provide macromolecules, such as asparagine, conferring protection to leukemic cells from chemotherapy toxicity (Iwamoto et al. 2007). Leukemic lymphoblasts are very sensitive to the depletion of exogenous asparagine and glutamine because of their low capacity to produce their asparagine supply, and the chemotherapeutic agent asparaginase depletes these amino acids. But MSCs, by increasing synthesis, lead to chemotherapy resistance. It has been shown that there is a complex network of metabolic interactions involving malignant cells and their neighbors in the tumor microenvironment and cancer cells could induce stromal cells to produce metabolites and nutrients to support their metabolism, among which glutamine is essential in sustaining the metabolism of proliferating cells and regulating redox homeostasis

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in leukemic cells in the hypoxic environment. It was demonstrated that AML blasts utilize glutamine as an alternative carbon source for energy production and are highly dependent on glutamine for proliferation and survival (Kokkaliaris and Scadden 2020).

Metabolic Dysregulation and the Role of Adipocytes in the Malignant Niche Metabolic factors and nutrient status are increasingly being recognized in cancer pathophysiology, pointing out the critical roles of dietary manipulation. Cancer cells consume high glucose levels, and the majority of them prefer glycolysis even in the presence of oxygen. On the other hand, the Krebs cycle utilizes substrates such as glutamine and fatty acids to generate intermediates for biosynthetic pathways and counteract oxidative stress. The metabolic stress sensor, checkpoint kinase AMP-activated protein kinase is implicated in protecting leukemia-initiating cells from oxidative stress and promoting leukemogenesis. Interestingly, leukemogenesis was profoundly suppressed when associated with dietary restriction (Saito et al. 2015). Studies have shown downregulation of nutrient-signaling pathways by dietary restriction of calories or macronutrients. The main pathway affected is the insulin/insulin-like growth factor 1 (IGF-1) system and its effectors, ERK, MAPK, and PI3K, known to modulate cell survival and proliferation pathways (Lu and Ashraf 2012; Longo and Mattson 2014; Klement and Fink 2016). These studies have led to consideration of dietary restriction and/or the use of insulin-lowering drugs to support cancer therapy. Fasting leads to significant alterations in lipid metabolism. Lipolysis rates are precisely regulated through hormonal and biochemical signals. In acute conditions, catecholamines bind to β adrenergic receptors on adipocytes and induce triacylglycerol hydrolysis, releasing fatty acids and glycerol that act as oxidative substrates to maintain energy requirements for other metabolic tissues. Chronic exposure to extreme nutritional states, such as obesity or starvation, also induces metabolic adaptations that include changes in lipolysis. Lipolytic products of adipocytes act as signaling molecules regulating metabolic processes in many non-adipose tissues (Yang and Mottillo 2020). Therefore, in addition to its roles in type 2 diabetes, fatty liver disease, and obesity, adipocyte lipolysis is a therapeutic target in malignancy (Munir et al. 2019; Koundouros and Poulogiannis 2020). Adipocytes have been implicated in favor of cancer cell survival, proliferation, and metastasis in solid tumors, such as breast, prostate, and ovarian cancers. Upon close interaction with malignant cells, these cancer-associated adipocytes undergo alterations and transport lipids to neighboring cancer cells to support tumor growth. Specialized transporters, fatty acid-binding protein (FABP4), fatty acid translocase (FAT/CD36), and fatty acid transport protein (FATP) have been identified as key proteins involved in this mechanism (Dirat et al. 2011). Cancer cells need ATP and macromolecules for proliferation and survival, among which fatty acids play a critical role in membrane biogenesis, energy production, and protein modification.

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Thus, lipolysis is an important metabolic process needed for cancer cell survival. Additionally, cancer-associated adipocytes, by releasing adipokines including leptin, adiponectin, IL6, CCL2, and CCL5, were shown to induce proliferation, angiogenesis, dissemination, invasion, and metastasis of breast cancer cell (Zhao et al. 2020). Cancer microenvironment is usually hypoxic with nutrient deficiency. To thrive under these changing and challenging conditions, cancer cells adapt their metabolism. Upon exogenous uptake of fatty acids from surrounding environment, cancer cells can perform de novo lipogenesis through which carbon atoms derived from carbohydrates such as glucose and amino acids including glutamine are converted into fatty acids. In normal tissues, de novo lipogenesis is restricted to hepatocytes and adipocytes; however, cancer cells may also reactivate this anabolic pathway. Thus, lipid metabolism remodeling is a metabolic hallmark of cancer cells and consists of alterations in fatty acid transport, de novo lipogenesis, storage as lipid droplets and β-oxidation for ATP generation (Koundouros and Poulogiannis 2020).

Marrow Adipose Tissue and Leukemia The solid tumor studies demonstrating the involvement of adipocytes in cancer pathophysiology in tissues rich in adipocytes, such as breast, prostate, and ovarian cancers, have reported the role of BM adipose tissue (MAT) in leukemia. The BM is another fat-rich tissue consisting of a unique type of fat (Fig. 3). The MAT has been a topic of interest in recent years due to its critical role in bone and BM metastasis of solid tumors, and the pathophysiology of hematological malignancies. It is also involved in metabolism regarding obesity, insulin resistance, metabolic syndrome, and cardiovascular defects. MAT is regarded as neither a white nor a brown adipose tissue carrying properties of both, thus suggesting it as a beige adipose tissue, or the fourth type of adipose tissue possessing some characteristics of each depending on the environmental signals. Despite increasing number of studies in MAT, there are many issues to be resolved. For example, there is a lack of standardized criteria for the definition, isolation, and characterization of MAT. Given the clinical implications and the uncertainty in definition and protocols involved therein, the International Bone Marrow Adiposity Society founded a working group to evaluate methodologies in BM adiposity research. Following their annual meeting in 2017, an article about reporting guidelines, review of methodological standards and challenges towards harmonization in BM adiposity research was published in 2020 (Tratwal et al. 2020). These developments point out the need for more studies on the BM microenvironment to understand the role of MAT in the pathogenesis of hematological disorders and pave the way for targeted therapies. In humans, in some studies, BM adipocytes were isolated by enzymatic digestion of specimens obtained during hip replacement surgery (Attané et al. 2020), whereas in others, in vitro differentiated adipocytes from MSCs were regarded as MAT. On the other hand, the fatty tissue of the BM obtained from the femur/tibia was used for isolation of marrow adipocytes in in vivo experimental models (Fazeli et al. 2013;

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Fig. 3 Summary of the close interactions between the marrow adipose tissue and leukemic cells. This results in metabolic remodeling in the leukemic cells leading to leukemic blasts (Created with BioRender.com).

Horowitz et al. 2017). Due to the heterogeneity in isolation methods and lack of standardization, it is difficult to establish characteristics of the MAT. The BM comprises the red and yellow marrow, the latter representing the fatty marrow in the long bones that increases with age and makes up 70% of the mass within all BM cavities in adulthood. The remaining hematopoietically active red marrow is highly vascularized (Scheller et al. 2015; Boroumand et al. 2020). Two subpopulations of MAT have been described: constitutive (cMAT) or the regulated marrow adipose tissue (rMAT). Distal marrow sites are filled with cMAT. It is a homogenous and stable marrow space in which adipocytes interact with other adipocytes, are larger in size, do not respond to environmental stressors, are relatively resistant to lipid loss and remodeling, and store monounsaturated fatty acids, i.e., myristoleic and palmitoleic acids (Sahebekhtiari and Tavassoli 1976). On the other hand, rMAT is present in the proximal, central, and endosteal skeletal sites that develop postnatally, are readily altered by environmental stimuli, maintain multicellular contact, and accumulate saturated fatty acids, i.e., myristic acid and palmitic acid. These adipocytes are smaller in size and interspersed among hematopoietic and other cell lineages in the marrow. They interact with various cell types (Craft et al. 2018) and affect cell fates. rMAT adipocytes are remodeled in response to endogenous and exogenous stimuli, and may show expansion in obesity

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(Scheller et al. 2015). It is suggested that (Horowitz et al. 2017) cMAT and rMAT represent different populations of MAT arising from diverse population of progenitors. BM adipocytes are morphologically similar to white adipocytes as they contain a single fat globule instead of multilocular, mitochondria-rich brown adipocytes. They also express brown adipocyte markers (uncoupling protein 1-UCP1), peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α-PGC-1α, PR domaincontaining protein 16-Prdm16, Forkhead box protein C2 (FoxC2), and β3-adrenergic receptor at low levels, and may show characteristics of beige adipocytes depending on the microenvironment needs. Another unique metabolic feature of MAT is the cholesterol-oriented metabolism (Attané et al. 2020) and altered lipolysis associated with a profound downregulation in the expression of monoacylglycerol lipase leading to monoacylglycerol accumulation. Although BM adipocytes show enrichment in proteins involved in cholesterol metabolism correlating with increased free cholesterol content, proteins involved in lipolysis were downregulated in both basal and induced conditions. It was shown that in obesity, through activation of PPARγ, rMAT undergoes hyperplasia, hypertrophy, and whitening (Scheller et al. 2019). Considering the crosstalk between adipocytes and marrow cells, it may be suggested that whitening of rMAT may affect hematopoiesis. There are conflicting reports in the literature about the role of BM adipocytes on hematopoiesis. The association of increased adiposity in patients with BM failure and experimental studies point out the negative role of MAT (Naveiras et al. 2009). In contrast, other studies suggest a hematopoiesis-supportive role (Mattiucci et al. 2018). It was shown that MAT could stimulate MSCs through LepR to skew their differentiation in favor of adipogenesis through a positive feedback mechanism, thus may affect hematopoiesis (Yue et al. 2016). These results demonstrate the highly active interaction between adipocytes and BM cellular elements in healthy and diseased states. As demonstrated in solid tumors, MAT also plays a role in cancer pathophysiology and is involved in BM metastasis of solid tumors (Liu et al. 2020a). It was shown that especially red marrow is a common site of metastasis. Excessive blood flow in red marrow, adipocyte synthesis of adipokines, chemoattractants, adhesion molecules, angiogenesis, and immune modulation contribute to establishing a pre-metastatic niche and facilitating BM metastasis (Paolillo and Schinelli 2019). The mechanisms involved include homing through E-selectin, SDF1/CXCR4 axis, activation of the PI3K/PTEN/AKT/mTOR signaling pathway, and establishing a microenvironment appropriate for dormant cancer cells. Micrometastases reside in specific BM niches that regulate their transit to and from the bone. These data suggest that MAT plays an important role in BM micrometastases of solid tumors, and the BM microenvironment can maintain tumor dormancy for extended periods (Price et al. 2016). Studies demonstrating the role of adipocytes favoring the survival of neighboring cancer cells and the contribution of MAT in BM metastasis of solid tumors (Dirat et al. 2011; Price et al. 2016) pointed out a critical role for MAT in hematological malignancies, which is being enlightened in recent investigations. As observed in

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solid tumors, free fatty acid supply to leukemic cells has aroused as a relevant mechanism providing support for cancer cell survival. It was shown that BM adipocytes were programmed by AML blasts towards the establishment of a protumoral microenvironment directing intracellular adipocyte metabolism into a lipolytic state, resulting in the release of fatty acids into the microenvironment to provide metabolic support to leukemic cells. Leukemic blasts program BM adipocytes to generate a protumoral microenvironment, and FABP4 is implicated as a fatty acid transporter protein providing lipolysis products to leukemic blasts (Shafat et al. 2017). It was shown that AML blasts induced phosphorylation of hormone-sensitive lipase to activate lipolysis and transfer of fatty acids from adipocytes to AML blasts. In co-culture of adipocytes and AML, FABP4 messenger RNA was upregulated in both; β-oxidation of AML blasts was activated, and inhibition of FABP4 prevented AML proliferation on adipocytes. In vivo experiments further showed increased survival upon knockdown of FABP4 and carnitine palmitoyltransferase, which is essential for ATP production from FA oxidation. A recent study has shown that BM-MSCs of patients with AML patients demonstrate adipogenic differentiation propensity with implications for leukemia cell support (Azadniv et al. 2020). Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs in which SOX9 expression was decreased. Their experiments showed that increasing the expression of SOX9 reduced the adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. Moreover, other studies demonstrated exosome-mediated remodeling of the leukemic niche and induction of lipolysis to support leukemic growth (Kumar et al. 2020). These studies have described significant alterations and remodeling of MAT upon exposure to malignant cells in an interactive microenvironment, pointing out cancer cell-driven alterations in the microenvironment and adipocytes. There is a need for further studies investigating the mechanisms of adipocyte-driven leukemia. In that regard, exposure of adipocytes to environmental stressors emerges as an interesting topic that may lead to identifying mechanisms suggesting a primary/critical role in cancer initiation.

Environmental Stressors and Adipose Tissue Toxic lipophilic substances are widespread in the environment. Many are resistant to degradation and persist in the environment and living organisms for long periods, particularly in adipose tissue due to the lipophilic nature of these compounds and many of their metabolites. Other organs also retain some of the materials, but the primary storage site for the most lipophilic substances is adipose tissue. There are studies in the literature about environmental toxic agents and adipocytes, but not specifically on BM adipocytes. Adipose tissue is an important protective organ against environmental agents, such as persistent organic pollutants (POPs). Typical examples of POPs include chlorinated compounds such as organochlorine pesticides, polychlorinated biphenyls, and dioxins. The primary source of external

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exposure to these chemicals is POP-contaminated food, especially fatty animal products such as fish, meat, and milk. It has been shown that once POPs enter the body, they are distributed through the lymph and blood to their primary deposition site, which is the adipocyte lipid droplets in adipose tissue. It is stated that compared with other critical organs, being a natural location for lipid storage, adipose tissue is a relatively safe organ for POP accumulation, decreasing the burden on other vital organs before elimination over several years (Lee et al. 2017). In addition to strong lipophilic POPs with long half-lives, less lipophilic chemicals with brief half-lives, such as polycyclic aromatic hydrocarbons (PAH), were also detected in adipose tissue. Therefore, adipose tissue is suggested as an organ storing various exogenous chemicals that are not easily metabolized and excreted from the body. In an experimental study, redistribution of hexachlorobenzene from adipose tissue to critical organs such as the brain and kidneys was reported upon weight loss, reversed after weight gain. These results provide evidence for the storage role of adipocytes for environmental toxic agents (Jandacek et al. 2005). However, the effects of this storage on adipocytes on cancer initiation are not clearly defined, both in the peripheral adipose tissue or MAT. It may be speculated that POP accumulation in adipocytes, even at low doses, upon chronic exposure may lead to establishing a dysregulated microenvironment, adipocyte inflammation, and play a cancer-initiating role. Epidemiological and experimental evidence has linked low-dose POP exposure to obesity-related metabolic dysfunctions such as type 2 diabetes and metabolic syndrome, suggesting dysregulation of adipocyte metabolism (Lee et al. 2014). However, there is a lack of data about POP accumulation in MAT and its possible effects on hematopoiesis and the development of leukemia. This issue may have clinical implications, particularly in diseases with increased susceptibility to mutagenic agents such as Fanconi anemia.

Conclusions Upon realizing the critical roles of the microenvironment and stem cell niches in maintaining homeostasis and the pathophysiology of diseases, many investigations have suggested tumor microenvironment as a novel target to treat malignancies. Being the main reservoir of stem and progenitor cells, and given its critical role in regeneration, the BM is frequently affected in pathological conditions. Most studies on MSCs were designed to elucidate the changes in phenotypic, molecular, secretory, functional characteristics in different diseases, including hematological malignancy. Leukemic celldriven alterations in MSCs were shown to contribute to leukemic progression. There is a lack of information for a leukemia-driver role for the BM microenvironment except for several recent reports describing the development of leukemia in experimental models, in which the induced defects in the MSCs and other microenvironmental components led to a dysregulated environment and malignant transformation. It has been shown that BM adipocytes, upon interaction with leukemic blasts, undergo alterations and provide free fatty acids to leukemic cells for their survival. However, MAT-driven leukemia is not described. Considering the role of adipose

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tissue as a depot for environmental pollutants, it may be speculated to have implications as a leukemia driver through the acquisition of epigenetic alterations upon chronic exposure. There is a need for studies focusing on BM adipocytes and leukemia pathogenesis. Fanconi anemia, a disease with DNA repair defect and increased susceptibility to toxic, mutagenic agents, appears to be an appropriate model disease for MAT investigations. Studies in other hematological pathologies and conditions, such as GCSF exposure, will contribute to understanding the role of MAT in disease pathogenesis. However, at first, there is a need for a better description of the MAT and standardization of methods for isolation and characteristics. Many different methods are being used in in vivo and in vitro experimental studies to identify the BM niche, ranging from studies on cells obtained by different isolation techniques to those using combinatorial approaches with next-generation technologies enabling simultaneous analysis of many BM subpopulations. In light of these studies, there is a need for more studies on human BM samples in relevant diseases and conditions for timely clinical translation of the accumulated scientific knowledge. This may contribute to the identification of therapeutic targets of the BM microenvironment, including the MAT.

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Renata Szydlak

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Therapeutic Potential of Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of Mesenchymal Stem Cells in Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSC-Based Therapies in Graft Versus Host Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cell-Based Therapies in Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cell-Based Therapies in Cardiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells-Based Therapies in Orthopedics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSC-Based Therapies in Neurology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential Risks of MSC-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

In recent years, stem cell-based therapy is being widely and intensively investigated. Nowadays modern treatment strategies with mesenchymal stem cells (MSCs) in translational medicine are met with great enthusiasm by scientists and clinicians. The extraordinary properties of MSCs that are better known and understood mean that new possibilities of their application are constantly being tested. Due to their ability to self-regenerate, secrete biologically active molecules and exosomes, differentiate into several cell types, and participate in immunomodulation, MSCs have become a promising tool in the development of modern treatment strategies. The readily available and enormous potential of MSCs allows for a variety of clinical applications in the treatment of many diseases that have hitherto been called “incurable.” Most of the results of administering MSCs in clinical trials confirmed the safety and showed promising beneficial R. Szydlak (*) Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_23

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results. The therapeutic effects of MSC-based treatments are still not spectacular, and many features of MSCs have not yet been thoroughly investigated, so MSCs continue to be the source of controversial opinion and much debate about these cells. In this chapter, we focus on summarizing the current state of knowledge about the complex nature of MSCs that can be applied to regenerative medicine. Keywords

iPSCs · Mesenchymal stem cells · MSCs-based therapy · Pluripotent · Stem cells · Transplantation · Regenerative medicine List of Abbreviations

ALS AT-MSCs BDNF BM-MSCs CD CFU-F CMV EMA EMT ESCs EVs GvHD HLA HO HSCs HSCT IPSCs ISCT MNC MPCs MSCs NGF OA OI SSCs WJ

Amyotrophic lateral sclerosis Adipose tissue-derived mesenchymal stem cells Brain-derived neurotrophic factor Bone marrow-derived mesenchymal stem cells Crohn’s disease Colony-forming unit–fibroblast Cytomegalovirus European Medicines Agency Epithelial-mesenchymal transition Embryonic stem cells Extracellular vesicles Graft versus host disease Human leukocyte antigens Heterotopic ossification Hematopoietic stem cells Hematopoietic stem cell transplantation Induced pluripotent stem cells International Society for Cellular Therapy Mononuclear cells Mesenchymal progenitor cells Mesenchymal stem cells Nerve growth factor Osteoarthritis Osteogenesis imperfecta Somatic stem cells Wharton’s jelly

Introduction Cell therapy is a modern therapeutic approach based on cells as therapeutic agents (Gálvez et al. 2011; Ciccocioppo et al. 2021). In regenerative medicine, examining and correctly determining the type of cell to be used in a particular treatment is

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essential to the success of therapy. Research to date suggests that stem cells can be used in regenerative medicine due to their unique features of self-renewal as well as differentiation (cell plasticity) into specialized cells with specific functions (Ratajczak and Suszyńska 2013). For this reason, their safety and the ability to repair, replace, or restore the biological function of damaged tissues and organs should be defined (Ciccocioppo et al. 2021). Currently, mesenchymal stem cells (MSCs) constitute the well-characterized and most used cell type in the clinical trials. Despite the low proliferative potential and limited plasticity compared to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), MSCs are easier to obtain from diverse tissues, and their manipulation is free from any ethical moral issues; they have a high in vitro expansion capacity and a low teratogenicity (Sancricca 2010; Trounson and McDonald 2015). All of these properties are in addition to their ability to produce cytokines, growth factors, and microvesicles loaded with bioactive molecules as part of their paracrine activity, migrate and home-in to the site of tissue damage to participate in the repair process, and exert an immunomodulatory effects (Haider and Ashraf 2005). Given these properties, the research and development of MSCs as a drug can help provide new therapeutic alternatives for of high potential in regenerative medicine and cell therapy for diseases that so far do not have effective conventional treatments (Nauta and Fibbe 2007; Gálvez et al. 2013). In this chapter, the advantages, disadvantages, and side effects of MSC-based therapies have been discussed. In particular, the therapeutic benefits of exogenously delivered MSCs have been discussed, focusing graft versus host disease (GvHD), Crohn’s disease, cardiovascular diseases, and orthopedic and neurological disorders.

Mesenchymal Stem Cells Friedenstein’s discovery of the presence of non-hematopoietic stem cells in the bone marrow of animals in the 1970s changed the current outlook on somatic stem cells (SSCs) (Friedenstein et al. 1970; Friedenstein et al. 1976). Friedenstein identified and reported a population of spindle-shaped cells similar to fibroblasts but with colony forming potential. He named these cells as colony-forming unit–fibroblast (CFU-F). Later, these cells were named MSCs or mesenchymal stromal cells (Horwitz et al. 2005). They constitute a heterogeneous group of cells, primitive, and with multilineage potential. The best-known source of MSCs is bone marrow (Wexler et al. 2003; Dominici et al. 2006); however, cells with similar morphology and characteristics can also be isolated from other tissues, including umbilical cord blood, Wharton jelly, placenta, peripheral blood, tissue adipose tissue, and skin (da Silva 2006). In all these organs and tissues, they are housed in specific niches, which are now being studied by high-throughput screening (Ghaemi et al. 2013). They are an integral part of the hematopoietic stem cells (HSCs) niche, where they offer both physical and chemical support to HSCs by secreting bioactive molecules (Crippa and Bernardo 2018).

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During in vitro culture, MSCs adhere to the plastic substrate, show high proliferative potential, and the ability to differentiate into cells of mesodermal origin, especially adipocytes, chondrocytes, and osteoblasts. To overcome the controversy regarding the functional potential of MSCs as well as the nomenclature, in 2006, the International Society for Cellular Therapy (ISCT) attempted to define the essential / basic criteria for their identification. In the resulting classification, MSCs must, in addition to adherence to the substrate and trilineage differentiation potential, express surface antigens considered specific for this population, i.e., CD105 (endoglin), CD73, CD90, but lack the expression of hematopoietic cell-specific antigens, i.e., CD34, CD45, CD14 besides the absence of CD11b, CD79a, CD19, and HLA class II antigens expression (Dominici et al. 2006). As our knowledge progresses, and with emerging controversy regarding their functionality, some researchers have started to believe that the criteria set by ISCT based on current literature data should be revisited (Lv et al. 2014). The high similarity of MSCs with fibroblasts in appearance and the lack of harmony in the expression of surface markers specific only for MSCs has led to doubts and difficulties in identifying “true MSCs” (Halfon et al. 2011; Kundrotas 2012; Lupatov et al. 2015). Hence, some researchers suggest that the minimum criteria proposed by ISCT for identifying MSCs are insufficient because MSCs isolated from various tissues represent a relatively heterogeneous population of cells for the expression of surface markers, the ability to proliferate and differentiate (Hass et al. 2011; Pevsner-Fischer et al. 2011; Maleki et al. 2014). Therefore, the ISCT paper issued in 2019 recommends using the acronym “MSC” but supplemented with the tissue source origin of cells, which would highlight tissuespecific properties of the cells being used (Viswanathan et al. 2019). These suggested criteria will help in interpreting the data and the difference in the properties of the cells from diverging tissue sources. As described earlier, MSCs constitute a heterogeneous population of cells that differ in their proliferative potential and differentiation capacity depending on their tissue location. Bone marrow-derived MSCs (BM-MSCs) can differentiate more efficiently into bone and cartilage as compared to their counterparts derived from the adipose tissue (AT-MSCs) (Im Il et al. 2005; Afizah et al. 2007; Pevsner-Fischer et al. 2011). Similarly, a head-to-head comparison revealed that BM-MSCs were superior in chondrogenic potential than umbilical cord blood-derived MSCs (Contentin et al. 2020). Hence, it is now generally believed that for applications in regenerative medicine, MSCs should be sourced depending upon the treatment outcome. It has now also been shown that in the MSCs population, only a fraction of cells meet the “parental” criteria, while the remaining cells may fulfill a helper function or are the cells capable of differentiation only in one direction (Siegel et al. 2013). Besides, MSCs may also contain a pool of much less advanced and immature cells that express pluripotent transcription factors such as Oct-4, Sox2, and Nanog, and are similar to ESCs (Kuroda et al. 2010; Ogura et al. 2014; Musiał-Wysocka et al. 2019). Reports on the pluripotent properties of the MSCs have not been fully explained. Similarly, a population of small juvenile cells is present in the bone marrow stromal cells with therapeutic potential (Okada et al. 2011).

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Mesenchymal Stem Cells Sources The data published during the last decade have revealed the possibility of obtaining a variety of stem cells from fetal and adult tissue sources, respectively grouped as fetal and adult stem cells, also known as somatic stem cells. The best known and identified source of stem cells by far is the bone marrow. It is inhabited HSCs fraction and MSCs. For 105 mononuclear cells total, it has been shown that there are approximately 1–4 MSCs (Pittenger et al. 1999). In one of the published studies, flow cytometric analysis of BM cells revealed as little as 0.0017 to 0.02% CD271+ CD45- MSCs which adhered to plastic surface and could undergo trilineage difference when challenged with appropriate cues (Alvarez-Viejo et al. 2013). The highest number of MSCs are found in the bone marrow of newborns, which significantly declines with age (D’Ippolito et al. 1999; Stolzing et al. 2008). BM harvesting protocols are invasive and inconvenient for the donor, and require anesthesia. Therefore, alternative sources of MSCs are being explored for use in cellbased therapy in regenerative medicine. In recent years, adipose tissue is a fairly popular source of MSCs (AT-MSCs). The prevalence of MSCs in adipose tissue is much greater than in the bone marrow, and 1 g of adipose tissue contains 500 times more MSCs than 1 g of BM (Fraser et al. 2006; Kitagawa et al. 2006). Adipose tissue-MSCs have a high proliferation rate and multilineage differentiation capacity within the mesodermal germ layer derivative cells, which renders them a favorable source for cell-based therapy from the clinical perspective. However, characterization of AT-MSCs reveals that the proliferation and differentiation potential of AT-MSCs was significantly influenced by the cell donor, his age, and BMI (Yang et al. 2014). Based on recent reports, the frequency of MSCs and limitations resulting from the amount of material available for collection, adipose tissue is considered a better source of therapeutic cells than BM (Fujimura et al. 2009). Tissues remaining after delivery, the postpartum tissues (umbilical cord blood, umbilical cord, placenta, membranes, and amniotic fluid) provide excellent primitive cells source. Obtaining tissue material from these sources for stem cell isolation is simple and does not require complex surgical procedures. Compared to MSCs derived from adult BM or AT, MSCs isolated from perinatal tissues are more primitive (Moretti et al. 2010; Lindenmair et al. 2012). Studies have shown that MSCs from postpartum tissues can differentiate into cells from all three germ layers, which indicates their greater differentiation potential, and therefore pluripotency and primitiveness (Guillot et al. 2007). However, obtaining cells from postpartum tissues is a time-constrained exercise and requires immediate isolation of cells or freezing tissues in biobanks. The procedure of obtaining MSCs from frozen UCB or umbilical cord deposits is still ineffective due to the lack of an optical protocol. Moreover, it is believed that UCB is a relatively unfavorable source for obtaining MSCs, in contrast to the rich fraction of other mononuclear cells (MNCs). Wharton’s jelly (WJ) – umbilical cord tissue is also a valuable source of MSCs. The WJ-MSCs collection procedure is technically simpler and a rich population of post-fetal MSCs can be efficiently isolated (Batsali et al. 2013; Nagamura-Inoue 2014). The

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Fig. 1 Summary of the possible tissue sources for mesenchymal stem cells

published data show that Wharton’s jelly contains much higher propensity of MSCs compared to the UCB (Zeddou et al. 2010; Pelosi et al. 2012). The possible tissue sources of MSCs have been summarized in Fig. 1.

The Therapeutic Potential of Mesenchymal Stem Cells Despite numerous in vitro and in vivo studies, as well as therapeutic benefits confirmed in clinical trials, the exact mechanism of therapeutic benefits of MSCs is still not fully understood. Based on the results obtained in preclinical and clinical studies, it can be concluded that MSCs certainly has a unique ability to immunomodulate, regenerate, and heal the damaged tissues. The available experimental data make it possible to dissect and understand some of the mechanisms responsible for the therapeutic potential of MSCs. The therapeutic efficacy of

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exogenously injected MSCs may result from both cell-cell interactions and the secretion of biologically active molecules. Hence, the therapeutic effect can be obtained exploiting the three main properties of MSCs: differentiation into several types of tissues, the immunomodulatory effect, and the ability to influence the intrinsic repair process through the paracrine secretion of appropriate cytokines and direct contact with other cells. MSCs act as a local coordinator of tissue repair in most cases. Their advantage over the use of other mediators (cytokines) is the cross talk between MSCs and other cells and tissue regeneration mediators. This close interaction MSCs with their local environment enables them to adapt well to the changing situation, e.g., inhibition of the inflammatory reaction in the first phase of regeneration and the production of stimulators for cell proliferation and differentiation in the next phase.

Application of Mesenchymal Stem Cells in Medicine The number of clinical trials using MSCs in regenerative medicine (ClinicalTrials.gov) is growing rapidly. There are currently ten approved MSCsbased therapies available for a variety of disorders in the pharmaceutical market worldwide (Table 1). The experimental in vitro data and the promising results from preclinical studies and translational studies show that cell-based therapy with MSCs therapeutic benefits for patients with various diseases. However, despite receiving encouraging data from these studies over the past decade or more, many questions related to the biology of MSCs and hence, their usefulness as choice cells remain open for discussion and further investigation. For example, there remain some uncertainties between the immunophenotype of MSCs with relevance to its functionality and the procedural hiccups, including posttransplant survival, route of administration, and type of transplant (autologous or allogeneic). Moreover, it also remains less wellexplored about the properties of cells, such as the potential for in vitro transdifferentiation, persist after transplantation.

MSC-Based Therapies in Graft Versus Host Disease Graft versus host disease (GvHD) accompanies allogeneic hematopoietic stem cell transplantation (HSCT) in many patients. Corticosteroids are used to treat GvHD; however, this therapy is ineffective in all patients (Martin et al. 2012). The immunomodulatory properties of MSCs described in experimental studies suggest their use in the treatment of GvHD (Mohanty et al. 2020). Many studies have proven that MSCs can modulate the function of the immune system and have found their use as companion cells in transplantation in the treatment of GvHD (Weng et al. 2010; Zhao et al. 2015). Indeed, MSCs transplantation and their derived exosomes have recently been performed to prevent or treat GvHD, especially in patients who do not respond to steroids (Le Blanc and Mougiakakos 2012; Elgaz et al. 2019;

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Table 1 MSCs-derived products with regulatory approval

MSC product (Company) Queencell (Anterogen Co. Ltd.)

Cellgram-AMI (Pharmicell Co. Ltd.)

Cupistem (Anterogen Co. Ltd.) Cartistem (Medipost Co. Ltd.) Prochymal, remestemcel-L (OsirisTherapeutics Inc., Mesoblast Ltd.) Neuronata-R (Corestem Inc.)

Temcell HS (JCR Pharmaceuticals) Stempeucel (Stempeutics Research PVT) Alofisel (TiGenix NV/Takeda)

Stemirac (Nipro Corp)

MSC tissuesource Autologous Human AT-MSCs Autologous Human BM-MSC Autologous Human BM-MSC Allogeneic Human UC-MSC Allogeneic Human BM-MSC Autologous Human BM-MSC Allogeneic Human BM-MSC Allogeneic Human BM-MSC Allogeneic Human AT-MSC Autologous Human BM-MSC

Indication Subcutaneous tissue defects

Approval granted (year) South Korea (2010)

Acute myocardial infarction

South Korea (2011)

Crohn’s fistula

South Korea (2012)

Knee articular cartilage defects

South Korea (2012)

GvHD

Amyotrophic lateral sclerosis

New Zealand (2012) Canada (2012) South Korea (2014)

GvHD

Japan (2015)

Critical limb ischemia

India (2016)

Complex perianal fistulas in Crohn’s disease Spinal cord injury

Europe (2018) Japan (2018)

Zhang et al. 2017). However, despite the use of MSCs in many clinical trials, there is still controversy about the benefits of such therapy. Although a reduction in inflammatory processes is observed after MSCs implantation, a reduction in the immune response may increase the risk of infection, especially in patients receiving immunosuppressive therapy after HSCT (Nauta and Fibbe 2007). It has been reported that infusion of MSCs may dangerously limit the antimicrobial immune response (Balan et al. 2014). A clinical trial published by Ning et al. showed that the incidence of acute and chronic GvHD in MSCs-transplanted recipients was lower than in the non-MSCs transplanted patients, but the episodes of severe infections were more significant in patients who received bone marrow-derived HSCT and MSCs than in the control group who did not receive the MSCs treatment. Among patients, two developed CMV interstitial pneumonia and/or fungal infection (Ning et al. 2008).

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Forslöw et al. suggest an increased susceptibility to pneumonia observed in patients with GvHD after MSCs infusion (Forslöw et al. 2012). High-peak CMV viral load was found in a retrospective study of patients with steroid-resistant GvHD receiving MSCs (von Bahr et al. 2012a, b). This contradicts the previous in vitro experiments, which showed that anti-CMV cytotoxic T cells were limited to the BM-MSCs effect (Karlsson et al. 2008). Recently, Thanunchai et al. postulated that in viral infections, human BM-MSCs might also act as viral transmitters (von Bahr et al. 2012a, b). There is a suggestion that MSCs may lose their immunosuppressive properties in mismatched settings, which has been shown in murine cells (Badillo et al. 2008). Moreover, the study by Muroi et al. showed that the transplanted BM-MSCs in the acute phase II/III GvHD study did not protect the development of chronic GvHD (Muroi et al. 2016). Based on the above studies, it should be emphasized that MSCs transplantation for the prevention or treatment of GvHD is relatively safe and effective in steroidresistant GvHD, but infections remain a major risk for patients. Moreover, it has been shown that MSCs transplanted due to established GvHD may cause increased relapse (Ning et al. 2008). In a recent study by Ringden et al., the authors mentioned several side effects following transplantation of residual placenta-derived MSCs in the treatment of GvHD. Among them, relapse, pneumonia, bacterial, viral, and fungal infection, and transplant failures have been listed (Ringden et al. 2018). A new strategy to support a high frequency of MSCs effects on GvHD with a little adverse effect on the patient appears to be warranted in large-scale randomized trials. Research by various laboratories focuses on developing new MSCs-based drugs. One of the first MSCs-based drugs approved for the treatment of GvHD was Prochymal.

Mesenchymal Stem Cell-Based Therapies in Crohn’s Disease Crohn’s disease (CD) is classified as a chronic inflammatory disease that mainly affects the gastrointestinal tract. In CD, the formation of an anal fistula is difficult to treat and is associated with a large number of complications, including the risk of bowel resection. Moreover, the perianal fistulas arising in the course of CD are difficult to treat with standard drugs and surgical procedures (Veauthier and Hornecker 2018). Despite significant advances in the techniques used, treatment of patients with CD remains a difficult task with a high risk of relapse (Gisbert et al. 2015). Recently, satisfactory therapeutic benefits have been obtained by administering MSCs. However, the indications for the use of MSC-based therapy in CD concern mainly perianal fistulas (Zhang et al. 2017). Based on the available results from preclinical and clinical studies, it can be assumed that the therapeutic mechanism of MSCs in this disease is mainly based on the immunomodulatory effect. MSCs have been shown to inhibit T cell proliferation mediated by indoleamine 2,3-dioxygenase (Wang et al. 2018). Thanks to their enormous regenerative potential, MSCs are currently used in the treatment of fistulas in Crohn’s disease and other etiologies. Data collected from the analysis of various

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clinical cases show that complete healing of the fistula can be achieved after several local administrations of MSCs either alone or in combination with infliximab and azathioprine (Forbes et al. 2014; Moniuszko et al. 2018). The first EMA-approved MSC-based drug for the treatment of complex perianal fistulas in CD was Alofisel. Recent advancement in this regard is the use of acellular products of MSCs based on the use of extracellular vesicles derived from MSCs. A direct comparison of cellbased and cell-free approaches based on MSCs and their derivative extracellular vesicles has been provided by Li et al. in an experimental mice model of DSS-induced colitis (Li et al. 2020) and reviewed by Ocansey et al. (2020).

Mesenchymal Stem Cell-Based Therapies in Cardiology Cardiovascular diseases are one of the most common causes of death worldwide (Virani et al. 2020). The currently used method of treating a “fresh” heart attack consists of administering a thrombus-dissolving drug as soon as possible and performing a cardiological intervention in the form of opening the lumen of a closed arterial vessel, which is aimed at limiting damage and then necrosis of the heart tissues (Peng et al. 2016). As the heart exhibits limited endogenous regenerative capacity, although the long-standing dogma about the heart has been challenged due to the presence of resident cardiac stem cells (Takamiya et al. 2011; Lu et al. 2013; Belostotskaya et al. 2015), cell-based therapeutic approach is currently the subject of much preclinical as well as clinical research. However, a significant focus of cell-based therapy is on MSCs as choice cells, due to their superior biological and functional characteristics, to demonstrate the effectiveness of using MSCs, either naïve or preconditioned or genetically modified (Changfa et al. 2017), to reduce postinfarction scars and restore normal contractile function in the infarcted heart (Haider et al. 2008; Kim et al. 2009; Haider et al. 2010; Ahmed et al. 2010; Afzal et al. 2010; Suzuki et al. 2010). Despite encouraging data, however, the exact mechanism by which MSCs contribute to myocardial regeneration is still not fully understood (Lpez et al. 2013; Zhao et al. 2015). Although MSCs show great potential for pro-chondrogenic, osteogenic, and adipogenic differentiation, several studies have provided evidence that under optimal in vitro culture conditions or in the cardiac microenvironment in vivo post engraftment, MSCs can also give rise to other highly specialized tissue types, including cardiomyocytes and endothelial cells (Shim et al. 2004; Aguilera et al. 2014; Haider et al. 2008) and lead to stable therapeutic benefits (Jiang et al. 2008). Given these findings, MSCs have been extensively tested as a source of cells to replace damaged myocardial tissue in vivo, in both acute and chronic cardiac injury, confirming the ability to transdifferentiate MSCs into cardiac and endothelial cells (Haider 2006; Dawn et al. 2009). However, the ability of MSCs to differentiate into functional endothelial and cardiac cells in vivo has not been fully established. Moreover, it is difficult to confirm the transplanted integrated cells in any tissue in vivo due to the lack of specific MSCs markers (Lin et al. 2013). However, the increase in recent evidence from many laboratories strongly indicates the

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overwhelming paracrine effect in MSCs after heart transplant to promote cell survival, proliferation, and differentiation by MSCs resulting from secreted bioactive factors and extracellular vesicles (EVs) (Nazari-Shafti et al. 2020; Lei and Haider 2017). Thus, these two main mechanisms, including (1) direct differentiation of MSCs into cardiac and endothelial cells and (2) paracrine activity mediated by soluble molecules and MSC-derived vesicles, are now considered to be the primary mediators of the beneficial effects of MSC-based therapies (Majka et al. 2017; Szydlak 2019; Nazari-Shafti et al. 2020). Most of the beneficial effects after MSCs injection are believed to be related to their paracrine effects on endogenous cells, resulting in increased vasculogenesis and angiogenesis, as well as increased cell survival (Haider and Aziz 2017). The role of the paracrine activity of MSCs is considered so dominating that cell-free therapy approach using MSCs conditioned medium rich in soluble factors (i.e., growth factors, cytokines) and insoluble factors (i.e., exosomes) is emerging as an alternative to cell-based therapy (Haider and Aramini 2020; Haider and Aslam 2018). Given their robust nature, MSCs have also been reprogrammed to iPSCs to achieve a continuous source of cardiac progenitor cells for use in cardiac repair in an experimental animal model (Buccini et al. 2012). Although MSCs, from adult tissue sources, represent one of the safest stem cell populations, with almost no risk of the endogenous teratogenic potential of normal pluripotent stem cells such as ESCs and iPSCs, in vivo application of MSCs to heart tissues could still potentially lead to some undesired effects post engraftment (Price et al. 2006; Breitbach et al. 2007). The few reported safety concerns for MSCs are related to their possible (1) pro-arrhythmic and (2) carcinogenic capacity in heart tissue, as well as (3) differentiation into undesirable tissue types (Price et al. 2006; Breitbach et al. 2007). Price et al. report that BM-MSCs administered intravenously to pigs with acute ischemia/reperfusion injury improves cardiac parameters and reduces adverse wall thickening but may also adversely affect the electrophysiological properties of the myocardium, suggesting the pro-arrhythmic potential of these cells. However, the beneficial effects of injected MSCs on cardiac function and anatomy observed in this study were greater than the recorded arrhythmic events, and ultimately the authors concluded the efficacy of MSCs in the heart repair model, however, with a note of caution (Price et al. 2006). On the other hand, numerous clinical studies in patients suffering from acute or chronic ischemic heart disease have shown very little or no adverse effect of MSCs on the electrical properties of the myocardium after transplantation, as summarized in several reviews and metaanalysis reports (Afzal et al. 2015). Consequently, reported events of pro-arrhythmic MSCs activity are somewhat rare and pharmacologically treatable; however, this should be considered and assessed, especially during clinical trials, as a potential risk identified in some animal studies (Menasché 2009). Some of the unresolved problems in clinical trials in cardiology, besides the source and quality of the cell preparation used during the trials (Shahid et al. 2016; Haider 2018), may also be partly related to an insufficient number of placebo groups. However, compared to other stem and progenitor cells used to repair the heart, including skeletal muscle myoblasts, widely studied in the early 2000s, MSCs can be considered cells with a limited risk of arrhythmia in the heart tissue (Haider et al. 2004; Kahn 2006).

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In summary, the positive therapeutic results of exogenously injected MSCs are probably due to several mechanisms of their action, namely the ability of MSCs to differentiate into cardiomyocytes, smooth muscle cells, vascular endothelial cells, and the ability of MSCs to secrete multiple cytokines and trophic factors including the insoluble factors, i.e., exosomes (Majka et al. 2017; Szydlak 2019). Due to their immunosuppressive properties, MSCs may also help to alleviate inflammation and stimulate endogenous repair mechanisms.

Mesenchymal Stem Cells-Based Therapies in Orthopedics Previous studies have shown a beneficial effect of MSCs primarily in the treatment of the osteoarticular system. Numerous studies with various animal models of orthopedic disease have documented the multipotential properties of MSCs, showing their ability to differentiate in multiple tissues such as muscle, bone, cartilage, and tendons (Kingery et al. 2019). Their use as an adjunct to orthopedic surgery is also being explored to ensure rapid wound healing (Murrell et al. 2015). However, contrary to the initial assumption that the therapeutic benefits of MSCs depend on their cell replacement capacity via transdifferentiation, recent studies have shown that the paracrine function of MSCs remains the primary mechanism by which they participate in the tissue repair post engraftment (Von Bahr et al. 2012a, b). MSCs have been reported to exhibit immunosuppressive and immunomodulatory properties by secreting specific factors that may modulate inflammatory responses following orthopedic trauma (Marcucio et al. 2015). However, the common mechanism of action of MSCs in orthopedic applications has not been fully established (Berebichez-Fridman et al. 2017). Many reports have summarized the role of MSCs in the treatment of osteoarthritis (OA). Several experiments in animal models of knee OA have shown that MSCsbased therapy may delay progressive degeneration of the joint (Shimomura et al. 2018; White et al. 2018). Most human studies support the notion that short-term use of MSCs is safe and feasible; however, further experimentation is necessitated. Importantly, we still need clear evidence to support the effectiveness of MSCs transplantation in OA patients (Jihwan et al. 2021). In randomized controlled clinical trials, injection of MSCs used to treat knee OA is effective (Lamo-Espinosa et al. 2016; Park et al. 2016). However, the results reported by Shim et al. and Pas et al. disclosed that after MSCs injections for the treatment of knee OA, only a few cells survived at the injection site (Shim et al. 2015; Pas et al. 2017). Moreover, the optimal therapeutic dose of cells, co-adjuvants, and uptake source has not yet been optimized (LamoEspinosa et al. 2016). The use of MSCs in cartilage repair has a significant placeborelated limitation because the tissue sampling procedure makes it difficult to perform a blind design study (Filardo et al. 2016). Therefore, new studies using MSCs for orthopedic patients must be performed with greater care and under controlled ex vivo preparation conditions to assess their therapeutic efficacy in these patients ultimately.

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Currently, there are attempts to research the use of MSCs in osteogenesis imperfecta (OI), a genetically determined disease associated with the production of an abnormal form of type I collagen (Horwitz et al. 2002). It has been shown that MSCs transplantation has a beneficial effect on the reduction of skeletal damage. In a clinical trial, prenatal transplantation of MSCs in 31-week-old fetuses with ultrasound confirmed diagnosis of osteogenesis imperfecta showed significant improvement in patients’ condition. MSCs transplantation in patients lowered the incidence of fractures and skeletal abnormalities (Götherström et al. 2014). Two patients underwent adjuvant transplantation to enhance the therapeutic effect, and at the age of 18 months after birth and 8 years of age, MSCs were re-transplanted. The effects of prenatal transplants in combination with postnatal transplantation resulted in clinical benefits. However, studies of a larger group of patients are needed to evaluate the effectiveness of the proposed therapy fully. Numerous studies suggest that apart from the potential effect of MSCs on tissue regeneration, these cells may also be significantly involved in the process of heterotopic ossification (HO), i.e., ectopic bone formation in tissues other than bone (Kan et al. 2017). Besides, stem cell-based therapies in orthopedic trauma have identified MSCs contributing to the high osteogenic differentiation (Agarwal et al. 2016). Also, the inflammatory response may stimulate the differentiation of mesenchymal progenitor cells (MPCs) into osteoblasts and osteoblast-like cells. If this process is localized in muscles or other soft tissues, it may directly contribute to the formation of HO (Winkler et al. 2015). It has also been reported that MSCs may be responsible for the recurrence of HO (after surgical resection). In turn, excision of HO may result in the re-emergence of the MSCs population and the signaling mechanisms observed in the original lesion (Agarwal et al. 2017).

MSC-Based Therapies in Neurology The concept of the clinical application of MSCs seems to be of great hope in the treatment of neurological diseases, both those of a neurodegenerative nature and damage to the central nervous system resulting from stroke or trauma. It seems that MSCs can be used in the direct regeneration of the cellular structure of the nervous system not only because of their immunomodulatory and neurotrophic functions but also due to their potential differentiation abilities and reparability. The neuroprotective and neuro-regenerative properties of MSCs transplantation can be associated with the production of numerous growth, anti-inflammatory, and antiapoptotic factors important for neurons. The observations of some researchers show that MSCs, due to their abovementioned functions, may be responsible for the protection of newly formed neurons, their proliferation, and maturation. Hence, co-transplantation of neuronal stem cells and MSCs has been used to promote neuronal stem cell survival, proliferation, and differentiation post engraft in experimental animal model of spinal injury (Hosseini et al. 2018). It has been reported that transplanted human MSCs in an experimental stroke or trauma model in animals can significantly improve motor functions

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(Gornicka-Pawlak et al. 2011; Sarnowska et al. 2013). The observed effect was associated with the anti-apoptotic action and production of factors by MSCs that stimulate the survival of neurons (Anbari et al. 2014; Gu et al. 2014; Yin et al. 2014). One of the major factors for successful cellular therapy of stroke is the route of cell delivery to the damaged part of the brain. For this reason, the circulatory system is considered correct. However, it is important to be aware of how the exogenous cells are administered to ensure the patient’s safety on the one hand and, on the other hand, to guarantee the maintenance of good quality therapeutic cells during the transplant procedure. Cui et al. disclosed that cell agglomeration before injection increased in proportion to the duration of the cells kept in suspension (Cui et al. 2016). Moreover, due to their size, MSCs can induce severe vascular occlusions after intravascular delivery. The size of MSCs in an in vitro monolayer culture increases with the number of passages; the solution can create a 3D nodular culture in vitro that will reduce MSCs again (Ge et al. 2014). Failure of a positive result after systemic MSCs administration in stroke was confirmed in another experimental study where the intravenous injection of human BM-MSCs in a mouse model of stroke contributed little to enhancing cell proliferation in neurogenic areas. Moreover, neither a detectable reduction in infarct size nor favorable clinical symptoms were observed (Steiner et al. 2012). Moreover, MSCs delivered intraarterially in a mouse model of ischemia did not improve functional recovery and may further promote the risk of brain damage (Argibay et al. 2017). These multifocal changes contributed to a significant decrease in cerebral blood flow as a result of small vessel obstruction by exogenous cardiovascular cells, while posing a deep risk of secondary embolism in the brain following stroke. A recent advancement in the treatment of stroke is the use of cell-free therapy approach in which MSCsderived secretome was injected intracerebroventricularly in an experimental animal model of ischemic stroke (Taei et al. 2021). Another example of a neurological disease in which MSCs-based therapies raise high hopes is amyotrophic lateral sclerosis (ALS). Phase I study results showed no significant positive effects of exogenous MSCs embedded in the spinal cord. The main conclusion from most clinical trials of ALS therapy with MSCs was limited to that of the safety of the treatment. In the study by Syková et al. in which BM-MSCs were transplanted intrathecal, the favorable outcome was seen only in a few patients and was limited to a short time after transplantation (Syková et al. 2017). This outcome may be due to the short survival time of the cells after implantation or the differentiation status of the transplanted MSCs. Repeated administration of cell doses can be crucial to achieving better prognosis; however, this may be challenging in terms of the manufacturing process. Another phase I study with an intrathecal autologous injection of BM-MSCs revealed mild adverse reactions immediately overcoming exogenous cell deposits, such as fever, pain, and headache; however, there were no major treatment effects (Oh et al. 2015). Staff and colleagues performed intrathecal injection of MSCs in the adipose tissue during ALS treatment. The authors did not observe any spectacular improvement in the treated patients and postulated a reduction in enthusiasm for the effectiveness of the therapy (Staffe et al. 2016). Moreover, MSCs transplanted intramuscularly and intrathecal, aspirated from the bone marrow, are

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safe and stimulate the release of neurotrophic factors; however, this approach contributed to some disease regression in only half of the patients over the next 6-month period (Petrou et al. 2016). Nevertheless, previous study by Karussis and colleagues in which BM-derived MSCs were injected intravenously and intrathecal did not bring any positive effects in ALS patients (Karussis et al. 2010). In the treatment of neurological disorders, neurotrophins may play a particularly important role. Neurotrophins play a key role in the differentiation and survival of neurons in the central nervous system and are also involved in synaptic plasticity underpinning learning and memory. The main source of neurotrophic factors are nerve cells, but recently it has been believed that they can also be produced by other cells, including MSCs (Sadan et al. 2012; Paczkowska et al. 2013) (Paczkowska et al. 2013; Sadan et al. 2012). Neurotrophins, in particular brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3, and neurotrophin 4/5, may have a neuroprotective function in multiple sclerosis, slowing the rate of atrophic changes and affecting the functional network of neural connections with improved cognitive functions in the patients. Evidence of the neuroprotective potential of neurotrophins has so far been obtained primarily in the studies conducted in experimental animal models (Lykissas et al. 2007; Gordon 2009). More and more reports suggest that neurotrophins secreted by MSCs may prove useful in many other neurodegenerative diseases: Parkinson’s disease, Alzheimer’s disease, in the treatment of patients after stroke and spinal cord injuries (Malgieri et al. 2010. Machaliński et al. 2012, Paczkowska et al. 2015).

Potential Risks of MSC-Based Therapies MSCs-based cell therapy represents a new promising approach in treating many diseases. However, data on the risks and possible long-term side effects of their use still lack despite their positive results. There is also a lack of data, including analyzes of long-term studies in the context of possible threats resulting from the possibility of neoplastic transformation. Although there are no reports to date about teratoma formation after MSCs transplantation, the long-term risk from cell therapy remains less well-studied and underreported in the published data. There are, however, some conflicting reports of spontaneous transformation of MSCs under in vitro culture conditions. Various effects were observed depending on the species used and the source of MSCs the cultivation techniques used, and the in vitro expansion time (Miura et al. 2006; Bernardo et al. 2007; Tang et al. 2013). The risk of spontaneous transformation of MSCs due to prolonged culture has been demonstrated in a study describing the population of cells from the bone marrow and blood (Tang et al. 2013). Bernardo and colleagues have shown that MSCs retained the correct phenotype and morphological structure during in vitro expansion in the optimal culture conditions, besides maintaining normal cell function over a more extended period. During 44 weeks of cultivation, no changes were observed in cell karyotype (Bernardo et al. 2007).

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Another problem that arises from MSCs transplantation, apart from direct transformation, may be the risk of stimulation of an already existing neoplastic growth by MSCs. Due to their migratory capacity, transplanted MSCs move to the site of neoplastic growth, stimulate tumor cell proliferation, promote angiogenesis, and support tumor metastasis. In this case, a prior precise diagnosis and selection of patients indicated for cell therapy are necessary. The results showed that the immunosuppressive environment created by MSCs also promoted tumor metastasis in the mechanism of the so-called epithelial-mesenchymal transition (EMT), facilitating cell migration (Ljujic et al. 2013). Studies by other authors have shown that the co-culture of cancer cells with MSCs accelerates tumor expansion (Xu-ting et al. 2009; Zimmerlin et al. 2011). Given the incredible enthusiasm for stem cell-based therapy, care should be taken to consider all the possible adverse effects.

Conclusions The more recent scientific analysis has shown limited therapeutic effects of treating MSCs, suggesting that the direct regenerative potential of these cells related to their ability to differentiate may not be as effective as previously expected. Several exogenous factors may significantly influence the biological properties of MSCs and ultimately on their therapeutic capacity, optimized protocols for MSCs isolation and ex vivo preparation for clinical use must be well-established and standardized. Such a comprehensive effort should be taken into account by the scientific community focusing on the practical applications of MSCs in tissue repair in terms of the optimal preparation of MSC-based products for more effective patient therapies. The advantages associated with the use of MSCs in tissue repair, i.e., their safety, relatively broad differentiation capacity, and high paracrine capacity, render these cells an important therapeutic option for further exploration and development as a novel cell-based therapy approach in the future. However, more in-depth and mechanistic research is needed at the preclinical and clinical levels. New research data on MSCs will help determine the effectiveness of cells administered to patients as part of a therapeutic approach. Additional research would also make a significant contribution to the overall biology of stem cells. Acknowledgments This work was supported by a research grant (STRATEGMED2/265761/10/ NCBR/2015) from the National Center for Research and Development in Poland.

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The Stem Cell Continuum Model and Implications in Cancer

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Theo Borgovan, Ari Pelcovitz, Rani Chudasama, Tom Ollila, and Peter Queseneberry

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Stem Cell Continuum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicle Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicle Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicle Pleiotropy and Regenerative Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles Affect Normal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles Affect Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An Introduction to Extracellular Vesicles and Hematological Malignancies . . . . . . . . . . . . . . . . . Extracellular Vesicles in Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles in Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An Introduction to Extracellular Vesicles and Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of TEVs in Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of TEVs in Breast Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of TEVs for Diagnostic and Predictive Markers in Breast Cancer . . . . . . . . . . . . . . . . . . . An Introduction to EVs and Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Vesicles in Primary CNS Lymphomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of EVs in Diagnosis and as a Biomarker in Primary CNS Lymphoma . . . . . . . . . Extracellular Vesicles’ Function on Lymphomagenesis and Lymphoma Treatment Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EVs’ Effects on Lymphoma Patient Outcomes and Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role or Target in Therapy for Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lymphoma-Specific EV Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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T. Borgovan (*) · A. Pelcovitz · R. Chudasama · T. Ollila · P. Queseneberry Division of Hematology and Oncology, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_24

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Abstract

The hematopoietic stem cell is constantly fluctuating through various intrinsic phenotypic potentials. It has dynamic plasticity and is intimately tied to cell cycle dynamics as well as numerous external stimuli. The salient studies conclude the stem cell exists in a continuum of transcriptional opportunity regulated by multiple variables, including biologically active nanoparticles termed extracellular vesicles (EVs). These bioactive mediators closely impact cellular function and phenotypic potential and play a critical role in the regulation of normal hemostasis, as well as in the development and evolution of various cancers. In this chapter, we explore the integral data explaining the stem cell continuum, the interplay between this model and EVs from various cell types, as well as the role of EVs in various solid and hematologic cancers. Finally, we evaluate the role of EVs as unique and reliable biomarkers across disease states ranging from traumatic brain injury to malignancy. Keywords

Biomarkers · Cancer · CSCs · Extracellular vesicles · Stem cells · Therapeutics Abbreviations

ALL BCR BM CAFs CD CLL CML ESCRT EVs FIH GPC1 HIF HUVEC hWJMSC IL LT-HSC MBC MHC miRNA MM MRP-1 MSCs MVB

Acute lymphoblastic leukemia B-cell receptor Bone marrow Cancer-associated fibroblasts Cluster of differentiation Chronic lymphocytic leukemia Chronic myeloid leukemia Endosomal sorting complex required for transport Extracellular vesicles Factor inhibiting Glypican-1 Hypoxia-inducible factor Human umbilical vein endothelial cells Human Wharton’s jelly mesenchymal stem cells Interleukin Long-term hematopoietic stem cell Metastatic breast cancer Major histocompatibility complex MicroRNA Multiple myeloma Multidrug resistance protein 1 Bone marrow mesenchymal stem cells Multivesicular bodies

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SDF1 TBI TD-MSCs TF

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Stromal cell-derived factor 1 Traumatic brain injury Palatine tonsil-derived MSCs Tissue factor

Introduction The Stem Cell Continuum Bone marrow hematopoietic stem cells (HSCs) have been exhaustively studied. Present dogma is that there is a dormant noncycling lineage negative (lin-) c-kit+Sca-1+CD150+ cell in the marrow, which represents this stem cell (Colvin et al. 2010, 2007). This cell is reported to have extensive differentiation potential, self-renews, and responds to microenvironmental cues. The system is hierarchical, with the stem cells giving rise to a diverse population of progenitor cells, which then differentiate into end hematopoietic cells. Our published data indicate that the bone marrow-derived pluripotent HSCs constitute a population of actively cycling cells which are characterized by continuously changing phenotypes (Colvin et al. 2007). This cell population exists as lin þ cells, discarded during conventional stem cell purification (Goldberg et al. 2014). Thus, a cell that is transiting cell cycle is continuously altering its nature. This is consistent with multiple reports of heterogeneity of bone marrow HSCs. Perhaps the most impressive observation is that heterogeneity is observed even with cyclesynchronized stem cells (Colvin et al. 2010). Critical observations have been reported that the long-term repopulating marrow stem cells alter hematopoietic differentiation potential as it transits the cell cycle (Colvin et al. 2007). In these experiments, highly purified lin-rhodamine low Hoechst low murine stem cells showed differentiation predilections as they transit a cytokine-induced cell cycle. Another critical component of the universal stem cell model is multiple observations of marrow “stem cells” forming non-hematopoietic cells after in vivo engraftment into lethally irradiated mice (Lagasse et al. 2000; Nilsson et al. 1999; Goldberg et al. 2014; Theise et al. 2000; Abedi et al. 2003; Badiavas et al. 2003). Krause and colleagues have demonstrated that single marrow cells can give rise to a wide variety of non-hematopoietic epithelial cells. And more recently, they have identified these cells as a very small embryonic-like stem cell (Krause et al. 2001; Kassmer et al. 2013). Put together, the observations on cell cycle status of long-term repopulation marrow stem cells, their differentiation potential including their capacity to differentiate into non-hematopoietic end cells, and their intrinsic heterogeneity suggest a new model of stem cell biology. We propose that there are no individual stem cells for different tissues; rather, a universal stem cell population exists that functions for most and possibly all tissues in the body. The universal stem cell is a cycling cell and thus is continuously changing phenotype. This includes its differentiation potential that is realized when the universal stem cell resides in the conducive environment. As shown in Fig. 1, the same universal stem cell at one point during the cell cycle in

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LT-HSC

G0

Progenitor #1

G1

Early S

LT-HSC

Mid S

Progenitor #2

Late S

M

LT-HSC

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Fig. 1 The cell cycle is a continuum. During hematopoiesis the long-term hematopoietic stem cell (LT-HSC, colored in dark purple) changes phenotype as it transits through cell cycle and fluctuates between different phenotypes (colored in blue, brown, and light purple) and functional potential. (Used with the permission of Chibuikem Nwizu, [email protected])

hepatic tissue will give rise to hepatic cells, while if it is in lung tissue environment, it will give rise to epithelial lung cells. Likewise, it will give rise to hematopoietic cells in the marrow, but the type of end cell differs depending upon cell cycle status; thus, it could differentiate into megakaryocytes, granulocytes, erythrocytes, or other end hematopoietic cells. This model can fit nicely into much of our current data on extracellular vesicle cellular modulation.

Extracellular Vesicle Basics At first glance, extracellular vesicles (EVs) seem simple, nano-sized, lipid membrane-enclosed particles that are reasonably ubiquitous and are released from essentially all cell types in the mammalian body. Their function unknown at the time, at first discovery they were merely dismissed as cellular waste products, shuttling cellular junk from predominantly red blood cells and platelets. Today, with the recent progress in the field of EVs, researchers have started to appreciate their complexity and robust biological potential. Scientific efforts have only just begun to architect framework taxonomy, unraveling their heterogeneity and pluripotency. Subsequent work has characterized and subdivided these entities on the basis of size, density, and morphology (Borgovan et al. 2019). Eventually, two basic types of vesicles were defined by differential ultracentrifugation purification: exosomes and microvesicles. Exosomes derived from multivesicular bodies range from 40 to 120 nm in diameter, while microvesicles derived as a result of membrane blebbing range from 50 to 1000 nm in diameter. Other vesicular entities were also defined, including apoptotic bodies as given in Table 1 and Fig. 2.

Extracellular Vesicle Functions The functional potential harnessed by EVs is immense. They allow for intercellular communication sans direct cell-to-cell contact, honing pertinent information to distinct cells in proximity to the effector cell, or cellular targets located in more distant vicinities.

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Table 1 Biological pathways of extracellular vesicles Vesicle type Exosomes

Microvesicles

Apoptotic bodies

Size (nm) 40–120

Origin Endocytic pathway

Content Proteins, lipids, and nucleic acids (mRNA, miRNA, and other noncoding RNAs)

50–1000

Plasma membrane blebbing Plasma membrane blebbing

Proteins, lipids, and nucleic acids (mRNA, miRNA, and other noncoding RNAs) Nuclear fractions, cell organelles

500–2000

Marker Alix, Tsg101, tetraspanins (CD81, CD63, CD9), flotillin Integrins, selectins, CD40 Annexin V, phosphatidylserine

Fig. 2 Extracellular vesicles are heterogeneous. The major populations include exosomes, microvesicles, and apoptotic bodies. Each subtype contains a unique surface architecture and carries distinct cargo. (Used with the permission of Chibuikem Nwizu, [email protected])

Being membrane-bound, they protect their rich cargo from degradation and allow for the shuttling and delivery of selectively packaged proteins, various types of RNA, bioactive lipids, and DNA (Borgovan et al. 2019). As highlighted in Fig. 3, once the vesicles reach a target cell, they impart various long-lasting downstream phenotypic and genotypic effects, which are likely mediated via intricate genetic and epigenetic variables.

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Fig. 3 Extracellular vesicles contain a large array of biologically active cargo. Once the vesicles reach the target cell, the released cargo imparts numerous downstream effects. (Used with the permission of Chibuikem Nwizu, [email protected])

Initial studies focused on EVs functional mechanisms have established the ability of vesicles to directly transfer protein into target cells, while modifying the phenotype of the recipient cells. The initial mediators of EVs’ function were primarily hypothesized to be protein and mRNA effectors. The biological effects of vesicles are inhibited after heat inactivation or pretreatment with RNase, suggesting the relevant involvement of protein and mRNA. However, the downstream functional and phenotypic effects observed in EV studies are persistent and long-lasting, spanning well beyond the degradation time expected for biologic proteins, thus suggesting other mechanisms at work. While most plasma miRNAs are bound to proteins, there is a smaller amount associated with extracellular vesicles. Further analysis has indicated that cellular phenotype alteration may be mediated by the transfer of transcriptional activators, possibly miRNA, and downstream modulation of epigenetic signals (Aliotta et al. 2015). Ratajczak et al. (2006) elegantly highlighted that embryonic stem cell-derived microvesicles successfully reprogrammed hematopoietic progenitors by horizontal transfer of their mRNA and protein payload (Borgovan et al. 2019). The analysis of EVs from embryonic stem cells has identified the expression of stem cell-specific molecules with established roles in stem cell proliferation and self-renewal.

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These vesicles also upregulated the expression of various early pluripotent HSCsspecific markers, including Oct-4, Nanog, Scl, HoxB4, and GATA 2. This mechanism of action was also supported by other research groups, highlighting RNA transfer and phenotypic change in different experimental models (Aliotta et al. 2006, 2007, 2015; Valadi et al. 2007). In these experiments, a genomic change could only occur once the vesicles had entered the target marrow cells allowing for the transfer of both mRNA and transcriptional regulators. As in other studies, the phenotypic effects were long-lived, well beyond the degradation of the transferred mRNA, once again suggesting that long-term expression of downstream proteins derived from the target cells (such as surfactants B and C, which were tested in these studies) represented a stable downstream epigenetic event (Aliotta et al. 2015).

Extracellular Vesicle Pleiotropy and Regenerative Potential EVs can be successfully isolated from virtually all bodily fluids and cells. EV populations harbor a functional endpoint specific to originator cell type and disease state. There are far-reaching implications in utilizing EVs toward clinical endpoints focused on disease identification, progression, and modulation. The recent focus has been on the capacity of EVs to restore injured tissue and treat disease (Wen et al. 2017). EVs packaging and cargo composition are constantly cycling, and it is in flux with the ever-changing homeostasis of the host’s cellular and physiological environment. As a result, EVs selectively package their cargo to address the functional endpoints at hand specifically. Moreover, as we will come to explore, EVs harness a potent regenerative ability in various disease states, including multiple types of solid and hematological malignancies, and can have direct protective and regenerative effects on perhaps the most important of cell regulators – the HSCs.

Extracellular Vesicles Affect Normal Stem Cells MSCs reside in the bone marrow and adipose tissue. They have multipotent differentiation capacity and, via direct co-transplantation, have been shown to support the HSCs directly by enhancing engraftment and improving bone marrow recovery from radiation in NOD/SCID mice. Wen et al. demonstrated that the functional mediators of these regenerative effects were attributed to the released EVs (Wen et al. 2016). Through elegant studies, his group successfully demonstrated the capacity of human MSCs-derived EVs to reverse radiation-induced damage to the bone marrow and gastrointestinal tissues of mice. EVs demonstrated a capacity to reverse radiation damage to the marrow and gastrointestinal tissues of mice, with the most potent effect on long-term engrafting stem cells (Wen et al. 2016). These results are in accordance with numerous other studies demonstrating how EVs can target and salvage damaged stem cells, promoting their differentiation, and proliferation, thereby reversing radiation-induced damage and apoptosis of the bone marrow cells.

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A common functional theme was once again observed in this interaction wherein mRNA was vital. Overexpression of multiple RNA populations involved in cell recovery, growth, radiation resistance, and promotion were observed, allowing for more efficient DNA repair and downregulation of apoptotic factors. Many of these effects were found to be closely integrated into the cell cycle status. In line with the stem cell continuum postulate described earlier in the chapter, Aliota et al. showed the functional effects of vesicles on marrow mRNA expression depended upon the cell cycle status of the target bone marrow cells and the condition of the originator lung cells, in this case either irradiated or not (Aliotta et al. 2012). The results showed that lineage depleted, Sca-1+ murine bone marrow cells showed peak pulmonary epithelial cell-specific mRNA expression in cell cycle phase G0/G1 when the vesicles were derived from irradiated lung tissue, while the peak was in the late G1/early S phase when the vesicles were derived from the nonirradiated lung.

Extracellular Vesicles Affect Cancer Stem Cells We have explored the evidence illustrating the bimodal interplay among stem cells and EVs. Unique populations of vesicles are released by healthy dividing stem cells (such as the MSCs described above), which in turn are received and employed by other stem cells, which may be stressed or damaged, toward a regenerative purpose (Wen et al. 2016). The vesicle “fingerprint” utilized by stem cells is subject to multiple variables as described in the studies above, including the tightly regulated and continuous phenotypic and genotypic flux the stem cell experiences as it moves through the cell cycle. Much of the reversible changes in short- and long-term engraftment, progenitor numbers, gene expression, and differentiation potential explored in the aforementioned studies are partly a direct result of EVs’ effects at various cytokine-induced cell cycle transits (Colvin et al. 2010, 2007; Goldberg et al. 2014). The regenerative potential of stem cells in which there is an injury-related conversion of bone marrow-derived stem cells toward different tissue cells in order to reconstitute damaged populations is, again, largely dependent on EV manipulation of the stem cell continuum during cell cycle. For instance, damaged irradiated lung tissue releases EVs which can transit to and enter the bone marrow stem cells, thereby modulating the stem cell continuum, driving stem cell differentiation, and leading to the bone marrow cell expression of lung-specific mRNA and protein (Aliotta et al. 2015). The fundamental postulates of the stem cell continuum theory and its applicability to healthy functional stem cells can be accurately extended to the biological mechanics that drive other “types” of stem cells. Cancer stem cells (CSCs) compose a relatively dormant subset of pluripotent cancer cells that have preserved the ability to differentiate, self-renew, and remain particularly resistant to chemo- and radiotherapeutic intervention (Hervieu et al. 2021). These cells have enhanced tumorigenicity and continuously adapt to the myriad of immunogenic and therapeutic threats

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they are taxed with, in order to maintain a pro-oncogenic microenvironment and ensure cancer growth (Margolin et al. 2015). Akin to their HSCs counterparts, CSCs are in a dynamic state of continuum, perpetually changing to respond adequately to its environment – this modulation is thought to be in part regulated by the effects of EV production and reprogramming. Kim et al. followed the expression of various CSCs-specific markers postulating that these aberrant CSCs were constantly reprogrammed during oncogenesis (Kim et al. 2013). Su et al. described these adaptations as a “continuum state,” showing that CSCs could transiently cycle through phenotypic (and likely epigenetic) states, with nearly 75% of their surface markers shared by adult or embryonic stem cells. Moreover, the remaining surface architectural composition showed significant variability and cycling, with the majority of these markers being found on differentiated normal tissue cells (Su et al. 2021). Mani et al. highlighted a similar link between EVs and the dedifferentiation process that a mature (or progenitor) cell undergoes when it acquires “tumor-initiating” properties (Mani et al. 2008). Both the research groups evidenced a critical role of EVs in reprogramming stem cell fate and modulating its microenvironment and stroma to harbor cancer growth. When following the expression of variable CSCs-specific markers, Wang et al. postulated that the observed phenotypic changes were linked to, and dependent on, EV manipulation (Wang et al. 2007). The effects were observed as bidirectional, with aberrant (cancer) stem cells promoting a microenvironment of the specific phenotypic state that contributed to the biogenesis, loading, and special packaging of EVs which promote CSCs propagation and further enhance oncogenesis.

An Introduction to Extracellular Vesicles and Hematological Malignancies EVs play an essential role in intracellular communication through active transcellular crosstalk and post-apoptotic residual messaging (Borgovan et al. 2019). The role of EVs in CSCs’ promotion, cancer pathogenesis, and their use in diagnosis, prognosis, and treatment remains an area of immense ongoing research. Herein, we will review the ongoing research surrounding EVs in hematologic malignancies with a special focus on the preclinical data exploring the role of the bone marrow tumor microenvironment and the utility of EVs in diagnostic and prognostic models.

Extracellular Vesicles in Leukemia The Leukemic Bone Marrow Microenvironment Leukemia is defined by the development of a clonal proliferation of malignant hematologic cells, with frequent bone marrow infiltration, as well as peripheral blood involvement. Recent research has expanded on this definition and identified

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some critical interactions between leukemic cells and the bone marrow microenvironment in cancer development and proliferation. Specifically, it is theorized that leukemic cells induce changes in the bone marrow microenvironment which are more favorable to leukemic cells’ growth and proliferation and less conducive for normal hematopoiesis (Duarte et al. 2018). EVs are thought to be one mechanism by which leukemic cells can induce microenvironmental changes (Kumar et al. 2018). Kumar and colleagues demonstrated that treatment of experimental mice with acute myeloid leukemia (AML)-derived exosomes successfully induced similar bone marrow changes in the recipient animals as were caused by typical AML cell engraftment in these mice populations. The exact mechanism by which leukemic EVs manipulate the bone marrow microenvironment remains less well-explored. In vitro studies with various AML cell lines and the plasma from mice-bearing AML xenografts have shown that the diseased animals released leukemic EVs laden with multiple stem cell-related microRNAs that target the downstream transcription factors involved in stem and progenitor cell growth and differentiation. Through the EV-directed epigenetic changes, AML can inhibit specific stem cell transcription factors with established roles in malignancy and other similar processes, necessary for the proper development of B-cell precursors (Borgovan et al. 2019). Peinado and colleagues demonstrated that melanoma-derived EVs would transfer the receptor tyrosine kinase MET, inducing its expression on bone marrow progenitor cells, which might create a more favorable metastatic environment (Peinado et al. 2012). Huan et al. identified similar bone marrow manipulation by AML cell lines. They demonstrated that AML cells transferred mRNA via EVs to the bone marrow stromal cells, thereby altering the biologic functions of these stromal and hematopoietic cells to create a leukemic microenvironment that would be more conducive for malignant growth (Huan et al. 2013). This intra-environmental EVs-based communication is not limited to AML. For example, Corrado et al. identified a similar paradigm in the growth and survival of chronic myelogenous leukemia (CML) cells (Corrado et al. 2014). Based on the published data, IL-8 plays a critical role in CML growth and survival, and treatment of the bone marrow-derived stromal cells with CML-derived EVs significantly increases the production of IL-8.

Predictors of Response and Promoters of Resistance in Leukemia While research is ongoing to identify further the role of EVs in the development and maintenance of bone marrow microenvironment homeostasis and as an emerging novel target for therapeutic intervention, preliminary data have demonstrated a potential role for leukemic cell-derived EVs in prognosis. Hong et al. measured the TGF-β1 level in EVs derived from patients with newly diagnosed AML and followed its level through the course of treatment (Hong et al. 2014). They found that reduction in the EV-derived TGF-β1 level was positively correlated with AML blast reduction during the treatment. While the TGF-β1 level was not predictive of relapse during consolidation, the level in patients with long-term remission (>2 years) was shown to be similar to control patients not diagnosed with AML.

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EVs play a critical role in predicting treatment response and are also explicitly involved in promoting drug resistance. Bouvy et al. showed the transfer of chemotherapy resistance between promyelocyte leukemic cell lines via direct cellular transfer of EVs (Bouvy et al. 2017). Chemosensitive cell lines treated with EVs derived from chemoresistant cell lines were shown to endocytose EVs into their intracellular compartments. The recipient cells subsequently showed increased resistance to daunorubicin compared to the untreated chemosensitive cells. A similar study was conducted by Bebawy et al. in which EVs from resistant cell lines, especially those with a high level of P-glycoprotein expression, were cultured with chemosensitive cells. The co-cultured chemosensitive cells subsequently not only expressed P-glycoprotein but also developed its functional mechanisms (Bebawy et al. 2009). Total plasma EVs isolated from leukemic patients may be exploited as a distinct biomarker. Machine learning-based algorithms have been employed to classify diverse leukemic EVs’ populations. Using these mathematical and statistical models to quantify and qualify various EV populations from a heterogeneous patient population at different stages of treatment has shown promising preliminary results toward the EVs use as theranostic biomarkers for both diagnoses and gauging therapeutic response (Borgovan et al. 2019).

Extracellular Vesicles in Lymphoma The Lymphoma Tumor Microenvironment The tumor microenvironment in lymphoma allows the growth and proliferation of malignant lymphocytes, a process facilitated by EVs-directed transcellular communication. Hansen et al. identified CD30 ligand expression in Hodgkin’s lymphoma-derived EVs, and these EVs were associated with the stimulation of IL-8 release, a pro-inflammatory cytokine implicated in the tumor growth (Hansen et al. 2014). They further demonstrated that CD30+ EVs formed a communication complex between the scattered malignant Reed-Sternberg cells and the surrounding immune meshwork. The group theorized that this might explain the therapeutic benefits of brentuximab vedotin, the antibody-drug conjugate directed against CD30, and that despite the meager propensity of CD30+ cells, they might be just a part of a more integrated EVs-mediated communication network supporting malignant cell growth and survival. On the same note, Dorsam et al. demonstrated how Hodgkin’s lymphoma-derived EVs communicated and manipulated the tumor microenvironment (Dörsam et al. 2018). They also documented the uptake of Hodgkin’s lymphoma-derived EVs by surrounding fibroblasts and significantly impacted fibroblasts’ migration. Uptake of EVs by the fibroblasts also resulted in a phenotypic change producing more cancerassociated fibroblasts. These findings again pointed to an elaborated network of communication and regulation within the tumor microenvironment in which EVs played a unique and central role.

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An Introduction to Extracellular Vesicles and Breast Cancer Breast cancer is one of the most common invasive cancers in women (Desantis et al. 2014). Despite advances in breast cancer therapy, the overall 5-year survival for patients with metastatic breast cancer (MBC) is only 28% (Desantis et al. 2014; Siegel et al. 2021). The clinical course and progression of breast cancer is a dynamic process and involves close interaction with the tumor microenvironment. There is a constant acquisition and regulation of signals, intercellular communication, as well as genetic and epigenetic alterations between cancer cells. Thus, the drivers that boost cancer evolution are essential in understanding and investigating disease progression and novel therapies. As discussed in the hematologic malignancies section, EVs are biomolecules released by cells to act as a carrier of various biomolecules to facilitate intracellular signaling and homeostasis between the communicating cells. Such cell-to-cell communication has been observed extensively in various solid tumor cell types, including breast cancer.

Role of TEVs in Breast Cancer Tumor-derived EVs (TEVs) can be retrieved from various bodily fluids to serve as potential biomarkers for theranostic applications (Kalluri 2016; Théry et al. 2002; De Toro et al. 2015; Van Niel et al. 2018). Studies have shown that TEVs play a critical role in metastasis, progression, and tumor initiation (Abak et al. 2018). TEVs produced from cancer cells have been shown to induce proliferation of the neighboring normal cells besides their ability to induce malignant transformation, proliferation, and oncogenic amplification (Jing et al. 2013). TEVs also help cancer cells evade immune surveillance and promote therapeutic resistance by regulating drug sensitivity via various modalities (Lowry et al. 2015). This extends beyond the standard chemotherapeutic resistance to immunotherapy. EVs have numerous suppressive effects across the population of immune cells that resides in the tumor microenvironment (Borgovan et al. 2019). Yu et al. have shown that breast cancer-derived TEVs could block the differentiation of myeloid precursor cells into dendritic cells. These data have opened an opportunity to use TEVs in cancer immunotherapy (Yu et al. 2007). As discussed elsewhere in this chapter, uptake of TEVs by the fibroblasts promotes their genetic and phenotypic transformation into cancer-associated fibroblasts, a phenotype known to cause immunosuppression. TEVs are also crucial in regulating metastases, and hence the treatments targeting TEVs may have significant utility in breast cancer as part of novel therapeutic intervention. Studies have shown TEVs derived from aggressive subclones of the triple-negative breast cancer cell line Hs578T were able to transfer their aggressive phenotype to a panel of breast cancer cells. By silencing one of the central regulators of TEVs secretion in breast cancer (GTPase Rab27a), there were a reduction in tumor growth and a significant decrease in metastatic dispersal (Bobrie et al. 2012). Other data have elucidated that TEVs can regulate the selective tropism of cancer

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cells to target specific organs and create sites of metastasis that are pro-oncogenic. This is achieved through differing integrin expressions. TEVs also target organspecific cells and alter integrin expression in that microenvironment to prepare the pre-metastatic niche. Various experiments have shown that CSCs and their microenvironment are affected by TEVs. Sansone et al. showed that the transfer of exosomal miR-221 to the luminal breast cancer cells, in the setting of hormonal therapy, induced plasticity, a stem cell-like state, and ultimately led to the development of resistant tumor cells (Sansone et al. 2017). TEVs have an impact not only on tumor promotion but can also inhibit tumor growth. We have explored the role of MSCs in the leukemic niche and have shown that EVs derived from healthy MSCs are negative regulators of the cell cycle and inhibit cancer cell growth. Ono et al. showed that bone marrow-derived MSCs secreted EVs, which acted as negative regulators of breast cancer growth (Ono et al. 2014). These findings are supported by various research groups who have reported that EVs from otherwise healthy (noncancerous) cells can promote breast cancer cell dormancy in metastatic sites (Bruno et al. 2013).

Use of TEVs in Breast Cancer Treatment TEVs are membrane-encapsulated small vesicular structures and have the ability to maintain their structural integrity and protect their cargo against external processes, such as proteases and other enzymes. We will review how TEVs can become a novel candidate as a “drug-loaded” delivery system for targeted cancer gene therapies (Théry et al. 2002). Various experimental studies have demonstrated that TEVs can be secreted at higher levels in the setting of cytotoxic chemotherapy and enhance the pro-metastatic capacity of cancer cells and allow for chemoresistance through the release and transfer of TEVs from chemoresistant to chemosensitive breast cancer cells (Keklikoglou et al. 2019; Wang et al. 2017). Conversely, TEVs have also been shown to play a role in overcoming treatment failure due to drug resistance in breast cancer. Some studies have also demonstrated a potential clinical use of TEVs to treat chemoresistant cancer cells. Zhang et al. (2015) highlighted the importance EVs have in the modulation of miRNA expression profiles that led to the reversal of drug resistance via the EVs-directed changes in multidrug resistance-related miRNAs. Researchers have postulated that manipulating drug efflux from the cells may successfully restore drug sensitivity in cancer and CSCs. This was confirmed by Kong et al. in the human breast cancer MDA-MB231 cell line (Kong et al. 2015). TEVs have also been investigated for targeted treatment of breast cancer based on their ability to deliver a specific cargo of bioactive molecules into the tumor microenvironment. Thus, they can potentially increase the option of targeted therapy to specific areas using donor-derived products as vectors. In theory, donor cells can produce TEVs with little or no immunogenicity and stable enough to be protected from destruction during delivery.

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The potential of TEVs as a delivery vehicle for certain molecules has been assessed in clinical settings. For example, Phase I clinical studies have used dendritic cell-derived TEVs to treat patients with various solid cancers (Escudier et al. 2005; Morse et al. 2005). In addition, Shin-Ichiro et al. have successfully demonstrated the use of breast cancer-derived exosomes to deliver specific miRNAs to EGFR-positive cancer cells (Shin-Ichiro et al. 2013). Many studies intend to use TEVs to encapsulate anticancer drugs/chemotherapeutic agents, such as paclitaxel and doxorubicin, for delivery across the blood-brain barrier and into the CNS (Yang et al. 2015). TEVs-mediated drug delivery has a broad range of future clinical applications and offers a new treatment strategy in targeting specific pathways such as HER2, PI3K/ AKT, and VEGF via direct and indirect modulation of these cellular pathways (Wang and Gires 2019). One example of TEVs application in the setting of targeted therapy is with HER2-expressing breast cancer cells. Studies showed that EVs released by HER2-positive breast cancer cells could bind with trastuzumab and resulted in the suppression of drug effects. These data indicated that TEVs could disrupt anticancer therapy, such as trastuzumab, and promote drug resistance (Marleau et al. 2012). However, by removing exosomes through the creation of a novel therapy called HER2osome (Aethlon Medical, San Diego, USA), which aims to reduce the quantity of circulating HER2 protein and breast cancer exosomes, the therapeutic effects of trastuzumab can be restored.

Use of TEVs for Diagnostic and Predictive Markers in Breast Cancer Given that TEVs play a vital role in tumor development and metastases, they have been studied for use as diagnostic and prognostic markers. Furthermore, there is promising data that TEVs can be detected before tumor detection or symptomatic appearance of the disease (Saraswat et al. 2015). Thus, the use of TEVs as biomarkers is a promising approach as it may serve as a minimally invasive diagnostic tool due to TEVs availability in various body fluids. Moreover, TEVs can be easily measured during course of treatment as a predictive biomarker for assessing response. Various research groups have reported specific TEVs associated with distinct disease phenotypes. For example, exosomal miR-373 and miR-939 are linked with triple-negative breast cancer and aggressive breast cancer phenotypes (Corinna et al. 2014; Di et al. 2017). Although the best modality for detecting and analyzing these TEVs is under investigation, high-sensitivity PCR and next-generation sequencing are emerging as promising possible options for their detection. There has been a significant advancement in breast cancer treatment over the last decade, with TEVs-based diagnostic and therapeutic applications giving encouraging results. Various research groups are analyzing a relationship between TEVs and breast cancer cells regarding their role in cell proliferation, invasion, and metastasis. TEVs are unique in their ability to transport bioactive molecules with protection from degradation both locally and throughout the body.

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An Introduction to EVs and Lymphoma There are over 80 types of lymphomas, including both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL) of B-lymphocyte and T-lymphocyte in origin, as well as indolent and aggressive lymphomas with an expanded spectrum. Even among the established lymphoma subtypes, there exists considerable genetic heterogeneity. Similar to the nature of the disease, therapeutic options and the standard clinical course also have vast heterogeneity. Standard therapy for the majority of lymphomas includes chemotherapy, and in B-cell lymphomas it is typically combined with an immunotherapy (such as the monoclonal anti-CD20 antibody, rituximab). In the treatment for aggressive lymphomas, therapy is of definitive intent, whereas in indolent lymphomas, patients can expect to have periods of therapy and remission. Research on how EVs may better guide diagnosis of lymphoma, assess and predict response to therapy, and ultimately serve as a therapeutic agent is underway.

Extracellular Vesicles in Primary CNS Lymphomas As in the case of other diseases and in normal physiologic states, lymphoma-derived EVs contain cell-specific cargo of bioactive molecules that includes DNA, RNA, and proteins (Navarro-Tableros et al. 2018; Trajkovic et al. 2008). Lymphoma-derived EVs and parent cells from which they originated express many common surface markers, such as CD19, CD20, and CD30 (Trajkovic et al. 2008; Yao and Wei 2015). As mentioned, compared to healthy tissues, cancer cells show increased release of EVs, making them particularly abundant in the disease state (Yu et al. 2005; Van Eijndhoven et al. 2017). EVs generally contain shared genomic materials, the majority of which is double-stranded DNA, but their cargo also includes numerous proteins, bioactive lipids, and RNA species that are capable of entering into target cells to alter the transcription and expression of genes and proteins related to numerous cellular functions (Navarro-Tableros et al. 2018). All of these aspects make the study of EVs integral to improved diagnosis, prognostic modeling, and possibly treatment of lymphoma.

The Role of EVs in Diagnosis and as a Biomarker in Primary CNS Lymphoma Liquid biopsies and cell-free DNA analyses continue to gain increasing relevance across several hematologic and solid malignancies. In lymphoma, excisional lymph node biopsy, when feasible, remains standard of care. With improved techniques and optimal protocols, both by interventional radiology and pathology, core biopsies will often suffice. However, with any biopsy, there is a relatively increased risk and discomfort to the patient due to the invasiveness of the procedure when compared to drawing a blood sample for use. In addition, sequential biopsies are at times

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necessary to confirm either response or progression but cannot occur routinely throughout treatment as a marker of response. Cell-free DNA sampling has emerged as a potential tool for assessing treatment response in DLBCL (Kurtz et al. 2015, 2018). Indeed, any liquid biopsy may serve as a biomarker of therapy response, ideally more accurate than imaging, or as a diagnostic tool when the proposed biopsy carries increased risk. An example would be in primary central nervous system lymphoma (PCNSL) and the morbidity associated with a brain biopsy or even lumbar puncture. Lymphoma-derived EVs make an appealing biomarker for liquid biopsy. They are abundantly present in most body fluids, including not just blood but cerebral spinal fluid, urine, peritoneal fluid, amniotic fluid, and even breast milk (Ofori et al. 2021). Of particular interest and a way to obviate a brain biopsy is the presence of neural-derived EVs in saliva (Guedes et al. 2020). Data from traumatic brain injuries demonstrate that the blood-brain barrier does not prevent EVs originating from the CNS from entering the saliva, and potential exists for EVs as a diagnostic tool in CNS malignancies (Guedes et al. 2020; Ramirez et al. 2018; Cheng et al. 2019, 2020). The use of EVs as the target in a liquid biopsy can also be helpful to discriminate between false-positive and false-negative results besides differentiating unclear diagnoses (Caivano et al. 2015). For example, HL-derived EVs express high levels of CD30 in contrast to CD20 and CD22 in the case of NHL. Tracking of CD20+ EVs also correlates well with the volume of B-cell lymphomas (Domnikova et al. 2013). In general, the actual number of circulating EVs has been correlated with disease progression in HL (Provencio et al. 2017). An elegant study by the Spanish Lymphoma Oncology Group assessed mRNA for C-MYC, BCL-XL, BCL-6, NF-κβ, PTEN, and AKT in EVs from B-cell lymphomas (Provencio et al. 2017). The authors concluded that the presence of BCL-XL mRNA and its increased level in BCL-6 mRNA in EVs might serve as suitable measurement targets for response and surveillance.

Extracellular Vesicles’ Function on Lymphomagenesis and Lymphoma Treatment Response EVs can enhance, but primarily impair, the immune response to malignancy through multiple methods, including effects on both innate and adaptive immunity (NavarroTableros et al. 2018; Théry et al. 2009). This has wide-ranging implications in disease progression and the efficacy of various therapies to target disease. EVs’ inhibition of regulatory T-cells (T-regs) plays an essential role in the immune escape of lymphomas (Navarro-Tableros et al. 2018). In doing so, T-regs decrease the efficacy of lymphoma-infiltrating T-cells and impair the immune response in HL (Marshall et al. 2004). In addition, EVs from EBV-positive lymphomas have been shown to incorporate into macrophages and induce an immune regulatory phenotype (Navarro-Tableros et al. 2018). PD-L1-expressing EVs suppress T-cell response within the lymph nodes (Poggio et al. 2019). EVs can also alter the function of

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natural killer (NK) cells in lymphoma (Navarro-Tableros et al. 2018). NK cells express the natural killer group 2, member D (NKG2D) receptor that plays a role in recognizing stressed self-molecules, similar to PD-1 in the T-cells (Nausch and Cerwenka 2008). EVs increase the NKG2D ligand, thereby activating a more robust immune response (Navarro-Tableros et al. 2018). In addition to supporting immune surveillance, EVs promote angiogenesis in lymphoma (Navarro-Tableros et al. 2018). Lymphoma-associated EVs deliver vascular endothelial growth factor (VEGF) and other angiogenic proteins within the lymphoma microenvironment (Yang et al. 2015; Chen et al. 2018). Regarding the treatment response, EVs also express CD20 and may “trap” antibodies relevant in therapy (i.e., rituximab) and thereby dampen the therapeutic effects (Navarro-Tableros et al. 2018; Aung et al. 2011).

EVs’ Effects on Lymphoma Patient Outcomes and Prognosis EVs have been studied as prognostic markers in lymphoma. Expression of EVs affecting specific markers, including HSP-70, c-Myc, Bcl-2, Mcl-1, xIAP, and Bcl-xL, has been associated with impaired response to therapy in aggressive lymphomas (Chen et al. 2018). In a study by the Spanish Lymphoma Oncology Group, expression profiling of multiple mRNAs in EVs had prognostic implications. EVs with AKT mRNA demonstrated an inadequate rituximab response (Provencio et al. 2017). High death rates were also observed in the presence of BCL-6 mRNA. Additionally, C-MYC mRNA predicted a short duration of response in follicular lymphoma. Like other malignancies, lymphoma patients are at increased risk of thrombosis, and EVs have been postulated to drive this hypercoagulable state (Navarro-Tableros et al. 2018; Litwińska et al. 2019). Systemically, EVs shed by the cancer cells are able to promote thrombus formation via the expression of tissue factor, which is a potent trigger of the coagulation cascade and thrombotic events. Moreover, various cancer and oncology treatments lead to the release and activation of platelet-derived EVs, further stimulating vascular remodeling and clot formation (Borgovan et al. 2019).

Role or Target in Therapy for Lymphoma As EVs clearly promote lymphomagenesis and resistance to therapy, therapeutic interventions directed toward lymphoma-related EVs create a novel avenue of research. As additional B-cell-specific antibodies are being studied or as in the case of anti-CD19 tafasitamab that has been already approved, their combined use may neutralize lymphoma-derived EVs and allow better therapeutic efficacy (Navarro-Tableros et al. 2018; Salles et al. 2020). Another approach is to block the release of EVs from lymphoma cells. As the generation and release of EVs are complex processes, targeting their formation and release by lymphoma cells presents

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a challenge. Several imidazoles and other agents interfere in pathways associated with EVs biogenesis and release (Datta et al. 2018). Other promising agents can impair Ca2+-dependent release of EVs (Savina et al. 2003). Additionally, EVs also provide a way to immunogenically prime and “educate” dendritic cells, which in turn could create T-cell expansion against various lymphoma epitopes and ultimately an anti-lymphoma response (Litwińska et al. 2019). Furthermore, dendritic cell-derived EVs-based methods have shown promise in mouse models when combined with multiple standard lymphoma therapies, such as cyclophosphamide (Taieb et al. 2006). Finally, dialysis-type approaches have been proposed to physically remove EVs, although no models have shown efficacy in malignancy so far (Navarro-Tableros et al. 2018).

Lymphoma-Specific EV Roles Diffuse Large B-Cell Lymphoma (DLBCL) The most common NHL, DLBCL, has emerged as a truly heterogeneous disease. Gene expression profiling first defined major subgroups, germinal center B-cell (GCB), activated B-cell (ABC), and not-classifiable more than two decades ago (Alizadeh et al. 2000). Since that time, immunohistochemistry-based algorithms have attempted to capture these groups with more straightforward techniques to varied success, with the Hans algorithm being the most accepted algorithm type (Hans et al. 2004). Several groups have recently used molecular profiling to define several more distinct subgroups, each with their prognosis, and possibly distinct treatment vulnerabilities (Chapuy et al. 2018; Reddy et al. 2017; Schmitz et al. 2018). EVs may serve as an adjunct to this taxonomy or may even offer a new, improved method for delineating DLBCL-specific gene expression without the need for biopsy (or repeat biopsy) and provide insight into therapeutic selection. Feng and colleagues using cell samples derived from patients undergoing treatment for diffuse large B-cell lymphoma found a specific high-level miRNA expression profile in EVs of patients with the chemoresistant disease compared to those with the chemoresponsive disease. They showed a clear difference in progression-free survival when patients were stratified based on low or high expression of this specific miRNA marker in EVs (Feng et al. 2019). Zare et al. showed a similar analysis of miRNA, demonstrating a significant difference in EVs levels in patients with refractory or relapsed disease compared to those who responded to standard chemotherapy (Zare et al. 2019). Virally Mediated Lymphomas In addition to EBV, Kaposi sarcoma-associated herpesvirus (KSHV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), human T-lymphotropic virus-1 (HTLV-1), and others have all been associated with lymphomagenesis through either directly induced transformation, chronic inflammation, or immunodeficiency and associated reduced surveillance (Xu and Wang 2017). In HTLV-1-positive cells, EVs contain the Tax protein, which is considered a main oncogenic protein that

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contributes to the development of adult T-cell leukemia/lymphoma (Pinto et al. 2019). Much like mammalian cells and bacteria, these viruses can also secrete EVs containing selectively packaged viral mRNA and oncogenes within the lymphoma microenvironment (Xu and Wang 2017). It appears that EBV+ Burkitt’s cells secrete pro-angiogenic EVs, unlike EBV- Burkitt’s cells which do not secrete pro-angiogenic EVs (Xu and Wang 2017; Yoon et al. 2016). Overall, EVs play an essential and central role in virally mediated lymphomas.

Hodgkin’s Lymphoma (HL) HL is a lymphoma characterized by scattered malignant Reed-Sternberg cells in an inflammatory milieu. EVs constitute a key component of this unique microenvironment. It is generally observed that direct contact dependent cell-to-cell communication supports tumor growth within Hodgkin’s lymph nodes. This current dogma is expanding as we begin to understand how EVs can provide a means of efficient noncontact dependent cell communication (Hansen et al. 2014). Surveillance in HL can be difficult, with positron emission tomography (PET) scans showing notably false positives during and after treatment. EVs-associated microRNAs in HL track well with treatment, response to treatment, and long-term follow-up. They can therefore serve as novel tumor biomarkers for response and surveillance. Pegtel et al. evaluated circulating EV-bound miRNAs in the plasma of HL patients, elucidating a significant decrease in various lymphoma-relevant miRNAs, which correlated well with the clinical metabolic response observed via PET. Of note, patients with progressive or relapsed disease had increasing levels of these circulating EV-bound miRNAs (Van Eijndhoven et al. 2017). A better understanding of the role of EVs in lymphoma offers hopes of ultimately improving patient care and outcomes through several methods. First, when considered as a form of liquid biopsy, lymphoma EVs may obviate the need for difficult or repeat biopsies, help clarify an unclear diagnosis, track response to treatment, and survey for relapse/recurrence. They may both prognosticate and ultimately guide treatment decisions. Either targeting or using EVs may improve the efficacy of therapy. Finally, with a better understanding of EVs, fundamentals of lymphomagenesis may be better elucidated.

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Toll-Like Receptor 3 A Multifunctional Regulator of Mesenchymal Stem Cells Mohamed Mekhemar, Johannes To¨lle, Christof Do¨rfer, and Karim M. Fawzy El-Sayed

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toll-Like Receptor 3 and Its Activation Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR3 Priming and MSC Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR3 Priming and MSC Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR3 Priming and MSCs’ Migration and Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR3-Mediated Apoptosis and Survival of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micro-RNAs as Modulators of TLR3-Mediated Response in MSCs . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Mesenchymal stem/progenitor cells (MSCs) are prospective cellular candidates for numerous regenerative and immunotherapeutic purposes. Their immunomodulatory potential and multilineage abilities allow them to be deployed for the treatment of various conditions. Nevertheless, depending on the local microenvironment, different biological tasks of MSCs can be adjusted. Toll-like receptors (TLRs) represent Mohamed Mekhemar and Johannes Tölle contributed equally with all other contributors. M. Mekhemar · J. Tölle · C. Dörfer Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, ChristianAlbrecht’s University, Kiel, Germany K. M. Fawzy El-Sayed (*) Stem Cells and Tissue Engineering Research Group, Faculty of Dentistry, Cairo University, Cairo, Egypt Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian Albrechts University, Kiel, Germany © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_25

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important bridges regulating the cross talk between MSCs and their microenvironment affecting diverse biological features of the cells. Toll-like receptor 3 (TLR3), a member of TLR family, recognizes double-stranded RNA (dsRNA) produced by DNA viruses and positive-strand RNA viruses. Its expression has been displayed by MSCs of various sources. Upon ligand identification inside the endosomes, TLR3 oligomerizes and recruits the Toll-interleukin-1 receptor domain-containing adaptor molecule-1 (TICAM-1), which triggers the production of NF-κB, β-catenin, IRF3, and AP-1, leading to the secretion of interferons and other immunomodulatory cytokines and results in pathogen clearance, as well as the recruitment of adaptive immune responses. It may also be involved in the cell-fate determination and cell cycle regulation in different developmental stages of the MSCs. Moreover, it has been shown to improve the therapeutic potential of MSCs by the promotion of multifunctional trophic factors. Priming of MSCs’ TLR3 within potential treatment procedures may serve as an additional step enhancing the required biological functions of the cells in different stages of the disease. Keywords

Stem cells · Toll-like receptor 3 · Stemness · Inflammation · Immunomodulation · Differentiation List of Abbreviations

AD AKT AP-1 AT-MSCs BM-MSCs C/EPB CFUS COX-2 DF-MSCs DP-MSCs DSRNA G-MSCs GSK3Β HGF HO IDO IKK INF1 IRAK1 IRF3/7 IΚB JNK LPS

Adipose tissue Protein kinase B Activator protein 1 Adipose tissue mesenchymal stem cells Bone marrow mesenchymal stem cells Ccaat/Enhancer-binding protein Colony forming units Cyclooxygenase-2 Dental follicle mesenchymal stem cells Dental pulp mesenchymal stem cells Double-stranded ribonucleic acid Gingival mesenchymal stem cells Glycogen synthase kinase 3 Β Hepatocyte growth factor Hemoxygenase Indoleamine-2,3-dioxygenase Iκb kinase Interferone type 1 Interleukin-1 receptor-associated kinase 1 Interferon regulatory factor 3/7 Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor C-Jun N-terminal kinase Lipopolysaccharides

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MAPK MIF MIRNA MKK MSC MTOR MYD88 NF-ΚB NK-CELLS NM-MSCs NO P38 PAMPS PD-L1 PGE2 PI3K PIP2 PMN POLY(I:C) HMW PRR PT RIP1 SCAP S-SCR SSRNA TAB1/2 TAK1 TBK1 TH1 TIR TLR T-MSCS TRAF TRIF UCB UCB-MSCS WJ

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Mitogen-activated protein kinase Macrophage migration inhibitory factor Micro-RNA Mitogen-activated protein kinase kinase Mesenchymal stem cell Mechanistic target of rapamycin Myeloid differentiation primary response 88 Nuclear factor kappa-light-chain-enhancer of activated B cell Natural killer cells Nasal mucosa MSCs Nitric oxide P38 mitogen-activated protein kinases Pathogen-associated molecular patterns Programmed death ligand-1 Prostaglandin E2 Phosphatidylinositol 3-kinase Phosphatidylinositol 4,5-bisphosphate Neutrophils High molecular weight polyinosinic-polycytidylic acid Pattern recognition receptor Placental tissue Receptor-interacting serine/Threonine-protein kinase 1 Stem cells from the apical papilla Cellular sarcoma molecule Single-stranded RNA Tgf-beta-activated kinase ½ Mitogen-activated protein kinase kinase kinase 7 Tank-binding kinase 1 Cd4+ T helper 1 Toll-interleukin receptor domain Toll-like receptor Mesenchymal stromal cells from human tonsils Tnf-receptor-associated factor Tir-domain-containing adapter-inducing interferon-Β Umbilical cord blood Umbilical cord blood mesenchymal stem cells Wharton jelly

Introduction Mesenchymal stem cells (MSCs) are promising contenders for many practices on the grounds of regeneration and immunotherapy (Han et al. 2019; Almeida-Porada et al. 2020). Their multilineage differentiation ability and immune regulatory aspects permit their potential uses to manage various pathologies and medical conditions (Han et al. 2019; Almeida-Porada et al. 2020). Nevertheless, the local

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microenvironment and inflammatory condition can modulate diverse immuneregenerative structures and characteristics of the cells (Han et al. 2019; Almeida-Porada et al. 2020; Li et al. 2019). Certainly, during their clinical application, MSCs may show interactive responses to their milieu through their expressed Toll-like receptors (TLRs). Hence, a comprehension of these TLR-mediated responses of MSCs and their effect on the cells’ immunobiology, potential MSC-based therapies can be better controlled and optimized (Abdi et al. 2018; Najar et al. 2017). TLRs link the acquired and innate divisions of the immune response, detecting pathogen-associated molecular patterns (PAMPs), in addition to the damage-associated molecular patterns (DAMPs). In recent years, ten functional extracellular and intracellular TLRs have been characterized for their functional contribution and regulatory involvement in different cellular processes (Henrick et al. 2019). As identifiers of diverse pathogens, they play a significant role in the progress and healing of inflammatory and infectious conditions (Farrugia and Baron 2017; El-Zayat et al. 2019). TLR3 as a member of the TLR family has the ability to identify DNA viruses’ double-stranded RNA (dsRNA) and positive-strand RNA viruses, initiating the acquired immune response and pathogen clearance (Chen et al. 2018; Mekhemar et al. 2018a). MSCs of miscellaneous origins have been shown to express TLR3 in definite patterns (Mekhemar et al. 2018a). Upon activation, TLR3 can control MSCs’ various functions including proliferation, immunomodulatory responses, and cell-fate determination (Mekhemar et al. 2020). Recent inquiries have shown that activation of TLR3 by Poly (I: C), a highly specific TLR3 ligand, augmented the immunosuppressive response of gingival mesenchymal stem cells (G-MSCs) (Mekhemar et al. 2018a) and human umbilical cord-derived mesenchymal stem cells (UCB-MSCs) (Zhao et al. 2014). These effects were mediated via downstream microRNA-143 inhibition. However, TLR3 activation also enhanced the production of different trophic factors of MSCs (Mastri et al. 2012), promoting stemness and differentiation-associated factors and modulating the cell-fate determination in several types of stem cells (Mekhemar et al. 2020). These effects are mediated by β-catenin and NF-κB co-activation as well as the regulation of Ca2+ signaling (Park et al. 2016; Jia et al. 2015; Yeh et al. 2016; Qi et al. 2014; Katoh and Katoh 2007) in a PI3K-AKT pathway controlled response (Zhu et al. 2019; Joung et al. 2011; Baker et al. 2015). Priming of MSCs’ TLR3 as the primary key of regulation within therapeutic procedures may enhance the required biological functions of the cells in diverse phases of the disease and optimize the understanding of MSC immunobiology.

Mesenchymal Stem Cells Human mesenchymal stem cells were first described as the plastic-adherent, nonhematopoietic cells, isolated from the bone marrow and able to self-renew and undergo multilineage differentiation in vitro (Friedenstein et al. 1970; Bianco and Robey 2001; Pittenger et al. 1999; Chamberlain et al. 2007). In their undifferentiated

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state, these cells can arise from diverse sources within the adult human body (Berebichez-Fridman and Montero-Olvera 2018). While bone marrow is recognized as the primary niche of adult mesenchymal stem cells (Mekhemar et al. 2018b), numerous researchers have tried to find other stem cell sources to achieve large amounts of MSCs with fewer morbidity and risks for the donors. These include placental tissue (PT), umbilical cord blood (UCB), adipose tissue (AD), and Wharton jelly (WJ), which have been defined as dependable niches of MSCs (Wu et al. 2018; Shree et al. 2019; Marino et al. 2019). MSCs can be similarly isolated from oral tissues as alveolar bone proper (Fawzy El-Sayed et al. 2017), gingiva (Mekhemar et al. 2020), periodontal ligament (Banavar et al. 2020), the dental follicle (Hong et al. 2020), and dental pulp (Noda et al. 2019). Despite the phenotypic similarity of MSCs isolated from different tissues, actions and biologic functions have shown differences, accenting the uniqueness of MSCs isolated from each niche (Mekhemar et al. 2020). MSCs self-renewal describes the process that enables MSCs to increase their number during development. This ability is vital for MSCs to promote their growth inside the tissues and presents a significant contribution in stem-cell-based treatments either alone or in combination with other cell types (He et al. 2009; Haider 2018; Hosseini et al. 2018). This property exhibited its reliance on the cells’ span of life, mostly limited to 44 weeks or 55 population doublings in vitro (Mekhemar et al. 2018b). Multilineage ability or multipotency of MSCs labels the potential of the cells to differentiate into other mesodermal cell lineages, including chondrocytes, osteocytes, and adipocytes. However, they can congruently differentiate to form cells of different embryonic lineages (Uccelli et al. 2008). Recently, MSC identification and “stemness” verification have been a challenging topic in many investigations (Pittenger et al. 2019). In 2006 MSCs’ plastic adherence upheld under basic culture conditions was demarcated (Dominici et al. 2006). Furthermore, the multilineage differentiation potential of the cells after stimulation by specialized media was explained by multiple studies (Mekhemar et al. 2018b). Another extensively described method for MSCs identification is the investigation of specific surface marker expression by the cells. Markers as CD29, CD44, CD71, CD73, CD90, CD105, CD106, CD120, CD124, CD 146, CD166, CD 271, Stro-1, and SSEA-4 display positive expressions, whereas CD86, CD11, CD80, CD14, CD18, CD31, CD34, CD40, CD45, and CD56 are faintly expressed or completely missing (Lv et al. 2014). Specific cellular colonies shaped by MSCs’ culture after isolation were described in further investigations as colony forming units (CFUs), and reported likewise as a method of MSCs’ recognition (Mekhemar et al. 2020).

Toll-Like Receptor 3 and Its Activation Pathways Pattern recognition receptors (PRR) carrying cells are called “sensor cells.” The TLRs’ expression allows them to recognize various pathogen-associated antigens (Gong et al. 2019). TLRs are single-pass α-helical transmembrane proteins

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(Choe et al. 2005). Dimerization strengthens antigen binding and induces signal transduction (Wang et al. 2010). Dimers can either consist of two identical monomers (homodimers, i.e., TLR3, TLR7, TLR8, and TLR9) or two discrete monomers (heterodimers, i.e., TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) (Janssens and Beyaert 2003). TLRs can be categorized based on two different features, including their localization in the cells (Kawai and Akira 2007b) and connection with intracellular signaling (Takeda and Akira 2004). Concerning localization, TLRs are mainly divided into intracellular or extracellular types. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR 10 are preferentially expressed on the cells surface membrane whereas TLR3, TLR7, TLR8, and TLR9 are mainly carried by intracellular endosomes (Fawzy-El-Sayed et al. 2016). Interestingly, some TLRs, such asTLR3 and TLR4, have the ability to switch their location (Bugge et al. 2017). It is believed that surface membrane expression is accompanied by fastened signal induction. Extracellular TLRs are sensitive to bacterial components like lipopolysaccharides (TLR4), peptidoglycans (TLR1, TLR2, and TLR6) and flagellin (TLR5). TLR10 ligand has not yet been identified, but its heterodimerization with TLR1 and TLR2 implies bacterial substrate, as well (Nagashima et al. 2015). Moreover, endosomal TLRs are nucleic acid sensing in nature and make up a defense against viral attacks. They are sensitive to dsRNA (TLR3), ssRNA (TLR7 and TLR8), and viral DNA (TLR9) (Miyake et al. 2018). On the other hand, TLRs can be classified based on their adaptor proteins interfacing receptor activation and intracellular signal transduction. Generally, there are two different adaptor proteins: myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). TLR1, TLR2, TLR6, TLR7, TLR8, and TLR10 are only capable of activating MyD88. On the contrary, TLR10 differs from its counterparts as TLR10 does not maintain the adaptor binding determinant. TLR4, TLR5, and TLR9 can use TRIF and MyD88-mediated signaling (Takeda and Akira 2004). TLR3 is the only receptor addressing TRIF standing alone among the other TLRs (Lundberg et al. 2013). TLR 3 is the first discovered antiviral TLR, which senses viral double-stranded RNA (dsRNA). Alexopoulou et al. (2001) described the induction of transcription factor NF-κB in response to TLR 3 stimulation. They also observed increased production of interferon type 1 (INF1) and various pro-inflammatory cytokines (Alexopoulou et al. 2001). This reaction is now considered one of the crucial and integral part of the antiviral defense and innate immune response activation (Kawai and Akira 2007a). Consequently, TLR3 is expressed by sensor cells, such as dendritic cells, monocytes, epithelial cells, fibroblasts, and neurons, thus ensuring fast detection of viral invasion (Kawai and Akira 2007a). Apart from that, El-Sayed et al. discovered TLR3 expression in mesenchymal stem cells like G-MSC (Fawzy-El-Sayed et al. 2016), dental pulp stem cells (DP-MSC) (Fawzy El-Sayed et al. 2016), and apical papilla stem cells (SCAP) (Fehrmann et al. 2020). This link between pathogen recognition and tissue regeneration has been proven by Mekhemar et al. (2020). They have

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reported a distinct connection between stem cell characteristics and their functionality, and TLR3 ligation. Primarily, TLR3 has an intracellular location in the endosomes, but some cells, such as dendritic cells and monocytes, have cell membrane surface expression of TLR3 as well. Such altered TLR 3 location renders the cells far higher sensitive for extracellular pathogen recognition and hence provides a potential target for pharmacological immune activation (Murakami et al. 2014). TLR3 is a single-pass α-helical transmembrane protein segmented into three parts: an extracellular/intraendosomal ectodomain, a transmembrane domain, and an intracellular adaperdomain, and mediates intracellular signal transduction via Toll-interleukin receptor domain (TIR) (Akira et al. 2006). The ectodomain is shaped like a horseshoe containing 23 leucine-rich sequences. Unlike the outer side, the inner side is glycosylated, thus allowing ligand binding on the outer side. After dsRNA binding, one TLR3 monomer forms a fragile complex while a second TLR3 monomer strengthens this connection, thus resulting in the formation of a dsRNA-TLR3 dimer complex (Choe et al. 2005; Wang et al. 2010). The exact mechanism of intracellular signal transduction remains less well understood. However, changes in TLR3 ectodomain structure during antigen binding or convergence of intracellular domains in dimerization have been extensively studied and discussed in the published literature (Liu et al. 2008; Gosu et al. 2019). TLR3 is the only TLR in which signaling occurs independent of My88 involvement, and it only involves TRIF (TIR-domain-containing adapter-inducing interferon-β). In trials under TRIF inhibited conditions, Yamashita et al. (2012b) recognized the effects of TLR3 stimulation via dsRNA. This discovery was ascribed to the cellular sarcoma molecule (s-Scr). Apparently, short-time TLR3 activation resulted in c-Scr phosphorylation and induction. On the contrary, longtime receptor stimulation caused c-Scr sequestration and inactivation. Although the exact pathway has not been fully revealed in MSCs, the effect of TLR3 stimulation appears multidirectional (Yamashita et al. 2012b; Mekhemar et al. 2020). Generally, the biphasic effect of TLR3 ligation has been proven in studies on G-MSCs (Mekhemar et al. 2020). TRIF acts as a checkpoint in TLR3 activation and regulation. TRIF is regulated via K27-linked polyubiquitination via RING E3 ubiquitin ligase complex Cullin-3Rbx1-KCTD10 complex. Deficiencies result in an obstruction in TRIF-TLR binding and TLR downstream signaling (Wu et al. 2019). TRIF works as a point of origin for four different pathways, finally affecting the transcription factor activation and gene transcription: β-catenin, IRF3/IRF7, NFκB, and AP-1 (Jiang et al. 2004). The distinct transcription factor activation profile depends on various cumulative factors influenced by numerous individually interacting pathways. As the most critical pathway in MSCs’ pluripotency and tumor development, β-catenin activation is influenced by two significant cascades: PI3K/Akt- and Wnt-Pathway. PI3K is induced via TRIF activation that converts the catalytic domain PIP2 into the activated PIP3 form. Binding with the PKB/Akt complex enables Akt phosphorylation and activation (Hemmings and Restuccia 2012).

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Akt as a kinase is now able to convert cytosolic β-catenin to its active form (Gantner et al. 2012). Akt activation undergoes inhibition via GSK3β but decreases Akt activity in a positive feedback loop. This suppresses GSK3mediated apoptosis and induces β-catenin-mediated proliferation (Endo et al. 2006). Additionally, Wnt pathway regulates total β-catenin accumulation as well as its nuclear translocation. During Wnt inactivation, β-catenin undergoes inactivation and proteasomal degradation (Fang et al. 2007; Matsumoto et al. 2018; Gantner et al. 2012; Gerlach et al. 2014). IRF3 and IRF7 are activated via TRIF-dependent TRAF3 activation. This requires a complex of TRIF and TBK1 which impairs K63-linked polyubiquitination of TRAF3 and TRIF (Cai et al. 2017). TRIF/TRA3 associates with IRAK1 and IRF3/7 activating kinases TBK1 and IKKε under TBK, and IRAK1 phosphorylation. This complex then phosphorylates IRF3 and IRF7 monomers. Continuously, both are capable of homo- and heterodimerization (IRF32, IRF72, and IRF3/7). Dimers are translocated through nuclear pores primarily inducing INF-1 activation. IRF activation requires PI3K/Akt/mTOR pathway activation. On the one hand, these studies revealed the significance of Akt-mediated phosphorylation of IRF, they also highlight that PI3K is critical in IRF2 nuclear translocation (Ning et al. 2011) through nuclear pores (Ullah et al. 2016). NF-κB and AP-1 are induced via TRAF6, wherein NFkB is influenced by TRAF6-TAB1/2-TAK1-RIP1 (Ko et al. 2015). The activity of this complex is guided via ubiquitin-mediated RIP1 regulation (Annibaldi et al. 2018). The IKK complex is now phosphorylated downstream, initiating the breakdown of the NFκB-IκB complex (Kinsella et al. 2018; Abujamra et al. 2006). The freed NFκB is then capable of trafficking into the nucleus, traversing across the nuclear membrane through the nuclear pores as part of the signal transduction. IκB can inhibit NFκB via two primary mechanisms: IκBα follows NFκB into the nucleus and resynthesizes NFκB-IκBα complex, thus resulting in its inactivation. Alternatively, negative feedback proceeds from the fast gene transcription. During RNA accumulation, another transcript IκBδ accumulates, which forms an NFκB-IκBδ complex, thus resulting in an inhibition of the ongoing gene transcription (Mitchell et al. 2016). On the other hand, TRAF6 can induce AP-1 via MAPK pathways and downstream phosphorylation cascades (Roy et al. 2016). Unlike NFκB activation, AP-1 is not activated via RIP1 induction but depends on GSK3β activity (Ko et al. 2015). It has been reported that GSK3β, TAK 1, and TRAF6 form a ternary complex supporting TRAF6-TAK1-TAB1/2 complex formation. On the contrary, GSK3β did not assemble with RIP1, indicating no influence on NFkB expression (Ko et al. 2015). The TRAF-TAK-TAB-complex phosphorylates MKK3, MKK6, MKK4, and MKK7 using TAK1 (Yamashita et al. 2008). MKK3, MKK4, and MKK6 together initiate p38 MAPK activation, and MKK4/MKK7 induces JNK phosphorylation. JNK and p38 MAPK activation finally result in AP-1 transcription factor activation and gene transcription initiation (Dainichi et al. 2019). Additionally, p38 MAPK modulates C/EPB transcription activity and post-transcriptional protein modification (Dainichi et al. 2019). Figure 1 shows the TLR3 signaling cascades in MSCs after ligation by dsRNA/Poly (I: C) are (Fig. 1).

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Fig. 1 Graph of TLR3 signaling cascades in MSCs after stimulation by dsRNA/Poly (I: C). TLR3 signals are transmitted to the PI3K-AKT pathway to regulate MSC functions through β-catenin, and to the TRAF pathways to regulate MSC functions via IRF, NF-κB, and AP-1 co-stimulation. TLR3 signaling pathway through the TRIF independent c-Src cascade contributes to the regulation of proliferation in MSCs. (Abe et al. 2020; Arnold et al. 2016; Bai et al. 2014; Baker et al. 2015; Chang et al. 2013; Choe et al. 2005; Dumitru et al. 2014; Fan et al. 2019; Fang et al. 2007; Ferreira et al. 2011; Gambara et al. 2015; Gan et al. 2018; Grandvaux et al. 2002; Harashima et al. 2012; He et al. 2018; Jia et al. 2015; Joung et al. 2011; Katoh and Katoh 2007; Kim et al. 2014; Kim et al. 2006; Krishnamurthy and Kurzrock 2018; Lathia et al. 2008; Leite et al. 2017; Liotta et al. 2008a; Liu et al. 2019; Lombardo et al. 2009a; Lu et al. 2015; Ma and Hottiger 2016; Matsumoto et al. 2018; Najar et al. 2017; Park et al. 2016; Qi et al. 2014; Sallustio et al. 2019; Sarkar et al. 2004; Shang et al. 2017; Wang et al. 2010; Yamashita et al. 2012a; Yamashita et al. 2012b; Yeh et al. 2016; Yoshizawa et al. 2008; Zhang et al. 2013; Zhu et al. 2019). TIR Toll-interleukin-1 receptor homology domain, c-Src Proto-oncogene tyrosine-protein kinase Src, TRIF TIR-domain-containing

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TLR3 Priming and MSC Immunomodulation Recently, the therapeutic abilities of MSCs by modulating immune responses have been reported in different studies (Weiss and Dahlke 2019; Saldaña et al. 2019). The unique interplay between activated MSCs and various immune aspects of the human body contributes significantly to restore and protect damaged tissues in inflammatory environments (Saldaña et al. 2019). TLR-specific ligands have exhibited associations with several inflammatory settings. Stimulation of TLRs is suggested to modify MSCs-regulated immunomodulatory effects. Earlier investigations proposed that the MSCs source and the triggered TLRs play essential role defining these immune biological processes (Mekhemar 2018). Immune cell migration and binding to MSCs’ environment is considered the foremost step for initiating immunomodulation responses after TLR ligation (Najar et al. 2017; Mekhemar 2018). By dynamically identifying their microenvironmental factors via TLR signaling, MSCs can consequently regulate the immune cells’ immunobiology from both branches of the immune system (Najar et al. 2017; Mekhemar 2018). This effect initiates the recruitment of MSCs to the injury sites. TLR3 ligation on bone marrow mesenchymal stem cells (BM-MSC) triggered signaling pathways, consequently inducing the cytokine and chemokine secretion that is primarily involved in cell migration (Tomchuck et al. 2008). Indeed, TLR3 ligand exposure promotes the BM-MSC stress migration response and consequently transforms BM-MSCs into strong chemotactic cells, thus enhancing the recruitment of immune-inflammatory cells and endorsing the secretion of cytokines including CCL5 (RANTES), IL-6, IP10, IL-1β, IL-8, and monocyte chemotactic protein (MCP)-1 through the NF-kB pathway (Romieu-Mourez et al. 2009). Previous investigations have also demonstrated that upon TLR3 ligation in BM-MSCs, a Janus kinase (JAK) 2/signal transducer and trigger of transcription (STAT) 1 pathway is started, and cytokine signaling (SOCS) proteins suppressor expression is augmented (Tomchuck et al. 2012). This suggests that TLR3 signaling can noticeably affect the MSCs’ response to danger signals and homing-in to injured sites. Moreover, Poly (I: C) activation of TLR3 increases the production of IL-6, in addition to MIF (macrophage migration inhibitory factor), the main factor for leucocyte recruitment (Kota et al. 2014). Human nasal mucosa MSCs (NM-MSCs) induced a strong release of pro-inflammatory cytokines (IL-6 and IL-8) and type I interferon after

ä Fig. 1 (continued) adapter-inducing interferon-β, PI3K phosphoinositide 3-kinase, AKT protein kinase B, GSK3β glycogen synthase kinase 3 beta, TRAF TNF-receptor-associated factor, RIP receptor-interacting protein, TAB TGF-beta-activated kinase 1 (MAP3K7) binding protein, TAK1 mitogen-activated protein kinase kinase kinase 7, IKK IκB kinase, NF-κB nuclear factor “kappalight-chain-enhancer” of activated B cells, IRF interferon regulatory factor, LEF T cell factor/ lymphoid enhancer-binding factor, p38 p38 mitogen-activated protein kinase, JNK c-Jun N-terminal kinase, AP-1 activator protein 1, EBP EBP cholestenol delta isomerase, and MMK mitogenactivated protein kinase kinase kinase

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activation of TLR3 (Dumitru et al. 2014). Furthermore, investigations have reported the solid immune cell chemoattractant profile secreting CXCL1, CXCL5, CXCL6, CXCL8, and CXCL10 in TLR3-activated mesenchymal stromal cells from human tonsils (T-MSCs) (Ryu et al. 2015). Indeed, TLR3 pre-activation with Poly (I: C) in BM-MSCs also significantly increases the number of leukocytes binding to MSCs via interacting with hyaluronic acid structures (Kota et al. 2014). After recruiting the immune cells to the MSCs surrounding area, diverse controlling mechanisms can participate in the progression of immune regulation. These mechanisms are complex but subtle to the microenvironment. In previous investigations, the results about TLR3 ligation and immunomodulation in this phase are contradictory and reported differently. Waterman and colleagues have described a new paradigm for MSCs immunomodulation following TLR3 priming (Waterman et al. 2010). TLR3 priming shifted the MSCs into a particular phenotype (MSC2), mainly expressing immunosuppressive mediators as TGF-β, IDO (indoleamine-2,3-dioxygenase), PGE2 (prostaglandin E2), NO (nitric oxide), hemoxygenase (HO), and hepatocyte growth factor (HGF), leading to T cell inhibition. Similarly, other investigators described the TLR3induced enhancement of immunosuppressive properties in BM-MSCs (Opitz et al. 2009) and G-MSCs (Mekhemar et al. 2018a) in response to the increased production of IDO1, as well as by an amplified expression of cyclooxygenase-2 (COX-2) in UCB-MSCs (Zhao et al. 2014) and by modulating the TGF-β and IL-6 production in the dental pulp (DP-MSCs) and dental follicle (DF-MSCs) (Tomic et al. 2011). On the other hand, TLR3 ligation was described to differentially affect the inhibitory functions of BM-derived MSCs, WJ-derived MSCs, and adipose-tissue-derived mesenchymal stem cells (AT-MSCs) (Raicevic et al. 2011). Notably, the immunosuppressive potential of WJ-MSCs and AT-MSCs remained unaltered, while BM-MSCs showed decreased potential to suppress lymphocyte activation. Another study showed that priming of TLR3 significantly reduced BM-MSCs’ ability to inhibit T cells proliferation (Liotta et al. 2008b). This effect was not associated with IDO and PGE2 pathway regulation, instead it involved the downregulation of Jagged-1 induced by TLR3 priming. It has been previously emphasized that MSCs-facilitated T cell inhibition arises via the production of galectins. One of the published studies has confirmed that galectin-9, which BM-MSCs do not constitutively produce, is significantly induced upon TLR3-MSC interaction and is imperative for the immunosuppressive properties of MSCs (Gieseke et al. 2013). Additionally, stimulation of TLR3 improved the suppressive effect of T-MSCs against Th17 differentiation by augmenting the expression of programmed death ligand-1 (PD-L1) (Cho et al. 2017). TLR3mediated preconditioning of the cells by Poly (I: C) also confirmed an enhanced efficacy of UC-MSCs through the TLR3-Jagged-1-Notch-1 pathway (Qiu et al. 2017). Following PGE2 and Jagged-1 upregulation, PGE2 then amplifies IL-10 production and permits the Treg cell differentiation. Furthermore, TLR3 ligation amplified IL-12p35, IL-6, IL-23p19, and IL-27p28 transcription in BM-MSCs (Raicevic et al. 2010). These IL-12-related cytokine members can initiate CD4+ T

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helper 1 (TH1) differentiation and support a T-cell-mediated immune reaction and induce pro-inflammatory changes in BM-MSCs. MSCs can also have immune-regulatory effects on cells of the innate immune branch. As stated in numerous published studies, TLR3 triggered BM-MSCs to support the function and survival of neutrophils (PMN) by enhancing the respiratory burst ability, CD11b expression, and the antiapoptotic effect in PMN (Cassatella et al. 2011). The biological properties regulated in PMN via TLR3-triggered BM-MSCs were facilitated by the combined function of IL-6, IFN-β, and GM-CS. TLR3-primed MSCs also showed enhanced suppressive functions against NK cells (Noronha et al. 2019). It seems that TLR3 and its ligands can assist as controllers of MSCs’ immunomodulatory functions. Still, the effects are conflicting and possibly dependent on investigational settings and the microenvironment of the cells.

TLR3 Priming and MSC Differentiation MSCs’ capacity for multilineage differentiation potential to adopt diverging tissue phenotypes has been often explained as an age-dependent process (Sallustio et al. 2019). Recent investigations underline the important functions displayed by TLR ligands in guiding MSCs’ maturation to form various cell phenotypes and in fate determination. The stimulation of TLR3 has been presented to affect osteogenic differentiation of MSCs and their maturation into osteocytes (Liotta et al. 2008b; Lombardo et al. 2009b; Pevsner-Fischer et al. 2007; Mekhemar et al. 2020). TLR-3 ligation via Poly (I: C) was described to endorse osteogenesis by supporting the BM-MSCs’ differentiation into osteoblasts (Qi et al. 2014). Similarly, another investigation (Lombardo et al. 2009b) has demonstrated that TLR3 Poly (I: C) activation of AT-MSCs augmented their osteogenic differentiation potential (Hwa et al. 2006). Poly (I: C)-activated G-MSCs showed a lower expression of ALP and RUNX as early osteogenic markers with a concomitantly increased mineral deposition observed by the alizarin red staining (Mekhemar et al. 2020). Synchronized to this effect on MSCs bone maturation, an interplay between downregulation of pluripotency factors, as well as proliferation potential, and colony-forming units of TLR3-ligated G-MSCs have been observed presenting a cell cycle shift from pluripotency to the stages of differentiation and maturation (Mekhemar et al. 2020). This modulatory process of differentiation and cell-fate determination through TLR3 activation in MSCs was explained to be involved via upregulated β-catenin with regulatory NF-κB co-activation (Jia et al. 2015; Katoh and Katoh 2007; Ma and Hottiger 2016; Park et al. 2016; Yeh et al. 2016) and further facilitated by phosphorylation of the AKT pathway, which contributes greatly to MSCs’ osteogenic differentiation and maturation (Baker et al. 2015; Joung et al. 2011; Qi et al. 2014; Zhu et al. 2019). However, some studies have reported that the activation of TLR3 inhibited the differentiation of UC-MSCs into osteocytes (Zhang et al. 2015) or did not affect the differentiation potential of BM-MSCs (Liotta et al. 2008a). Although TLR3 has shown a similar supporting role in promoting

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adipogenic and chondrogenic maturation of MSCs in some investigations so far, opposing results occur regarding the effect of TLR3 on MSCs’ maturation into these lineages (Mekhemar et al. 2020). As reported by multiple studies, TLR3 ligation outlined a fragile adipogenic modulation of MSCs, mediated by the phosphorylation of β-catenin and GSK3β inhibition leading to a partly reverse arrangement between the induction of MSC osteogenic and adipogenic differentiation (Mekhemar et al. 2020; Hwang et al. 2014; Lombardo et al. 2009a). Furthermore, additional studies clarified the stimulatory effect of β-catenin pathways in response to TLR3 stimulation on MSCs. They showed increased chondrogenic and osteogenic differentiation of MSCs with an attenuated adipogenic lineage effect (Mekhemar et al. 2020; Galli et al. 2013; James 2013; Li et al. 2008). It appears that TLR3 activation can function as a modulator of differentiation potential and may determine the cell fate besides maintaining their multipotency.

TLR3 Priming and MSCs’ Migration and Proliferation Continued growth and maintenance of MSCs are important characteristics much needed for their therapeutic effect as they eventually endure replicative senescence after prolonged periods of normal growth (Ciria et al. 2017). MSCs possess the significant ability to relocate to the areas of the inflammatory response, ischemia, and mechanical damage or even to sites of growing tumors (Kholodenko et al. 2013). TLRs have been suggested to moderate MSCs’ proliferation, immunomodulation, as well as migratory activity, and differentiation potential (Sallustio et al. 2019). Various studies have shown that TLR3 is a key mediator in migratory responses of BM-MSCs (Tomchuck et al. 2008). Still, this consequence seems to be connected to the exposure time: After one-hour incubation, TLR3 activation promoted migration, while after 24 h of incubation, the rate of migration and invasion of the treated MSCs became suppressed (Liotta et al. 2008b; Waterman et al. 2010). Furthermore, abrogation of TLR3 expression using knockdown plasmids reduced the migration potential of the nonactivated MSCs to the half (Tomchuck et al. 2008). Nonetheless, Poly (I: C) ligation of the transfected cells caused an improved migration potential when associated with unligated controls (Waterman et al. 2010). These different reactions of MSCs migration, reliant on marginal changes in the cell setting, support the TLR3 regulation of MSCs migration by multifaceted and generally unraveled molecular processes. On the level of TLR3-mediated MSCs proliferation, TLR3 activation and expression was linked to suppressed proliferation in neuronal stem/progenitor cells (NPCs) (Sallustio et al. 2019; Lathia et al. 2008) and UCB-MSCs (Sallustio et al. 2019). Correspondingly, TLR3-activated G-MSCs displayed a decreased proliferative potential (Mekhemar et al. 2020), resembling a prior designated antiproliferative effect of Poly (I: C) on BM-MSCs (Choumerianou et al. 2010). As the TLR3 pathway begins with TRIF involvement, the IRF3 pathway consequently gets triggered, leading to cell proliferation inhibition (Dumitru et al. 2014; Lathia et al. 2008; Mekhemar et al. 2020). Additionally, the TRIF-independent branch of TLR3

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signaling may similarly add to the suppression of cell proliferation via the c-Src pathway (Yamashita et al. 2012b).

TLR3-Mediated Apoptosis and Survival of MSCs In cancer research, TLR3 ligands have become promising pharmaceuticals in immune-based chemotherapy. Investigations dealing with different tumor entities revealed direct apoptotic induction after TLR3 activation (Otkur et al. 2018; Zhao et al. 2012). Similar effects have been reported in fibroblast-like synoviocytes (Karpus et al. 2016). This direct induction is mainly based on TLR3-dependent TRAF6 and NF-κB activation, leading to INF-induced apoptosis (Chiron et al. 2009). Similar effects have been shown in adipose cells, which make TLR3 an attractive target in chronic fatigue treatment (Strayer et al. 2012). Besides its direct influences, TLR3 provides immunomodulatory effects on NK-cells as key players during innate immune response (Petri et al. 2017). Remarkably, Giuliani et al. (2014) showed protective effects of TLR3 primed MSCs against NK-cell killing (Giuliani et al. 2014). TLR3 stimulation also increases the therapeutic potential of MSCs and causes 40% less apoptosis and improved regeneration in heart failure models (Mastri et al. 2012; Shabbir et al. 2009). However, the effect of TLR3 activation on MSCs under pro-inflammatory and regeneration conditions have not been completely unveiled yet and offer promising targets in tissue regeneration studies in future.

Micro-RNAs as Modulators of TLR3-Mediated Response in MSCs Micro-RNAs (miRNAs) comprise noncoding RNA regulating gene expression by coupling with messenger RNAs (mRNAs) and inhibiting their translation to form proteins. These minor RNA molecules are mainly transcribed inside the cell nucleus and transformed into originator miRNA and then conveyed to the cytosol to be altered to developed miRNA by Argonaut and DICER proteins (Gulyaeva and Kushlinskiy 2016). Every sequence of miRNA can bind to and inhibit the expression of multiple mRNAs (Avraham and Yarden 2012), making them greatly important in various cellular functions and hence for the study of human cellular disorders and their treatments (O’connell et al. 2012). Given MSCs’ extensive and critical involvement in diverse cellular functions, they are being induced by preconditioning of stem cells (Suzuki et al. 2010) or through genetic manipulation with the miRNA of interest to enhance their functionality (Kim et al. 2012a, b) and reparability (Haider and Aramini 2019). Pharmacological preconditioning has also been shown to enhance their survival and angiomyogenic potential via phosphorylation of PI3K, AKt, GSK3b, and nuclear NFkB (Afzal et al. 2010). By interfering with the cellular functions in the immune cells and their precursors, miRNAs regulate numerous functional features of innate and acquired immune reactions. In pre-T and pre-B lymphocytes, inhibition of

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DICER protein revokes differentiation and growth of these cells (O’connell et al. 2012). miRNAs can moderate the role of immune cells by directing diverse intracellular signaling pathways, i.e., the Wnt pathway in dendritic cells (Luo et al. 2015), as well as the Akt signaling in B lymphocytes and the TLR pathways (He et al. 2013; O’connell et al. 2007; Fehniger 2013). Recently, many studies have recognized a collection of miRNAs that control the characteristics of MSCs, as multilineage differentiation and immunomodulation (Clark et al. 2014). These miRNAs can impact the communication between MSCs and the cells of the immune system by affecting the mechanisms involving TLR signaling pathways, as several molecules in TLR signaling, their related cytokines, and transcription factors are involving in there (Clark et al. 2014). Therefore, the molecular exchange between miRNAs and the TLR pathways in MSCs may promote inhibition or stimulation of the TLR signaling pathway and responses in MSCs (Abdi et al. 2018). For example, miR-143 has been reported as a critical regulator of MSCs’ cell cycle activity via transcriptional regulation of cyclin D (Lai et al. 2011, 2012). In MSCs-TLR3-mediated signaling, miR-143 plays an effective role as a negative regulator. In human umbilical cord MSCs, TLR3 ligands significantly amplified the secretion of cytokines and chemokines as COX2, IDO, and PGE2, IL-6, and IL-8, which was linked to a miR-143 downregulation (Abdi et al. 2018). When upregulated, miR-143 abrogated cytokine production and reduced the immunosuppressive effects of TLR3-activated MSCs, besides reducing the beneficial properties of MSCs in an experimental animal model of sepsis (Zhao et al. 2014). In these studies, COX2 and TAK1 genes were directly targeted and downregulated by miR-143. These data show that TLR3 stimulation moderates immune effects of UCB-MSCs through inhibition of miR-143. Nevertheless, more future investigations are required to determine the modulatory role of miRNAs in MSCs involved in cell-fate determination and regulatory functions of immune reactions including T cell proliferation and macrophage polarization in the context of TLR3 activation.

Conclusion Exploring TLR-mediated effects on MSCs’ immunobiological and regenerative functions remains an area of immense importance to facilitate effective therapeutic utilization of MSCs. In this chapter, we have discussed the involvement of TLR3 in regulating diverse MSC functions. The listed studies in the chapter provide strong evidence that TLR3 priming could critically impact the cell-fate determination, multilineage potential, hematopoietic support, immunomodulatory ability, as well as the apoptosis, migration, and proliferation of MSCs from different tissue sources as main thus impacting the outcome of various MSCs-based therapies. Consequently, preconditioning of MSCs to be employed clinically may be treated with TLR3 ligands to enhance their therapeutic effect post-engraftment (Delarosa et al. 2009). In this regard, TLR3 stimulating agents are also being studied as potential vaccine adjuvants for cancers, infectious diseases (Seya et al. 2015; Lebedeva et al. 2018), or as a highly specific novel treatment strategy against tumors (Le Naour et al. 2020).

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Likewise, inhibitors of TLR3 signaling have shown considerable potential in the treatment of inflammatory disorders (Dunne et al. 2011). Notwithstanding a large amount of information gained about TLR3-mediated effects on MSCs, the data emanating from these studies remain inconsistent. These discrepancies and inconsistencies observed in the published data have been attributed to the multiplicity of experimental settings and protocols employed to learn the impact of TLR3 activity level on MSCs. Notably, the influence of tissue source, culture conditions, purity level, etc., may play an essential role determining the outcome of the studies. In this context, we need to well design and standardize the investigations to better understand the mechanism of action of TLR3 on MSCs and precisely acquire the needed information to control TLR3 and its downstream pathways to modulate the needed MSCs characteristic features in the pathological environment. These data would certainly help in improving the safety and therapeutic potential of MSCsbased therapy in the future.

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Targeting Cancer Stem Cells: New Perspectives for a Cure to Cancer

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Beatrice Aramini, Valentina Masciale, Giulia Grisendi, Federico Banchelli, Roberto D’Amico, Massimo Dominici, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Cancer Stem Cells (CSCs) in the Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Markers in CSCS: A New Approach Against Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Hedgehog Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Notch Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Wnt Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting ALDH as the Drug-Detoxify Enzymes Against CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting Drug Efflux Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting the CSC Niche and the Quiescent State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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B. Aramini (*) Division of Thoracic Surgery, Department of Experimental, Diagnostic and Specialty Medicine– DIMES of the Alma Mater Studiorum, University of Bologna, G.B. Morgagni–L. Pierantoni Hospital, Forlì, Italy e-mail: [email protected] V. Masciale Division of Thoracic Surgery, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected] G. Grisendi · M. Dominici Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected]; [email protected] F. Banchelli · R. D’Amico Center of Statistic, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_31

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An Update on the Most Used Target Therapies Against CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Adenovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Herpes Simplex Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Virus and Its Effects in Recent Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Adenoviruses and Pancreatic Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Herpes Simplex Viruses and Pancreatic Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . Stem Cell-Derived Exosomes as Therapeutic Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer stem cells (CSCs) are present in many solid tumors. Their signaling pathways and functions may be the key to developing new anticancer strategies against cancer. Several studies have linked the signaling pathways’ (e.g., Wnt, Notch, and Hedgehog pathways) aberrant activation to the development of numerous cancers. These signaling pathways hence provide attractive targets for further study toward new therapies. CSCs show several characteristics, such as self-renewal, differentiation, high tumorigenicity, and resistance to anticancer drugs. Drug resistance is the most useful in further evaluating the possibilities of reducing tumor mass or eliminating cancer by discovering new therapies. One of the key questions concerns the necessity of identifying superficial as well as intracellular markers, which are essential if the drug is to respond positively to CSCs. In recent years, CD133, CD44, ABCG2, and ALDH have been identified as biomarkers for certain forms of CSCs. However, recent studies have contributed to a better understanding of CSC-specific antigens; to date, there is no univocal characterization of antigens for CSCs. Our chapter aims to highlight the importance of identifying new markers to develop new therapeutic strategies against cancer. Keywords

Cancer · Cancer stem cells · Chemotherapy · Radioresistance · Tumor List of Abbreviations

ABCG2 ALDH1 CSCs Gli GCSF HCS HDECC HMGA2 IFN-α MDR RNS ROS

ATP-binding cassette subfamily-G member 2 (ABCG2) Aldehyde dehydrogenase1 Cancer stem cells Glioma-associated oncogene homolog Granulocyte colony-stimulating factor High-content screening High drug efflux cancer cell inhibitors High mobility group AT-hook 2 Interferon-α Multidrug-resistant proteins Reactive nitrogen species Reactive oxygen species

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Introduction Role of Cancer Stem Cells (CSCs) in the Tumor One of the recurring problems that remain unresolved about cancer is the possibility of developing local and/or distant recurrence (Sopik et al. 2018; Hung et al. 2012). The tumor relapse comes from its multiple cell types’ composition, which contributes to our capacity to abolish tumor to replication. CSCs partially control the cancer heterogeneity CSCs described for the first time in 1928 by Krebs and colleagues. They discovered similarities in the mechanisms involved in tumor development and the development of embryos (Krebs 1947). However, the first real CSC model was reported in a small population of cells considered the leukemia promoter (Terstappen et al. 1991; Bao et al. 2013; Schulenburg et al. 2015). The subsequent studies have shown that the only cell type capable of forming tumors was CSCs. This aspect was demonstrated in different types of cancers when CSCs were implanted into experimental animal models. Only in 2005, the scientists were able to test the presence of these tumor-initiating cells in solid cancers, such as in breast, brain, and intestine cancer, and melanoma using a transgenic mouse model (Cho et al. 2008; Alcantara Llaguno et al. 2009; Barker et al. 2009; Fang et al. 2005). CSCs have been extensively studied and characterized during the last decade. CSCs show similar characteristics to normal stem cells, such as the capacity to grow, self-renew, and disseminate to other tissues and organs during the metastatic process (Bonnet and Dick 1997). CSCs show resistance to apoptosis besides possessing the ability to escape immune surveillance. This involvement of CSCs and lymphocytes has been already discussed in the recently published literature (Ravindran et al. 2019). In a study published by Masciale et al. (2019a), the authors have reported a correlation between cytotoxic tumor-infiltrating lymphocytes CD3+ and CD8+ and lung CSCs, which could be useful for the development of future therapies in the field of precision medicine, tailored on the specific marker besides the different roles of lymphocytes against lung cancer. These data show that lymphocytes may have a strategic role in tumor development and cell-cell interactions. In summary, CSCs interact with tumor epithelial cells, tumor microenvironment, and tumor-infiltrating lymphocytes. They are considered as one of the most important causes of resistance to chemotherapy besides their involvement in radioresistance and loss of the DNA repair of the tumor cells inside the tumor caused by radiations. These also induce the destruction of mitochondrial membranes and functions by reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Schulz et al. 2019; Pilié et al. 2019; Azzam et al. 2012; Schieber and Chandel 2014). The physiologic intracellular mechanisms can maintain ROS’ presence, which may protect the cells against the oxidative stress induced by radiations (Trachootham et al. 2009). Several studies have attempted to identify the leading causes of resistance due to CSCs. They found that many intracellular pathologic factors such as quiescence, dormancy, transporter accumulation, cell reprogramming, interruption of apoptosis, and metabolic pathways may reinforce the tumor resistance to chemotherapy and radiotherapy of this subpopulation of cells. The commonly used treatment for

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oncologic patients, including surgical resection, may reduce the tumor mass but fails to destroy all the cells with the cancer relapse (Arruebo et al. 2011). For this reason, scientists believe that the development of new targeted drugs able to target CSC will be beneficial to overcome tumor resistance. The possibility of CSC-targeted treatment options will provide a novel “door opening door” for the development of more personalized medicine in the field of solid tumors. Clinical trials are underway to study the pharmacokinetics, safety, and efficacy of combination treatment; however, there are no drugs available for lung cancer that specifically could target CSCs (Aramini et al. 2020a). The combination of “old treatments” associated with targeted CSC-specific agents will probably set a more personalized oncologic medicine that may be more promising in terms of response than the existing anticancer therapies.

Markers in CSCS: A New Approach Against Tumor One of the most debated aspects of CSCs is their existence and identification of tumors (Prager et al. 2019). The scientists have attempted to characterize several solid tumors for the presence of CSCs for their surface markers, although the discussion is still open for the complexity of their identification (Sun et al. 2019). Identifying CSCs in human tissue or cell lines is essential to set effective new target therapies against cancer. As CSCs have many characteristics in common with normal stem cells, the scientists believe that specific markers, as superficial markers, are especially useful to identify them (Franco et al. 2016). For their innate characteristics, as the capacity to form non-adherent spheres and their intracellular mechanisms, several assays have been found to identify CSCs (Dou and Gu 2010; Alison et al. 2011; Ghani et al. 2011; Zhang et al. 2011; Dou et al. 2007). In particular, in vitro studies demonstrated that markers as aldehyde dehydrogenase1 (ALDH1), CD133, and others might be suitable for identifying CSCs (Zhao et al. 2017; Kim and Ryu 2017; Klonisch et al. 2008). Besides surface markers, specific signaling pathways, sphere-forming assays, and other relevant specific assays are being developed for CSC identification and characterization (Table 1). However, to demonstrate CSCs’ presence, in vivo experiments are being used to augment previously reported data from the in vitro studies. These data are significant to characterize CSCs in terms of tumor development and dissemination. For this reason, scientists have opted to transplant CSCs into experimental animal models. Despite the importance of these models, which are in use since 1902 for genetic studies on mice (Cheng et al. 2010; Aiken and Werbowetski-Ogilvie 2013), new technologies have emerged during the last decade. For example, 3D culture technologies, which aid preclinical studies for drug testing and promote the tumor-sphere formation, represent the first step in simulating the in vivo model (Ericsson et al. 2013; Zhang et al. 2020; Candini et al. 2020). The determination of this malignant cell subpopulation, CSCs, in the tumor mass remains a complicated process. Surface markers are the most commonly and frequently used for identifying CSCs in many solid tumors (Xia 2014; Sullivan et al. 2010).

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Table 1 Therapeutic attempts to target CSC Target ALDH

ALDH

ALDH1A1

ALDH STAT3

CD133

CD133

Hedgehog

Hedgehog

Cancer type Melanoma

Effector Preclinical result Nanodisc Nanodisc vaccination (ND) vaccination against ALDHhigh CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine mode NSCLC DIMATE, an In lung cancer xenografts irreversible with high to moderate inhibitor of cisplatin resistance, ALDH1/3 combination treatment with DIMATE promoted strong synergistic responses with tumor regression Ovarian cancer Solanum Notch1 and FoxM1 were incanum extract downregulated, which resulted in increased chemotherapeutic sensitivities Ovarian cancer Disulfiram (DSF) DSF enhances cisplatin cytotoxicity in ALDH + cells Gastro/intestinal Napabucasin BBI-608 target and inhibit cancer (BBI-608, gene transcription induced Boston by STAT3 and cancer cell Biomedical Inc., stemness properties MA, USA) Hepatocellular SS2 virus, an SS2 inhibits in vivo growth carcinoma, oncolytic HSV-1 of tumors in subcutaneous colorectal mouse model for cancer, and melanoma, hepatocellular melanoma carcinoma, and colorectal cancer Testicular and BST204-a Treatment with BST204 embryonal fermented (25, 50, and 100 μg/mL) carcinoma ginseng extract inhibited the proliferation of NCCIT cells in a dosedependent manner Bladder Cyclopamine Tumor formation was suppressed via inhibition of GALNT1 that mediates SHH signaling GDC-0449 GDC-0449, an inhibitor of NSCLC and small-cell lung the hedgehog pathway, cancer demonstrated its efficacy in both NSCLC and small-cell lung cancer via suppression of stemness-related features

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Table 1 (continued) Target Hedgehog

Cancer type Ovarian cancer

Effector GDC-0449 (vismodegib derivative) and sonidegib (LDE225)

KLF5

Breast carcinoma

Metformin

KLF4/PI3K/ Akt/p21

Prostate cancer

MicroRNA-7

Wnt/β-catenin Breast cancer

Pyrvinium pamoate

Wnt/β-catenin Breast cancer

Resveratrol

YAP1

Verteporfin

Head and neck squamous carcinoma cells

Preclinical result Since the SHH pathway plays an important role in self-implantation of CSC and other characteristics of these cells, its inhibition may disrupt CSC stemness through differentiation of these cells Metformin suppresses TNBC stem cells partially through the inhibition of KLF5 miR-7 is as a suppressor of prostate CSCs and via suppressing the KLF4/ PI3K/Akt/p21 pathway CD44+CD24
/low and ALDH+ cells were suppressed by downregulating NANOG, OCT4, and SOX2 Resveratrol could eliminate breast CSCs in primary xenografts and consequently abrogate the regrowth of tumors in secondary mice Verteporfin suppresses the proliferation, epithelialmesenchymal transition, and stemness of head and neck squamous carcinoma cells

References Keyvani et al. (2019)

Shi et al. (2017)

Chang et al. (2015) Rodriguez et al. (2019)

Fu et al. (2014)

Liu et al. (2019)

The most established surface markers to identify CSCs are CD133, CD24, and CD44 besides intracellular enzyme aldehyde dehydrogenase (ALDH), which relates well to both solid tumors and lung cancer (Qiu et al. 2019). The main issue regarding surface markers is to discriminate between their isoform expressions in various organs. For example, Al-Hajj et al. found that CD44/CD24 surface marker expression was related to the cells’ capacity to form a tumor mass, while its other isoforms did not show this potential (Al-Hajj et al. 2003). Similarly, Li et al. described similar results have for other organ cancers, i.e., in human pancreatic cancer (Li et al. 2007). Regarding lung cancer, for example, Sullivan et al. 2010 tested ALDH in several cell lines and human tissue samples (Sullivan et al. 2010). In 2019, Masciale et al. compared the isolated ALDHhigh population with the marker CD44+. They found that these two subpopulations did not match exactly and found a broader population

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in CD44+ than in ALDHhigh (Masciale et al. 2019, 2020b). The comparison between ALDHhigh and CD44+/EpCAM+ identified a remarkably similar cell population. This study represents the first attempt demonstrating how markers that seemingly identify CSCs do not always identify similar populations. In summary, the consideration of surface markers is not enough to isolate CSCs and, therefore, necessitate combining surface markers with other mechanisms to identify and target them.

Targeting CSCs The Hedgehog Signaling Pathway CSC formation seems to primarily derive from the dysregulation or the aberrant activation of intracellular signaling pathways which are numerous (Matsui 2016). The most well studied of these signaling pathways include Hedgehog pathway, Notch, Wnt/b-catenin, the high mobility group AT-hook 2 (HMGA2), Bcl-2, and Bmi-1, among many others (Pelullo et al. 2019). Further in-depth knowledge of these mechanisms will allow the researchers to exploit them to find novel anticancer therapies. Signaling pathways generally comprise the expression of a network of genes and their encoded proteins that can regulate diverse intracellular processes. These pathways are significantly involved in self-renewal, maintenance, proliferation, differentiation, and cell migration. In contrast, the aberrant expression of the genes/ protein involved therein or their dysregulation can lead to the development of CSCs, which are the inductors of tumorigenesis (Olivares-Urbano et al. 2020). For example, the Hedgehog signaling pathway controls several biological activities in the normal cells and stem cells’ development, and it is central to the regulation of different solid tumors (Olivares-Urbano et al. 2020; Petrova and Joyner 2014). Hedgehog signaling’s dysregulated activation signaling is crucial for tumor initiation, infiltration, dissemination, and chemoresistance. In particular, the activation of Hedgehog signaling is initiated by the interaction between the Hedgehog protein and the cellular transmembrane patched (Ptch), resulting in a gene cascade control that is crucial for several cell functions as the cells proliferate, disseminate, and self-renew (Jia et al. 2015). The Hh signaling’s abnormal activation induces cellular alteration processes with the consequent transformation of normal cells into cancer cells. In particular, recently published data show that Hedgehog signaling forms the basis of CSC equilibrium in many solid tumors (Pietrobono et al. 2019; Gotschel et al. 2013; Filbin et al. 2013; Gulino et al. 2010), and hence it seems to be one of the leading causes of resistance against cancer therapy (Gurung et al. 2013). Recent studies have studied the effect of inhibiting the Hedgehog signaling pathway (Pietrobono et al. 2019; Gotschel et al. 2013; Filbin et al. 2013; Gulino et al. 2010; Gupta et al. 2010). For example, the drugs, which inhibit the molecule smoothened (SMO), i.e., cyclopamine and GDC-0449 (vismodegib), do not perform well against cancers harboring lesions that lie downstream of the SMO (Gupta et al. 2010). Other drugs seem to be effective against the glioma-associated oncogene

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homolog (Gli) proteins and the Gli transcriptional activity or the combination of different factors with the inhibition of CSCs’ main characteristics, for example, the capacity to self-generate (Gurung et al. 2013).

The Notch Signaling Pathway The Notch pathway has a vital role in the communications that occur among adult stem cells and during embryo development (Yu et al. 2008; Nguyen et al. 2006). Four receptors (Notch-1–Notch-4) and their ligands Deltalike1, Deltalike3, and Deltalike4 and Jagged 1 and Jagged 2 activate the Notch with a cascade of multiple genes (Serneels et al. 2005; D’Souza et al. 2010; Kovall et al. 2017). The Notch pathway’s prominent role is in stem cell evolution, from initiation to apoptosis, but it also has an oncogenic/oncosuppressive role, depending on the tumor microenvironment (Nowell and Radtke 2013). Besides its pivotal role in intercellular communication, Notch signaling’s most interesting characteristic is its double function as an oncogenic protein in several solid tumors. In contrast, it acts as a tumor suppressor in others (Dotto 2008). Given this differential role, researchers have started to consider it a potential novel target to eliminate CSCs (Wang et al. 2010, 2011; Pannuti et al. 2010; Qiao and Wong 2009; Fan et al. 2010). Tetering et al. have reported that abrogation of the Notch pathway blocks the proteolytic process (Van Tetering and Vooijs 2011). Lim et al. used g-secretase inhibitors (GSIs) to reduce primary stem cell characteristics (Lim et al. 2015). They asserted that blocking the Notch pathway in glioblastoma helps establish new target therapies to counter CSCs. Treatment with GSI MRK-003 destroys cancer stemlike cells, with the consequent blocking of the tumor and dissemination into the body (Venkatesh et al. 2018; Grudzien et al. 2010; Ng et al. 2019). The only limitation is that GSI MRK-003 is nonselective as it could block four Notch ligands and several substrates of the g-secretase. Despite interesting data, further mechanistic investigations are required to understand its toxic effects on human organs.

The Wnt Signaling Pathway Another critical pathway subject of recent interest and useful for developing future generations’ targeted therapies against CSCs is the Wnt signaling pathway, which is involved in all the steps about the stem cell generation, progression, up to differentiation (Ng et al. 2019; Komiya and Habas 2008). The most rigorously defined Wnt pathways are the Wnt/b-catenin signaling pathway that starts when a Wnt glycoprotein binds with a cell receptor to initiate the activation of a cascade (Macdonald et al. 2009; Franch-Marro et al. 2008). The role of the Wnt pathway is crucial for stem cell regeneration and the preservation of CSCs; however, mutations in the signaling molecules or inactivation of cell processes may induce the dysregulation of this pathway with the induction of cell proliferation and tumor dissemination (Huang and He 2008; Itasaki et al. 2003; Itoh et al. 2005).

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Nevertheless, mutations can interfere not only with the Wnt signaling pathway but also with the tumor microenvironment. For example, biologic inhibitors or drugs may block or prevent the interactions between the ligand and the receptor. These small molecules can inhibit the Wnt target enzymes, reduce CSCs’ drug resistance and proliferation, etc. (Jamora et al. 2003; Artem et al. 2020). A study published in 2017 by Wiese et al. (2017) showed that the drug ICG-001 might positively effect tumorigenicity of pediatric glioma tumorigenicity by a Wnt-independent regulation of genes. Another technique used for inhibiting Wnt signaling is the RNA interference technology to downregulate the betacatenin expression, resultant reduction in several CSC properties, and the silencing of stemness genes as OCT-4 (Hadjimichael et al. 2015). In summary, targeting the Wnt pathway is another practical and effective approach in developing and investigating the future of targeted treatment approach for solid tumors. This capacity to target this mechanism will also be essential to better control normal stem cell activities, differentiating CSCs from normal ones in tumors (Takahashi-Yanaga and Kahn 2010; Yang et al. 2020; Vassalli 2019). This aspect would help target only the cancerous cells in the tumor.

Targeting ALDH as the Drug-Detoxify Enzymes Against CSCs ALDH is the most used marker for the identification of CSCs. During these past 10 years, several publications described its role as a useful and reliable predictive marker for CSCs (Masciale et al. 2019, 2020; Vassalli 2019; Ho and Weiner 2005; Walcher et al. 2020; Croker et al. 2009; https://clinicaltrials.gov/; Singh et al. 2013; Croker et al. 2017). In particular, in 2010, Sullivan et al. showed that ALDH-positive cells had the characteristics of cancer stemlike cells in NSCLC lines (Sullivan et al. 2010) and lung primary cell cultures. ALDH has also been described in benign diseases (Ho and Weiner 2005) although it is highly expressed in lung epithelial cancer cells. This aspect is exciting and opened the door of recent studies on ALDH as a NAD(P)þ-dependent enzyme catalyzing the aldehydes’ oxidation into carboxylic acids (Ho and Weiner 2005). ALDH superfamily encompasses different isoenzymes, but the most used in detecting CSCs are ALDH1A1 and ALDH1A3. However, the possibility of targeting to set personalized therapy for CSCs is still debated (Walcher et al. 2020). For example, Masciale V. et al. recently demonstrated that the ALDHhigh cell population does not correlate with CD44+ cells in lung adenocarcinoma. However, the ALDHhigh cells are positively correlated with CD44+/EpCAM+ cell subpopulation (Masciale et al. 2020). Croker et al. (2009) demonstrated a subpopulation of cells identified by the ALDH marker, among others, might be the driver of metastasis in breast cancer and the cause of resistance to traditional chemotherapies or radiotherapy in epithelial cancer cell lines. However, although some clinical trials on ALDH (disulfiram and DIMATE) are running at the moment trying to identify a drug which can be effective against solid tumors (Singh et al. 2013; Croker et al. 2017), there is not enough clarification about ALDH isoform markers in terms of their predictive value and

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targeted therapy against cancer. Several future studies are needed to increase the knowledge about this important family of enzymes.

Targeting Drug Efflux Pumps The efflux systems to get rid of the xenobiotic substances in the cells are involved in diverse CSCs’ processes similar to their involvement in normal stem cells. Their presence protects the cells from xenobiotic molecules such as chemotherapeutic agents, toxins, and drugs in general. Many ABC transporters’ family members mediate the efflux of cytotoxic chemotherapeutics and are called multidrug-resistant (MDR) proteins. MDR proteins are the ATP-binding cassette subfamily-G member 2 (ABCG2) described in breast cancer resistant cells (BCRP) involved in CSC compartments as some MDR transporters are much more prominent in CSCs than cancer cells and healthy cells (Prabavathy et al. 2018). ABCG2 overexpression maintains stem cell compartments by supporting their ability to counteract chemotherapeutic agents (Xiao et al. 2006). ABCG2 is a characteristic feature of CSCs akin to other non-CSCs with self-renewal, lineage capacity, tumorigenicity, and expression of stem cell-specific markers (Xiao et al. 2006). To visualize and identify potent high drug efflux cancer cell inhibitors (HDECC), scientists applied an image-based high-content screening (HCS) starting from 1280 pharmacologically active molecules (Xia et al. 2010; Ansbro et al. 2013). This assay showed that these inhibitors could overcome multidrug resistance, increasing the chemotherapy’s efficacy or reducing the tumorigenicity of cancer cells that might affect stemlike cancer cells. The ABCG2 seems to be fundamental in detecting the side population of various cancer types. It is essential to consider that these abovecited markers equally identify CSCs and normal healthy stem cells. Information should be taken into account during targeted therapy to find the best treatment, avoiding side effects (Wijaya et al. 2017). Therefore, the combined use of chemotherapy and inhibitors precisely targeting ABC drug transporters of CSCs may serve as an effective strategy for eradicating CSCs.

Targeting the CSC Niche and the Quiescent State The stem cell niche provides an ideal tissue microenvironment conducive to the growth and maintenance of stemness (Morrison and Spradling 2008). Regarding this aspect, both CSCs and normal stem cells reside in their respective CSCs’ niche or a stem cells’ niche. The conducive niche microenvironment provides nourishment and adequate signals to regulate, first, self-renewal and, second, normal physiological processes such as those of inflammation, epithelial to mesenchymal transition (EMT), hypoxia, and angiogenesis (Morrison and Spradling 2008). In particular, stemness depends on extrinsic factors disposed of by the cancer microenvironment (Cabarcas et al. 2011), as happens in the colon CSC population, described by Vermeulen et al., in which the Wnt pathway regulates the colon CSCs (Vermeulen

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et al. 2010). Moreover, it is a condition in which the brain tumor’s microvasculature was crucial for niche and consequently for the support of CSCs (Seidel et al. 2010). For that reason, the vascular niches should be targeted for future therapeutic strategies to affect CSCs. Hypoxia has a crucial role in tumor growth since the hypoxic tumor microenvironment also sustains CSCs (Seidel et al. 2010). Regarding hypoxia, antiangiogenic therapies promote hypoxia within the CSCs’ niche, causing radioresistance to the CSCs, as occurring with the use of sunitinib and bevacizumab, as described by Conley and Wicha (2012). In this study, there was an intra-tumoral hypoxic environment created that leads to a stimulation of CSCs. Consequently, the study suggested that antiangiogenic treatments in combination with CSC-targeted drugs may be a better and more effective option. For example, it is essential to note the positive anticancer effects produced by the inhibitors for hypoxia-inducible factor1a (HIF-1a) that could serve as a basis for effective therapy (Zhong et al. 2000). Indeed, targeting the CSCs’ niche in combination with chemotherapy will be a therapeutic intervention approach for eradicating these cells. This is fundamental for decision-making during cancer therapy because CSCs are resistant to traditional chemotherapy due to their quiescent state (Iwasaki and Suda 2009; Cheung and Rando 2013). Current treatment strategies can inhibit tumor growth or DNA synthesis but are ineffective as a long-term response to cancer. Unlike other cancer cells, CSCs can remain resting, shielded from the external stimuli to prevent their replicative potential (He et al. 2017; Cheung and Rando 2013). CSCs are slow-cycling cells, a characteristic that helps them protect against chemoradiotherapy agents (Kusumbe and Bapat 2009). Therefore, strategies that induce CSCs to enter the cell cycle will enhance their susceptibility to chemotherapy (Plaks et al. 2015). Recent research has shown interferon-α (IFN-α), granulocyte colony-stimulating factor (G-CSF), or As2O3 is useful in this regard (Han et al. 2013). Therefore, the combination of IFN-α, G-CSF, or As2O3 with chemotherapeutic factors may effectively target the dormant CSCs. The future advances in anticancer treatment may be characterized by therapeutic methods that can target the stem cell niche, affecting the primary source of nourishment and intracellular signal for CSCs (Han et al. 2013).

An Update on the Most Used Target Therapies Against CSCs One of the main areas necessitating innovation in cancer treatment is studying the mechanism of the development of resistance and relapse of the disease. This is generally considered a kind of failure in anticancer therapy development due to an apparent lack of specific targeting that directly attacks the tumor. During the past 10 years, stem cells have attracted interest in the scientific community for biological characteristics such as self-renewal, the capacity to migrate, and the modulation of cell interactions, among others. The scientists have mainly focused their attention on discovering new markers to better identify these cell populations (Plaks et al. 2015; Han et al. 2013; Xuan et al. 2019). Diverse approaches have been considered for

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Fig. 1 Advanced cancer therapy. New perspectives in cancer therapies consist of different approaches using the great potential of the MSC by using genetic engineering, oncolytic virus, and nanoparticles releasing drugs or exosomes. Moreover, immunization technique with both vaccines and chimeric antibody is also investigated to enhance the immune response against cancer

stem cell therapy: HSC transplantation, MSC infusion, stem cells as carriers or generators of immune cell interactions, and vaccine production (Fig. 1). In the last decade, biological characterization of CSCs has remained an area of immense interest, including their potential for self-renewal, the capacity to migrate, the modulation of cell interactions, etc. (Plaks et al. 2015; Han et al. 2013; Xuan et al. 2019). In particular, scientists have focused their attention on discovering new markers to identify these cell populations. There are ongoing difficulties in identifying unique markers. In particular, several vital drugs are not always effective in anticancer therapy, which is why patients have a frequent relapse and eventually succumb to the disease. One of the most urgent problems that require the immediate attention of researchers is drug resistance, which remains a challenge for modern medical oncologic treatments (Xuan et al. 2019). In particular, scientists discovered a small population of cells during the past two decades, similar to stem cells in their intrinsic characteristics but derived from the tumor itself. These cells are defined as CSCs and known for their self-renewal, differentiation, and tumorigenicity: these traits seem to be what causes cancer relapse and tumor dissemination (Han et al. 2013; Xuan et al. 2019; Kuczynski et al. 2019). Several interventional approaches have been tested to target this population of cells; however, currently, the two most utilized techniques in targeting CSCs are virotherapy through the oncolytic virus and miRNAs as CSC targets (Fig. 1). An oncolytic virus (OV) destroys tumor cells with mechanisms that work differently from traditional medical treatments (Jain and Stylianopoulos 2010). These

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mechanisms involve specific genes against cancer cells. However, when defining the possibilities and advantages of virotherapy, we should be aware and conscious of the limitations that hinder the development of new therapies for solid tumors. Scientists are attempting to exploit specific characteristics of the virus, i.e., tropism, the viral capacity of diffusion into the body, antiviral immunity, the delivery platforms, etc. (Maroun et al. 2017). However, the unique obstacles hindering their development include the physical barriers that prevent viral diffusion into the tumor and its passage through the endothelium (Kuczynski et al. 2019). A significant barrier for the OVs can be the host’s immune surveillance mechanism. Bioactive factors, including cytokines and chemokines, and growth factors secreted by the tumor cells activate immune cells against OVs. Additionally, the OVs may develop a robust innate response with an uncontrolled reaction against cancer but also against other tissues (Jain and Stylianopoulos 2010; Maroun et al. 2017; Hagedorn and Kreppel 2017; Guedan and Alemany 2018; Sharp and Lattime 2016; Nayyar et al. 2019; Oh et al. 2018; Vaupel 2004). It is pertinent to mention that OVs are capable of stimulating an effective immune response, both specific and innate, and the massive release of coagulation factors together with the complement proteins (Jain and Stylianopoulos 2010; Maroun et al. 2017; Hagedorn and Kreppel 2017; Guedan and Alemany 2018; Sharp and Lattime 2016; Nayyar et al. 2019; Oh et al. 2018; Vaupel 2004). It remains undetermined how many OVs reach the tumor-specific site, as they are very likely to be eliminated by the immune system (Hagedorn and Kreppel 2017; Guedan and Alemany 2018; Sharp and Lattime 2016). Once OVs target the tumor, the most crucial aspect is that they maintain intrinsic properties, such as the capacity to induce immune suppression of the TME to kill cancer cells and block tumor cell dissemination. Unfortunately, current studies are not setting out how to improve virotherapy’s effectiveness against cancer in terms of tumor targeting, virus delivery, and dissemination. Therefore, it is imperative to distinguish the best virus species for OVs to reduce the events of tumor-targeting failures (Zheng et al. 2019).

Oncolytic Adenovirus During the past two decades, oncolytic adenovirus (Ad) has gained popularity for cancer gene therapy primarily due to its ability to precisely infect and replicate in tumor cells. Given that the oncolytic antitumor activity is inadequate to eradicate tumors, different strategies based on genetic engineering have been developed to improve the efficacy of the cancer-targeting gene-virotherapy (Liu et al. 2012; Zhang et al. 2003). There are examples of genetically modified Ad eliminating E1B protein which blocks a p53-independent apoptosis mechanism or a portion of E1A involved in binding the retinoblastoma (Rb) protein since the significant part of CSCs has a defect in Rb and/or p53 pathway (Wang et al. 2015b; Lei et al. 2013). For this transcriptional target therapy, a specific cancer promoter has been used to control the

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initial phases of the viral replication. Recently, GP73 protein and its promoter were used to regulate the activity of the E1A and E1B proteins to treat hepatocellular carcinoma (Mao et al. 2010; Fimmel and Wright 2009; Wang et al. 2015; Zhang et al. 2016). For example, the ZD55 and GD55 oncolytic viruses were tested for efficacy against lung CSCs. The result, much higher with GD55, was a significant increase in apoptosis rate due to the cytotoxic effect and oncolysis. Indeed, GD55 could represent a hopeful agent to develop target therapy for lung CSCs. Regarding ZD55, as reported by Zhang and colleagues (2003), the increased acetylcholinesterase activity induced by the OV is responsible for a substantial antitumor effect against gastric cancer. This result is achieved by inhibiting the tumor growth in the gastric cancer cells and stem cells, whereas a low dosage of the ZD55 OV affects apoptosis (Zhang et al. 2003; Xu et al. 2014). Instead, Yano et al. investigated the efficacy of OB-301 telomerase-specific OV against gastric cancer cells (Yano et al. 2013). The oncolytic adenovirus allowed an efficient response by the killing of CD133+ cells responsible for chemoresistance and radioresistance. The CD133+ cells contributed to protection through their ability to remain in a dormant state were pushed to enter the cell cycle and then became a target to be killed by OBP-301.

Oncolytic Herpes Simplex Viruses The oncolytic herpes simplex virus (OncoHSV) belongs to the neurotropic virus category, which is primarily effective against cancers of the nervous system (Li et al. 2020). For example, the FDA has authorized a phase III trial to treat melanoma using the oncolytic virus named T-VEC, which is administered either alone or in combination with other medical therapies (Bommareddy et al. 2017). Even if the OncoHSV is specific for neurological malignancies, their efficacy has also been tested in colon cancer with encouraging data. There was a selective killing of the cancer stemlike cells in vitro and in vivo (Bommareddy et al. 2017; Hill and Carlisle 2019). As for the cancer of the nervous system, the designed OncoHSV named YE-PC8, which regulates the cell cycle, was responsible for 80% of the tumor regression in glioma (de Sostoa et al. 2019; Kahramanian et al. 2019; Dmitrieva et al. 2011; Mckee et al. 2006; Guedan et al. 2010; Desjardins et al. 2018). Regarding metastatic gastric cancer resisting the standard chemotherapy treatments, the use of OncoHSV NV1020 was considered as a novel treatment option and led to an overall improved survival during a phase I clinical study due to reduced tumor growth (https://clinicaltrials.gov/ct2/show/NCT00149396). Also, another OncoHSV directed against gastrointestinal cancer showed promising results avoiding side effects (Yang et al. 2016). A recent study has demonstrated that another OncoHSV was potent in the killing of glioma CSCs (Kambara et al. 2005). This oncovirus, developed by the Kambara laboratory, allowed nestin expression in the embryonic neuroglial cell inhibiting tumor growth (Kambara et al. 2005).

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Oncolytic Virus and Its Effects in Recent Clinical Trials Recent preclinical studies have investigated OVs’ role in solid tumors using an experimental pancreatic model (Eissa et al. 2018). Besides that, these models are complex and are only used for a specific virus; the oncolytic ONYX-015 adenovirus has already been tested in phase I and phase II clinical trials. ONYX-015 (dl1520) is an E1B-55 kDa gene-deleted virus replicating selectively in p53-mutated tumor cells (Mulvihill et al. 2001). During the phase I clinical trial, 23 patients with inoperable pancreatic cancer were enrolled to receive ONYX-015 at 4 weeks by a computed tomography injection. Most of the patients responded well and gave encouraging data, except for one patient who developed pancreatitis. No adverse effects were reported in the study; however, no objective response against the tumor was observed during the phase I study. In order to enhance the effectiveness of ONYX-015 therapy, phase II clinical trials were designed involving the use of a combination of ONYX015 with gemcitabine (Mulvihill et al. 2001). A total of 21 patients with advanced and metastatic pancreatic adenocarcinoma were enrolled in the phase II study (Mulvihill et al. 2001). Phase II studies reported two partial regressions and two minor responses without any severe side effects. Some more clinical trials are currently underway to assess VCN-01 and LOAd703, preparations either alone or in combination with nab-paclitaxel/gemcitabine in metastatic pancreatic cancer (NCT02705196, NCT02045589, and NCT02045602).

Oncolytic Adenoviruses and Pancreatic Clinical Trials Preclinical studies investigating the role of OVs in solid tumors have been recently set. In particular, the pancreatic model has been used. Althought these models are complex and developed only for a specific virus, the oncolytic ONYX-015, specific for p53-mutated cells (Mulvihill et al. 2001). In a phase I dose-escalation clinical study, 23 patients have been enrolled with a diagnosis of inoperable pancreatic cancer. ONYX-015 was administered every 4 weeks by a computed tomography injection, and the results were generally satisfying except for one patient who developed an episode of pancreatitis. Although the results seem to be without adverse effects, no objective response against the tumor was observed in the phase I clinical study; however, most of the patients showed low level of cellular immunity (indicated by low number of CD4 cells). Moreover, no viral replication was observed in any of the patients receiving ONYX-015. In the light of these data, a combination of ONYX015 and gemcitabine was used during a subsequent phase II clinical study involving 21 patients with advanced and metastatic pancreatic adenocarcinoma without liver metastasis (Hecht et al. 2003). The trial was designed to ascertain the safety, feasibility, and tolerability of the combinatorial therapy approach of chemovirotherapy. The authors reported partial regression of the injected tumors in two patients, while another two patients showed minor responses with no severe side effects. The remaining patients either had a

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stable disease progression or had to leave the study due to treatment intolerance. Other two clinical trials are running at the moment (VCN-01 and LOAd703), either alone or in combination with nab-paclitaxel/gemcitabine in metastatic pancreatic cancer (NCT02705196, NCT02045589, and NCT02045602).

Oncolytic Herpes Simplex Viruses and Pancreatic Clinical Trials Concerning therapies using herpes simplex viruses, seven viruses were selected for their assessment during phase II clinical trials for different solid tumors (Table 2). Only HF10 and T-VEC were studied during these trials to treat pancreatic cancer. During a single-arm, open-label phase I clinical study, eight patients were enrolled to receive HF10 intra-tumoral injection of HF10 during surgery. Each patient received injection every 2 weeks for up to 4 weeks. The results of the study showed that three patients achieved stable condition; one showed partial remission, while other showed a progression. Three patients showed an increase in the tumor-specific marker CA19.9. Histological studies showed that HF10 activated macrophages as well as natural killer (NK) cells (Hirooka et al. 2018; Nakao et al. 2011). However, the results failed to show any significant remission of the disease despite that molecular connections are being identified between CSCs and macrophages as part of the novel strategies to treat cancer (Aramini et al. 2021). Another clinical trial studying unresectable pancreatic cancer is in progress to test the safety and efficacy of HF10 in combination with gemcitabine, nab-paclitaxel, or S-1 (tegafur/gimeracil/oteracil, TS-1 ®) (NCT03252808) (https://clinicaltrials.gov/ct2/show/NCT03252808). Two more clinical trials were designed after encouraging data from phase I clinical trial

Table 2 MicroRNAs and oncolytic viruses for possible future therapy in solid tumors: preclinical results Organ Lung

Breast

Brain

miRNAs [Ref.] miRNA-106b-5p Yu et al. (2017) miRNA-7 Zhao et al. (2015) miRNA-99 Yu et al. (2015) miR-218-5p Zhu et al. (2016) miR-142-3p Troschel et al. (2018) miRNA-150 Isobe et al. (2014) miR-203 Muhammad et al. (2016) miR-8084 Gao et al. (2018) miR-21, miR-221 Wang et al. (2015)

Thyroid

miR-21, miR183, miR-375 Chu and Lloyd (2016)

Ovary

miR-628-5p (171)

Oncolytic virus [References] Ad5D24-CpG Garofalo et al. (2018) VSV-IFNβ Patel et al. (2020) MYXV Kellish et al. (2019) H101 Lei et al. (2015) MG1 Bourgeois-Daigneault et al. (2016) G47Δ-mIL12 Ghouse et al. (2020) MRB Mullins-Densereau et al. (2019) NDV Al-ZIyadi et al. (2020) DNX-2401, PVS-RIPO, Toca 511 Martikainen and Essand (2019) H-1PV Geletneky et al. (2017) dl922-947 Heise et al. (2000) NV1020 Jiang et al. (2018) VB-111 Reddi et al. (2011) CRAd-S-pk7 Mooney et al. (2018)

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with T-VEC in 17 patients with pancreatic cancer. T-VEC doses were well tolerated, however, without an expected objective response (Conry et al. 2018).

Stem Cell-Derived Exosomes as Therapeutic Carriers During the last decade, novel approaches in cancer cell targeting have been found and mainly involved microRNAs (miRNAs) and CSCs (Wang et al. 2011; Khan et al. 2010). Despite the successful outcome from several studies, a secure target regarding miRNAs or CSCs in solid tumors has not been identified yet. MiRNAs are small noncoding molecules able to regulate at least 60% of human genes, their expression, and hence their downstream signaling that affects diverse cellular functions ranging from metabolism to proliferation and differentiation. Hence, they are being used as part of the theranostic strategies (Haider and Aramini 2020). Like any other cell type, they are critical determinants of stem cell functions encompassing survival to proliferation of stem cells (Kim et al. 2010, Lai et al. 2011, 2012). Changes in miRNA profile also enhance solid tumors’ induction and alter CSC stemness properties (Ruggieri et al. 2019; Aramini et al. 2020b). As previously described, CSCs appear to be involved in many processes regulating tumor and are often associated with poor clinical outcomes (Aramini et al. 2020b). One of the most exciting aspects recently studied miRNAs’ role in CSC regulation and preservation through pathways as wingless (WNT)/β-catenin, Notch, JAK/STAT, PI3/AKT, etc. (Wang et al. 2011). These data are reckoned as a milestone for the identification of new target treatments against cancer. In particular, we have observed the association between miRNAs and their effects on CSCs’ characteristics: self-renewal capacity, ability to differentiate, involvement in the metastatic process, etc. These data seem to be linked with several solid tumors. More recently, Khan et al. have reported an altered mRNA profile with a selective reduction in the number of miRNAs associated with CSCs, which may be a vital aspect in discovering new treatment targets (Khan et al. 2010). Several miRNAs have emerged as potential oncogenic targets with a double role as tumor suppressors and tumor inductors. In breast cancer, miR-200 interacts with the tumorigenic process negatively, while members of miR-34 facilitate cell processes and apoptosis. In different solid cancers, downregulation of miR-34 and let-7 is responsible for oncogenesis, although their upregulation stimulates the suppression of cancer development (Khan et al. 2010; Ruggieri et al. 2019; Aramini et al. 2020b; Diana et al. 2019). A similar oncogenic activity has been confirmed for other miRNAs, i.e., miR-181 and miR-155, which seem to promote self-renewal, the formation of niche, and tumor development and progression. In prostate cancer, downregulated expression of miRNAs, i.e., miR-34a, let-7b, miR-106a, and miR-141, has been observed in CSCs, whereas other miRNAs, i.e., miR-301 and miR-452, were highly expressed (Wang et al. 2011; Khan et al. 2010). Moreover, altered expression of miRNAs has been reported in breast cancer CSCs, with downregulation of miR-203 and miR-375 and concomitant upregulation of

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miR-100, miR-221, miR-222, and miR-125b (Diana et al. 2019; Howard and Yang 2018; Ding et al. 2019). We summarize here the most active miRNAs involved in CSC regulation in different solid tumors (Table 2). These clinical trials focused on pancreatic cancers, and only HF10 and T-VEC were studied. During phase I clinical trials, eight patients were enrolled to receive an intra-tumoral injection of HF10 during surgery, with a stable condition in three patients, partial remission in one, and a progression in the others. Three patients showed an increase in tumoral marker CA19.9. Histologically, HF10 activated macrophages besides the activation of NK cells (Lim et al. 2019). However, the results do not show an improved remission of the disease. Another recent trial in unresectable pancreatic cancer is in progress to test the safety and efficacy of HF10 plus gemcitabine and nab-paclitaxel and S-1 (tegafur/ gimeracil/oteracil, TS-1 ®) (NCT03252808). The other two trials have been set without particular results, as expected, as phase I trial with T-VEC in 17 patients with pancreatic cancer. T-VEC doses were tolerated well; however, no objective response has been shown (Nakao et al. 2011).

Conclusions and Future Directions Besides our knowledge about the significance of miRNAs, CSCs, and OVs for developing future targeted anticancer therapies, the prospect for these treatments’ efficacy remains less well defined. This is primarily attributed to the nonavailability of optimal and standardized isolation and purification protocols and the shortage of specific CSCs’ specific markers. Secondly, the unreliability of virotherapy continues to be problematic. Thirdly, there are many instances wherein both upregulation and downregulation of the same miRNAs occur in different types of tumors, signifying the importance of altered miRNA profile for specific miRNAs. The scientific community has a big responsibility in optimizing these new approaches for future generations of patients who will develop solid tumors. Of course, new innovative thinking is desirable; nevertheless, improved life quality in terms of duration and well-being must be considered in the clinical process. Several clinical trials are underway regarding the possibility of finding a practical approach in treating advanced cancers. However, the future of the oncological field is mainly focused on the prevention of cancer recurrence at early stages and prediction when and if cancer will return. In conclusion, the actual results regarding the role of CSCs in oncogenesis are currently less than satisfactory and therefore necessitate further in-depth investigations in the future.

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Bioengineering Technique Progress of Direct Cardiac Reprogramming

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A New Perspective from Microbubbles and UTMD Dingqian Liu, Khawaja Husnain Haider, and Changfa Guo

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pluripotent Stem Cells in Myocardial Regeneration and Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . The Strategy of Direct Reprogramming to Generate Cardiomyocytes . . . . . . . . . . . . . . . . . . . . Observations and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis and Characterization of CMBs and Lentivirus-Binding Capacity . . . . . . . . . . . . . . Reprogramming of Rat CFs into iCMs by the GMT Lentivirus Vectors In Vitro . . . . . . . . UTMD-Mediated Localized Delivery of Direct-Reprogramming GMT Lentivirus–CMB Hybrids In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of the Combinatorial Approach of UTMD with CMB-Lentiviral Hybrid . . . . . . CMB–Lentivirus Hybrids in Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages of Lentiviral Vectors for Direct Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improvement of Reprogramming Factors in Direct Cardiac Reprogramming . . . . . . . . . . . . Ultrasound-Mediated Localized Direct Reprogramming via Lentivirus Delivery for Cardiac Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Summary of the Experimental Methods and Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vectors and Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Creation of CMBs and Microbubble–Virus Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct Reprogramming of CFs In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Western Blot Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of Experimental Rat Model of MI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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D. Liu · C. Guo (*) Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China e-mail: [email protected]; [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_27

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Delivery of Microbubble–Lentivirus Hybrids Using UTMD In Vivo . . . . . . . . . . . . . . . . . . . . . Histological Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac Magnetic Resonance Imaging (cMRI) and Echocardiography . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The combination of heart-specific transcription factors GATA4, MEF2C, and TBX5 (GMT) has been proven to have the ability to directly reprogram cardiac fibroblasts; this approach is considered a promising regenerative technique. Meanwhile, research on microbubbles as biological vectors has made great progress in recent years. This study describes the loading of GMT lentiviral vectors on cationic microbubbles and the release of these direct-reprogramming vectors into an infarcted myocardium by ultrasound targeted microbubble destruction (UTMD) to repair the cardiac tissue. Lentivirus which encode GATA4, MEF2C, and TBX5 transcription factors were generated via a lentiviral production system and were confirmed to have a direct reprogramming ability in vitro. Combined with the cationic microbubbles, UTMD-mediated gene delivery was evaluated, and the gene transfection efficiency was optimized in an in vitro experiment on rat cardiac fibroblasts. With UTMD-mediated direct reprogramming, the viral vector particles were directly deposited in cardiac tissue and repaired the infarcted myocardium. An immunofluorescence assay and histological examination confirmed newborn cardiomyocytes and neo-angiogenesis after a 4-week follow-up of the treated rats. All treated groups showed ventricular-function improvement according to cardiac magnetic resonance imaging and echocardiography. This chapter reports a novel strategy for the delivery of direct-reprogramming lentiviral vectors to a target acute myocardial infarction zone by using UTMD as a tissue repair therapy. Keywords

Bioengineering · Cardiac · Cationic · Direct reprogramming · iPSCs · Ischemic heart disease · Microbubbles · Myocardium · Stem cells · UTMD Abbreviations

CFs cTnI cTnT GMT H&E HDAC HIV iCMs IHD

Cardiac fibroblasts Cardiac troponin-I Cardiac troponin-I Gata4, MEF2c, and TBX5 Hematoxylin and Eosin Histone deacetylase Human immunodeficiency virus Induced cardiomyocyte-like cells Ischemic heart disease

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Induced pluripotent stem cells Left anterior descending Left ventricle LV-ejection fraction Microbubble ultrasound contrast agents Myocardial infarction Ultrasound-targeted microbubble destruction

Introduction Ischemic heart disease (IHD) is the leading cause of heart failure (Moran et al. 2014). According to the recently published report by the American Heart Association, cardiovascular diseases incur one of the highest health and economic burdens globally (Virani et al. 2021). Although the contemporary surgical and pharmacological options provide symptomatic relief to the patients in most cases (Santucci et al. 2020), these treatment options fail to recover the ischemic damage incurred at the structural and cellular levels in terms of massive cardiomyocyte loss. Heart transplantation is widely used as a gold-standard treatment option; organ transplantation still has disadvantages, including an insufficient number of donor organs and the need for long-term immunosuppressant drug therapy (von Dossow et al. 2017). Given that adult cardiomyocytes have only limited regenerative capacity (Bergmann et al. 2009; Mollova et al. 2013), the intrinsic repair mechanism by the resident cardiac stem cells to recover the massive loss of functioning myocytes is only restricted and less than optimal. Although protocols have been reported to reenter the surviving cardiomyocytes in the peri-infarct region into the cell cycle or stimulate the resident stem cells to participate more efficiently in the repair process, the success has been limited (He and Zhou 2017). Attempts have been made to directly reprogram cardiac fibroblasts by myocardial delivery of GATA4, Mef2, and Tbx5 to adopt cardiomyocyte phenotype in experimental animal studies (Qian et al. 2012). Alternatively, stem cell-based therapy has a promising future in cardiac regenerative medicine. Since the publication of the first report of cell-based therapy in a 57-year-old patient undergoing coronary artery bypass grafting (Menasché et al. 2001), the novel stem cell-based intervention has progressed from bench to bedside, advancing to multicenter randomized Phase II and Phase III clinical trials, primarily focusing on the use of bone marrow-derived stem cells (Heldman et al. 2014; Mathiasen et al. 2015; Hare et al. 2017). Despite the well-established safety aspects of cell-based therapy for myocardial repair in preclinical and clinical studies, the efficacy in most cases, especially in clinical studies, has been modest. These modest clinical data have been ascribed mainly to the cell-type selection and the quality of the cells used for transplantation (Shahid et al. 2016; Haider 2018). In this regard, pluripotent stem cells, mainly induced pluripotent stem cells (iPSCs), have given new impetus to cell-based therapy.

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Pluripotent Stem Cells in Myocardial Regeneration and Repair From among the pluripotent stem cells, undifferentiated embryonic stem cells (ESCs) and their derivative cardiomyocytes have been extensively studied in experimental animal models for myocardial repair and regeneration. The transplanted cells differentiate into morphofunctionally cardiomyocytes, endothelial cells, and vascular smooth muscle cells as part of their mechanism to regenerate infarcted myocardium and preserve global cardiac function (Min et al. 2002; Nelson et al. 2006; Caspi et al. 2007; Rufaihah et al. 2007, 2010; van Laake et al. 2008). However, ESCs have not progressed beyond preclinical experimental studies due to moral and ethical issues associated with their availability and use, and tumorigenic potential. Pre-differentiation strategy of ESCs to form cardiomyocytes rather than using undifferentiated ESCs has been pursued to address the tumorigenicity concerns. However, the contaminant population of undifferentiated or incompletely differentiated cells still remains a concern for in vivo use. Hence, various genetic and nongenetic methods of selection and enrichment of pluripotent stem cell-derived cardiomyocytes have been reported (Lin et al. 2010; Ban et al. 2013, 2015). However, all this is still in experimental phase. In 2006, Takahashi and Yamanaka were the first to successfully generate iPSCs from adult somatic cells by integrating-virus-based delivery of a classic combination of four transcription factors, i.e., Oct3/4, Sox2, cMyc, and Klf4 (Takahashi and Yamanaka 2006). The reprogrammed cells were pluripotent and were considered as surrogate ESCs due to similarity in biological and stemness characteristics, however, without ethical and moral strings associated with the production and application of ESCs (Ibrahim et al. 2016). Although this groundbreaking discovery gave new impetus to stem cell-based therapy, tumorigenicity during preclinical assessment raised serious concerns about this progress to the clinical use in the patients (Ahmed et al. 2011; Buccini et al. 2012). Various strategies have been adopted to generate iPSCs to fully exploit their theranostic applications from drug development to disease modelling in vitro. They are also suitable for patient use in the clinic. These strategies include the use of a smaller number of transcription factors, mainly the exclusion of cMyc (Nakagawa et al. 2008, 2010; Giorgetti et al. 2009), the use of non-integrative viral vectors (Chou et al. 2011; Macarthur et al. 2012), and nonviral vectors for transcription factors’ delivery (Huangfu et al. 2008; Qu et al. 2012). Somatic cell reprogramming protocols have also used a combination of small molecules and pluripotency-related transcription factors to overcome the low efficiency of classical transcription-based protocols. For example, DNA methyltransferase and histone deacetylase (HDAC) inhibitors, i.e., valproic acid, the treatment improved the efficiency of reprogramming by 100-fold (Huangfu et al. 2008; Pasha et al. 2011). Similarly, Woltgen et al. successfully used TGF-β inhibitors E-616452 (25 mM), E-616451 (3 mM), or EI-275 (3 mM) for induction of pluripotency in mouse embryonic fibroblasts (MEF) (Woltjen and Stanford 2009).

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Although the overexpression of transcription factors for reprogramming is the most prevalent method for cellular reprogramming, search for novel meth ods with higher efficiency and safety index continues. Given their essential participation in major cellular signaling pathways involved in various cellular functions, an exciting strategy for somatic cell reprogramming is based on miRNA or a set of miRs manipulation to achieve cellular reprogramming. For example, Anokye-Danso and colleagues reported that expression of miR-302/ miR-367 cluster, ESCs-specific miRs highly expressed during the embryonic development, successfully and efficiently reprogrammed both human and mouse somatic cells such that reprogramming efficiency were two orders of the magnitude more efficient than the classical transcription based-method (Anokye-Danso et al. 2011). Also, there was no difference in the quality and characteristics of the iPSCs generated from the two methods. More importantly, the strategy alleviated the need to deliver transcription factors to the cells. An important step forward in cellular reprogramming is the application of CRISPR/ Cas9 technology, a gene editing technique which works as molecular scissors (Shakirova et al. 2020).

The Strategy of Direct Reprogramming to Generate Cardiomyocytes Direct reprogramming is an emerging new approach which is being used to derive cardiomyocytes from differentiated somatic cells without passing through pluripotency status (Kelaini et al. 2014). The strategy generally involves developing partially induced multipotent progenitors which are then directed to differentiate into cardiomyocytes (Kelaini et al. 2014). For example, Ieda et al. successfully used direct reprogramming strategy to convert mouse cardiac fibroblasts (CFs) into induced cardiomyocyte-like cells (iCMs) using three heart-specific transcription factors: GATA4, MEF2C, and TBX5 (GMT) (Ieda et al. 2010; Kurotsu et al. 2017). This bioengineering technique is an exciting approach free from the limitations of the existing technologies, such as exogenous stem cell injection or iPSCs. Similar approach has also been used by other research groups using different sets of heart-specific transcription factors (Song et al. 2012; Protze et al. 2012; Mathison et al. 2012). Microbubble ultrasound contrast agents (MCAs) have become an effective tool for gene delivery (Negishi et al. 2016). MCAs’ biocompatible shells make them stable carriers of transfection vectors for gene delivery (Sun et al. 2013; Panje et al. 2012; Geis et al. 2012). With a compressible gas core, MCAs have a unique feature that allows them to be flexible according to different phases of ultrasound waves (Shapiro et al. 2016). In addition, the cell membranes and vascular endothelial integrity can be trespassed by the ultrasound shock waves. Hence, MCAs can penetrate vessels in an ultrasound field and serve as therapeutic modality for the circulatory system (Castle and Feinstein 2016; Chen et al. 2016). Recent research

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yielded a reliable method for fabricating controlled-release vectors loaded with MCAs; this method is ultrasound-targeted microbubble destruction (UTMD) (Zhou et al. 2017). High-amplitude oscillations leading to microbubble destruction can be induced by high-mechanical-index ultrasound at its resonance frequency (Liao et al. 2014). This sonoporation phenomenon improves localized tissue deposition of bioactive substances. Additionally, the features mentioned above, UTMD generates high-velocity fluid microjets that produce transient nanopores in cell membranes and enable higher capillary permeability (Haber et al. 2017). Considering UTMD as a feasible targeted delivery method, some authors have successfully loaded vectors, including viral vectors and plasmid DNA to treat a wide range of diseases in different animal models (Zhang et al. 2018; Lin et al. 2018). The virus-mediated expression cloning methods include retroviral, Sendai virus, and lentiviral vector-mediated gene delivery systems. The characteristics of these viral systems, i.e., high transduction rates, make them popular in stem cell research (Sharon and Kamen 2018; Gandara et al. 2018; White et al. 2017; Miyamoto et al. 2018; Isomi et al. 2021). Nowadays, the HIV-1–derived thirdgeneration lentiviral vector is the most commonly used manufacturing system, which originates from the laboratories of Didier Trono and David Baltimore (Kotterman et al. 2015). After deletion of two-thirds of the HIV-1 viral genome and preservation of the elements critical for gene transfer, this viral packaging system is free from replication competence risk. As a result, the biggest advantage of this system is replication deficiency, making lentiviral vectors safe for further applications in mammalian tissues. Many clinical trials using lentiviruses for gene therapy have been conducted in recent years (Houghton et al. 2015; Liu et al. 2014; Aiuti et al. 2013). The results proved that lentiviral vectors could facilitate the delivery of genetic material and its stable long-term expression in a target tissue without severe inflammatory response. In polar media, viral vectors also have a pH-dependent surface charge. As a result, the electrostatic charge of virus particles, also known as the isoelectric point, has an important property in an electric field (Michen and Graule 2010). This property determines the viral electrostatic adsorption capacity, which is critical for further virus sorption processes. Based on this property of viral vectors, injection of cationic microbubbles (CMBs) loaded with viral vectors affords an improved gene transfection effect in vivo (Li et al. 2009; Xie et al. 2010). Some studies have also reported successful delivery of MCA–virus hybrids to the cardiac tissue, indicating the feasibility of organ-specific targeting of viral vectors (Michen and Graule 2010). Inspired by direct-reprogramming strategies and the research advances in MCAs, we have attempted to synthesize CMBs so that lentiviral vectors encoding GMT for direct reprogramming could be loaded on the surface of microbubbles (Fig. 1). Instead of direct intramyocardial injection, the microbubble–lentivirus hybrids were injected through the jugular vein, and direct-reprogramming relevant vectors were then released into the targeted myocardium by UTMD. This chapter is based on our novel data and discusses in-depth the superiority of this novel approach in regenerative medicine.

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Fig. 1 A schematic of ultrasound-targeted CMB destruction–mediated localized delivery of the direct-reprogramming GMT-encoding lentivirus for cardiac repair in ischemic heart disease. The lentivirus encoding heart-specific transcription factors, GATA4, MEF2C, and TBX5 (GMT) have the capacity to directly reprogram CFs into iCMs in vitro. The synthesized CMBs are loaded with an anionic GMT lentiviral vector so that the microbubble/lentivirus hybrids could be then injected via the jugular vein, and direct-reprogramming vectors are released into the targeted myocardium by the UTMD technique for cardiac repair in vivo

Observations and Data Analysis Synthesis and Characterization of CMBs and Lentivirus-Binding Capacity During the study, CMBs were developed using DPPC, DPPE, and DOPE in chloroform, followed by rotary evaporation to form a lipid film (Fig. 2a–c). After sufficient rehydration with PBS and emulsification with glycerine, the resulting emulsion was sonicated with a gas (SF6). Thus, the CMBs with a sulfur hexafluoride gas core and a lipid shell were fabricated. The concentration of CMBs was approximately 4.0
109/ml. The average microbubble diameter was 966.6 nm (Fig. 2d), and the zeta potential of the CMBs was 28.3  1.4 mV (Fig. 2e). The initial mean microbubble count in this experiment was 6.1  0.2
107. Given the surface of CMBs was cationic and the lentivirus capsids were anionic, the

Fig. 2 Design and characterization of CMBs. (a) A schematic of CMB synthesis. The CMBs consist of DPPC, DPPE, and DOPE in chloroform, and the CMBs with an SF6 gas core and a lipid shell are fabricated. (b) The shape and concentration of CMBs were evaluated microscopically, and CMBs appeared as gas-filled spheres under a microscope. (c) Microbubbles loaded with the control GFP-expressing lentivirus were labelled with the FITC-conjugated anti-p24 antibody, revealing a strong fluorescence signal on the microbubble surface, which indicated that the GFP-expressing lentivirus was bound to the surface of microbubbles. (d) Log-normal size distribution and (e) zeta potential of the CMBs as measured by a Zetasizer NANO ZS system

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loaded microbubbles could be distinguished from the unloaded ones by fluorescence to prove viral-vector binding. As microbubbles have low stability upon exposure to repetitive flotation or gentle shaking, some of the original microbubbles could be retrieved after all the washing steps. After incubation with the FITC-conjugated antip24 antibody, the loaded microbubbles showed a strong fluorescence signal on their surface, thus proving that anionic capsids of the GFP-expressing lentivirus were efficiently bound to the microbubbles’ surface (Fig. 2c).

Reprogramming of Rat CFs into iCMs by the GMT Lentivirus Vectors In Vitro Amplified and cloned sequences of Gata4, Mef2c, and Tbx5 in the plasmids for lentiviral packaging were confirmed by restriction enzyme identification and gene sequencing (Fig. 3a). After packaging, the titer of GATA4 and TBX5 viral vectors were 3.05
108 and 3.02
108 TU/ml, respectively, according to the results of 293 T cells infection. The titer of the MEF2C vector was 3.25
108 TU/m as confirmed by an RT-PCR assay (Table S1). After infection of 293 T cells, the infected cells showed the corresponding fluorescence signal and puromycin resistance, which confirmed the successful construction of reprogramming vectors (Fig. S1).

Fig. 3 Optimization of in vitro reprogramming. (a) Restriction enzyme identification and gene sequencing confirmed amplified and cloned sequences of Gata4, Mef2c, and Tbx5 in the plasmids for lentivirus packaging. (b) At the fixed parameters (1.5 MHz, duty cycle 20%, duration 30 s), the EGFP-positive cells of the 1.5 W/cm2 ultrasound group were detected by fluorescence microscopy. (c) A representative Western blot showing overexpression of all three transcription factors GATA4, MEF2C, and TBX5, and cardiomyocyte-specific marker cTnT at 7 and 14 days after lentivirus infection (*P < 0.05 vs. control)

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Table S1 Real-time PCR sequences for detecting Mef2c lentiviral vector titer WPRE sequence Forward primer (1277F) Reverse primer (1361R) Probe(1314P) β-actin sequence Forward primer Reverse primer Probe

CCGTTTCAGGCAACGTG AGCTGACAGGTGGTGGCAAT FAM-TGCTGACGCAACCCCCACTGGT-TAMRA GCGAGAAGATGACCCAGCTC CCAGTGGTACGGCCAGAGG FAM-CCAGCCATGTACGTTGCTATCCAGGC-TAMRA

Figure S1 Electrocardiogram (ECG) of the left anterior descending coronary artery (LAD) rat model. Line 1: ECG of shame group (n ¼ 8) before and after LAD ligation; Line 2: ECG of saline group (n ¼ 8) before and after LAD ligation; Line 3: ECG of GMT group (n ¼ 8) before and after LAD ligation; Line 4: ECG of GMTþCMBsþUS group (n ¼ 8) before and after LAD ligation; Line 5: ECG of GMTþCMBsþUTMD group (n ¼ 8) before and after LAD ligation

After in vitro cell transfection with the GMT viral vectors, Western blotting revealed overexpression of all three reprogramming transcription factors after 7 days. The expression level remained stable until day 14 of observation (Fig. 3c, d). In addition to the increased expression of the three reprogramming transcription factors, overexpression of the cardiomyocyte-specific marker cTnT was confirmed by Western

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blotting after the transfection. In contrast to the infected CFs, those infected with the control vectors did not show the same expression levels of transcription factors or cTnT. Therefore, the fabricated GMT viral vectors were capable of in vitro reprogramming of CFs, which expressed the typical marker of cardiomyocytes.

UTMD-Mediated Localized Delivery of Direct-Reprogramming GMT Lentivirus–CMB Hybrids In Vivo Histological Assessment of Post-Infarct Ventricles After UTMD Therapy Neomyogenesis around the infarct area after GMT and UTMD therapy via potential cardiomyocyte proliferation was demonstrated by immunofluorescence staining specific for α-actin and histone H3 phosphorylation (PHH3) expression, a marker of cell mitosis. Twenty-eight days after the surgical procedure, an increase in the PHH3-specific immunofluorescence signals were detected in the peri-infarct area in groups GMT, GMT þ US, and GMT þ UTMD treatment groups (Fig. 4, panels-2 & 4). Superimposition of immunofluorescent images of α-actin (Fig. 4, panel-1) and DAPI stained images (Fig. 4, panel-3), showed that treatment with GMT, GMT þ US, and GMT þ UTMD induced cardiomyocyte proliferation after myocardial infarction (MI). According to statistical analysis (Fig. S2), there were significantly more PHH3-positive and α-actin–negative cells in the peri-infarct zone in all the treated groups; these data fit the characteristics of reprogrammed fibroblasts under the conditions of MI. The TdT-mediated dUTP nick-end labelling (TUNEL) analysis of the peri-infarct myocardium in groups GMT, GMT þ US, and GMT þ UTMD manifested a lower percentage of apoptotic cardiomyocytes than that in the saline treated group of animals (P < 0.05). Groups GMT, GMT þ US, and GMT þ UTMD showed no statistically significant differences among themselves (Fig. 5a, panel-3, & Fig. 5d). It was observed that the damaged myocardium in the saline group was replaced by a fibrous scar (Fig. 5a, panel-2), and ventricular-wall thickening could be observed at 28 days after infarction. Masson trichrome staining and HE staining were also used (Fig. 5a, panels-2 & 3) to measure the scar size and quantitatively analyze interstitial fibrosis (Fig. 5b, c). Compared with the saline treated group of animals, myocardial fibrosis leading to scar formation and left ventricular remodeling were significantly attenuated in all the treatment groups. The total area of fibrosis and the number of scar-producing myofibroblasts were significantly reduced observed after the administration of the GMT vectors at 28 days of observation. In addition, angiogenesis, which is important for restoration of regional blood supply (including capillaries and arterioles), was significantly improved in the peri-infarct area. In the groups GMT, GMT þ US, and GMT þ UTMD, there was significant increase in capillary density after immunostaining for cluster of differentiation 31 expression (CD31; Fig. 5a, panel-5 and Fig. 5e), and arteriole counts [according to combined staining for alpha-smooth muscle actin (α-SMA) in the peri-infarct zone] than the saline treatment group (Fig. 5a, panel-4, and Fig. 5f). Although without statistical significance, immunohistochemical

Fig. 4 Neomyocytes around the infarct area after in vivo reprogramming. Panel 1: Immunofluorescence of α-actin (red fluorescence). Panel 2: Immunofluorescence of PHH3 (green fluorescence). Panel 3: DAPI staining showing cardiomyocytes nuclei (blue fluorescence). Panel 4: Merged images showing that newborn cardiomyocytes can be detected via PHH3 expression by fluorescence microscopy 28 days after the operation (scale bar ¼ 50 mm)

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Figure S2 Screening experiments results of GMT lentivirus (a) The packaging plasmids of Mef2c consist of puromycin gene which provides transfected cells puromycin resistance. Line 1: Puromycin resistance experiment for fibroblasts. Line 2: Puromycin resistance experiment for fibroblasts transfected with GMT lentiviral vectors. (b) Cardiac fibroblasts infected by 108 TU/ml GFP control lentivirus. (c) Gata4 and Tbx5 lentivirus tilters counting by infecting 293T human embryonic kidney cell. Line 1: 293T cells infected by GFP control lentivirus; Line 2: 293T cells infected by GFP-enhanced Gata4 lentivirus; Line 3: 293T cells infected by RFP-enhanced Tbx5 lentivirus

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Fig. 5 Histological examination of ultrasound-mediated in situ direct reprogramming in the rat heart model. (a) Histological analyses. Panel 1: HE staining images depicting the basic tissue structure of the histological tissue sections 28 days post-surgery; Panel 2: HE staining illustrating the basic tissue structure in histological tissue sections 28 days post-surgery (scale bar is ¼ 50 mm); panel 3: Masson trichrome staining showing a scar (blue) and viable (red) tissue sections 4 weeks post-surgery (scale bar is ¼ 50 mm); panel 4: TUNEL staining showing apoptotic cardiomyocytes (brown) 1 day post-surgery (scale bar ¼ 50 mm); panel 5: immunohistochemical images of arterioles stained for α-SMA (brown; scale bar ¼ 50 mm); panel 6: immunohistochemical images showing capillaries stained for CD31 (brown; scale bar ¼ 50 mm). (b) Quantitative analyses indicating that the scar size in groups GMT, GMT þ US, and GMT þ UTMD at 4 weeks postsurgery was smaller than that in any other group. (c) Quantitative analyses indicating that the fibrosis percentage in animal groups GMT, GMT þ US, and GMT þ UTMD 4 weeks post-surgery was less than the saline treated animal group. (d) Quantitative analysis suggesting that the apoptotic-cardiomyocyte percentages in the peri-infarct myocardium of groups GMT, GMT þ US, and GMT þ UTMD 28 days post-surgery were lower as compared to the saline treated group of animals. (e) Quantitative analysis showing that capillary density at 4 weeks post-surgery in the periinfarct myocardium of groups GMT, GMT þ US, and GMT þ UTMD was higher than the saline treated animal group. (f) Quantitative analysis indicating that arteriole counts in the peri-infarct myocardium of groups GMT, GMT þ US, and GMT þ UTMD at 4 weeks post-surgery were higher than the saline treated animal group (*P < 0.05 vs. saline group)

staining of CD31 and α-SMA indicated an increase in angiogenesis in the peri-infarct area at 4 weeks post-surgery in UTMD and GMT lentiviral-vector–treated groups. Histological examination of related organs including lungs, the liver, kidneys, and spleen in all the groups was also performed 28 days after the intervention. HE staining showed delicate alveolar walls of the lungs without any signs of pathological changes such as edema or hemorrhage (Fig. 6, panel-1). The liver in all the groups examined appeared normal, divided into lobules with the central vein and peripheral triads (Fig. 6, panel-2). There was no enlargement of the white pulp or a

Bioengineering Technique Progress of Direct Cardiac Reprogramming

Fig. 6 Histological examination of various organs in all the groups at 28 days after respective intervention. Panel 1: HE staining images revealing the basic lung tissue structure of mass sections; panel 2: HE staining images showing the basic liver tissue structure of mass sections; panel 3: HE staining images revealing the basic spleen tissue structure of mass sections; panel 4: HE staining images depicting the basic kidney tissue structure of mass sections (all images scale bar ¼ 1 cm)

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reduction in the red pulp composed of many splenic sinusoids in the spleen (Fig. 6, panel 3). The structures of glomeruli were also normal in all the kidneys (Fig. 6, panel-4). Immunohistochemical staining of GATA4, MEF2C, and TBX5 was also performed in the heart, liver, spleen, lung, and kidney tissues 28 days after the surgical procedure (Fig. S3), which revealed off-target expression in the liver and kidneys (Fig. S4). This was attributed to the high tropism of the lentivirus towards the liver and kidney cells.

Cardiac Function Improvement After UTMD-Mediated Direct Reprogramming Therapy To assess the therapeutic effectiveness of GMT, GMT þ US, and GMT þ UTMD on global LV remodeling and function, serial echocardiographic analyses and cardiac MRI scans were performed on days at 2 and 28 after surgery in all the groups (Fig. 7a). MI via ligation of the left anterior descending coronary artery (LAD) induced a gradual reduction in LV-function, as evidenced by a declining LV-ejection fraction (LVEF), which is the critical event in the progression of adverse post-infarct remodeling. Two days post infarction, the results revealed similar baseline heart function among all the groups with induced infarction (p > 0.05). Compared with baseline (LVEF 46.3%  0.7%), the saline group showed significantly lower LVEF % (47.46%  2.9%) post infarction. On the other hand, treatment groups, including GMT, GMT þ US, and GMT þ UTMD had higher LVEF (54.01%  3%, 59.6%  1.9%, and 56.9%  1.98%, respectively; p < 0.01 vs. control group). However, all the treated groups showed no statistically significant difference in LVEF improvement among themselves (Fig. 7b–d). Therefore, cardiac function analysis from the baseline values showed that the LVEF after MI declined over time. As illustrated in the figure, the mean change in the LVEF in groups MI-CMB/ GMT þ UTMD, MI-CMB/GMT þ US, and MI-CMB/GMT was more significant (p < 0.05) compared with the sham or saline group from the time point of lentivirus administration to the follow-up until 28 days of observation (Fig. 7b–d).

Significance of the Combinatorial Approach of UTMD with CMBLentiviral Hybrid This data signifies the combinatorial approach of using UTMD with CMB–lentiviral hybrids to transfer direct-reprogramming GMT factors into rat CFs in the ischemic myocardium (Fig. 1). These data shows the high efficiency of ultrasound-targeted CMB destruction–mediated localized delivery of the direct-reprogramming GMT-encoding lentivirus for cardiac repair in ischemic heart disease. The lentivirus encoding heart-specific transcription factors, GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed the non-cardiomyocytes population of cardiac cells, i.e., CFs, into iCMs in vitro successfully. The most significant findings of the presented data include successful neomyogenesis in and around the infarcted myocardium, increased blood vessel density, attenuated fibrogenesis and infarct size, and preservation of LV contractile function.

Figure S3 Histologic examination of ultrasound-mediated in situ direct reprogramming in rat model. (a) Histological analyses. Line 1: Immunohistochemistry images showing cTnT (brown), Scale bar ¼ 50 mm; Line 2: Immunohistochemistry images showing Gata4 (brown), Scale bar ¼ 50 mm; Line 3: Immunohistochemistry images showing Mef2c, Scale bar ¼ 50 mm; Line 4: Immunohistochemistry images showing Tbx5 (brown), Scale bar ¼ 50 mm. (b) Quantitative counting showing that cTnT positive counting 4 weeks post-surgery in peri-infarct myocardium of GMT, GMT+US, and GMT+UTMD groups are higher than saline group. (c) Quantitative counting showing that Gata4 positive counting 4 weeks post-surgery in peri-infarct myocardium of GMT, GMT+US, and GMT+UTMD groups are higher than saline group. (d) Quantitative counting showing that Mef2c positive counting 4 weeks post-surgery in peri-infarct myocardium of GMT, GMT+US, and GMT+UTMD groups are higher than saline group. (e) Quantitative counting showing that Tbx5 positive counting 4 weeks post-surgery in peri-infarct myocardium of GMT, GMT+US, and GMT+UTMD groups are higher than saline group

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Figure S4 Immunohistochemistry stained by Gata4 in lung, liver, spleen, and kidney. Line 1: Immunohistochemistry images stained by Gata4 (brown) in lung tissue, Scale bar ¼ 50 mm. Line 2: Immunohistochemistry images stained by Gata4 (brown) in liver tissue, Scale bar ¼ 50 mm. Line 3: Immunohistochemistry images stained by Gata4 (brown) in spleen tissue, Scale bar ¼ 50 mm. Line 4: Immunohistochemistry images stained by Gata4 (brown) in kidney tissue, Scale bar ¼ 50 mm

CMs play a pivotal role in the structural repair maintenance of myocardial structural integrity of the infarcted myocardium by transforming into myofibroblasts. However, their excessive activation has been reported to support HF progression via fibrotic activity that contributes to the replacement of the dead cardiomyocytes with fibrosis, scar tissue formation, and modulation of cardiomyocyte structure and function (Nagaraju et al. 2019). Therefore, suppressing their activation (Meng et al. 2018) or involving them in neomyogenesis via direct reprogramming strategy may be exploited to add them into the repair process. Lentiviral vectors have been extensively used for transgene delivery into various cell types, including the poorly dividing cardiomyocytes, owing to the stable, heritable gene transfection in both dividing and nondividing cells without inflammatory/immune response (Kim et al. 2001; Zhao et al. 2002). These advantages render them undeniably more appealing as transgene delivery vectors as compared to the other vectors. Attempts are underway to optimize protocols to generate enhanced viral vector titter to achieve transfection efficiency (He et al. 2021). However, one of the limitations of lentiviral vectors is their lack of site-specific uptake when delivered systemically, thus delivering the transgene to nontarget cells and tissues. Hence, the lentiviral vector is being combined with other strategies to increase its transduction efficiency. In recent years, CMBs and MMBs (magnetic microbubbles)-hybrids with lentiviral vector combined with UTMD has gained popularity for efficient therapeutic gene delivery as UTMD causes increased sonoporation of the cells at the target

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Fig. 7 In vivo heart function assessment by the 7 T MRI system for small experimental animal models. (a) Panel 1: MRI showing a cardiac cross-section at 2 days after surgery; panel 2: MRI of a cardiac cross-section on 28 days post-surgery; panel 3: M-mode echocardiography images depicting a cardiac cross-section on day 28 after surgery. (b) Quantitative analyses of LVEF on days 2 and 28 after surgery. (c) Quantitative analyses of LVEDV at 2 and 28 days post-surgery. (d) Quantitative analyses of LVESV on days 2 and 28 after surgery. (LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-systolic volume)

sites to facilitate the genetic material uptake (Yang et al. 2011, Cool et al. 2013). These data successfully demonstrated the applicability of combining lentiviral vectors with CMBs, which allowed the viral vector’s high binding capacity to achieve high gene transfection efficiency. In both the in vitro and in vivo experiments, direct-reprogramming GMT factors proved capable of cardiac repair. With UTMD, viral vectors were directly deposited in cardiac tissue and successfully repaired the infarcted myocardium. The immunofluorescence assay and histological examination confirmed neomyocytes and neo-angiogenesis during a 4-week followup in the rat model of MI. All the treatment groups showed significant LV-function improvement as compared with the control treatment group.

CMB–Lentivirus Hybrids in Gene Delivery These data describe the synthesis of CMBs from DPPC, DPPE, and DOPE (Fig. 2a). The lentivirus–CMBs conjugation was confirmed visually by green fluorescence (Fig. 2c). It is important to mention that the characteristics of the synthesized

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microbubbles, including their lipid composition, size distribution, residual charge, etc., significantly impact their genetic material loading capacity. Similarly, CMBs synthesis protocols involve the use of different types of cationic lipids integrated onto the lipid microbubble shell (Cao et al. 2017a; Negishi et al. 2016; Yang et al. 2018). It is pertinent to mention that the chemical composition of the cationic head groups are the significant determinants of the physiochemical properties of the CMBs besides remaining as the major determinants of their capacity to hybridize with the lentiviral vectors carrying the transcription factors of interest for delivery. Similarly, the inclusion of helper lipids contributes to the physiochemical properties as well as their transfection efficiency. As a result, the CMBs showed high loading capacity and transfection efficiency because of their charge coupling. In these experiments, the prepared CMBs yielded a mean zeta potential of 28.3  1.4 mV. This cationic lipid has turned out to be an efficient and popular method for gene transfection and drug delivery. Because of the electrostatic adhesion between CMBs and anionic lentivirus particles, CMB–lentivirus hybrids could be a promising noninvasive platform for direct reprogramming in rats.

Advantages of Lentiviral Vectors for Direct Reprogramming Besides their ability to transduce both dividing and nondividing cells with equal efficiency and low immunogenicity, the third-generation lentivirus packaging system involves several safety measures (Gill and Denham 2020). The viruspackaging system relies on four separate plasmids to produce lentiviral vectors; this arrangement decreases the chance of wild-type virus recombination events (Lukashev and Zamyatnin 2016). Moreover, the third-generation lentivirus packaging system deletes the crucial viral transcription activator gene tat and its regulatory region, thus generating self-inactivating (SIN) vectors (Sharon and Kamen 2018). Additionally, this system separates viral packaging genes, including gag, pol, and rev on two plasmids (Stellberger et al. 2017). Considering other features of this lentiviral vector system, including the deletion of accessory genes vif, vpr, vpu, and nef, which are vital for the pathogenesis of the virus, and replacement of the native HIV-1 envelope protein by heterologous envelope protein VSV-G, the third-generation lentivirus system maximizes the biosafety for its applications (Shearer and Saunders 2015). For the production of the vectors, the lentiviral vector transfer plasmid for GMT overexpression and three plasmids containing packaging and envelope genes (pRsvREV, pMDlg-pRRE, and pMD2G) were transfected into a HEK293T producer cell line, which then assembled the virus and released it into the culture medium. By restriction enzyme identification, PCR, and sequence analysis, the successful construction of the recombinant lentiviruses was confirmed, and there were no HIV-1 recombination events detected (Fig. 3). The lentiviral vectors were then concentrated to a high titer and tested for key characteristics before their use for direct cardiac reprogramming and UTMD application.

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Improvement of Reprogramming Factors in Direct Cardiac Reprogramming As discussed earlier, the CFs have a crucial role in the vicious cycle of LV remodeling post-MI. The adult functioning cardiomyocytes undergoing apoptosis and necrosis in the infarcted myocardium are replaced by fibrotic scar tissue as a part of the intrinsic repair process. Given the pivotal role of CFs in both reactive and reparative fibrotic activity (Hara et al. 2017), the strategy to involve them in neomyogenic repair of the infarcted myocardium has gained momentum that is based on their direct reprogramming into iCMs to restore cardiac function (Fu and Srivastava 2015). Although several combinations of transcription factors with or without microRNA manipulations, the published data so far provides mounting evidence confirming that GMT is an effective combination of cardiac-specific direct reprogramming factors both in vitro or in vivo for the generation of iCMs from CMs (Sadahiro et al. 2015; Jayawardena et al. 2015; Boon and Dimmeler 2015; Sinagra and Fabris 2016). Tronolab lentiviral production system was successfully used to generate reprogramming lentivirus, and GMT viral titers achieved were 3.05
108, 3.25
108, and 3.02
108 TU/ml. During the transfection protocol in the experiment, 8 μg/ ml polybrene was added to neutralize the electrostatic interaction between the cells and the viral particles that significantly contributed to improving the transfection efficacy. The successfully reprogrammed CFs yielded high expression of GATA4, MEF2C, TBX5, besides the expression of cardiac-specific cTnT (an important cardiac marker) at 48 h after sequential transfection with the GMT virus (Fig. 3). In vitro experimental data strongly support that the GMT lentivirus can directly and efficiently reprogram CFs, which is in agreement with other published studies (Fu et al. 2013; Inagawa and Ieda 2013; Song et al. 2012; Ieda et al. 2010). However, the success of direct reprogramming and its efficiency is determined by the proper reprogramming factors selection and well-cultured fibroblasts (Adams et al. 2021). Besides the triad of GMT, several other combinations of cardiac-specific transcription factors (López-Muneta et al. 2020) and strategies can directly reprogram mouse and human fibroblasts into iCMs (Hashimot et al. 2016). Given the pivotal role of miRNAs in diverse signaling pathways regulating the cellular functions (Haider et al. 2015), they are being used for stem cell-based therapy to promote various functions, i.e., stem cell survival, differentiation, and to modulate their paracrine behavior, etc. (Haider et al. 2009; Kim et al. 2009; Haider et al. 2010; Kim et al. 2012a, b; Lai et al. 2012). They are also being used in various direct cardiomyogenic reprogramming protocols. For example, cocktails including GMT plus Hand2 (GHMT), and transient expression of four microRNAs, i.e., miR-1, miR-133, miR-208, and miR-499 (together termed as miRcombo), and chemical enhancement of direct reprogramming; therefore, further exploration for the optimal combination of reprogramming factors is still being pursued (Nam et al. 2014; Tani et al. 2018; Mohamed et al. 2017). The presence of miRNAs increased the rate of cardiomyogenic reprogramming and needed a shorter reprogramming period (Alfar

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et al. 2018). Paoletti et al. used transient expression of miRcombo to directly reprogram human adult cardiac fibroblasts to develop iCMs (Paoletti et al. 2020). They have reported up to 11% of fibroblasts to adopt cardiac phenotype by day 15 after manipulation, as evaluated by flow cytometry for cTnT expression. And by day 30, the transdifferentiated cells exhibited spontaneous calcium transient. Manipulating these cocktails of reprogramming factors can increase the efficiency of direct cardiomyogenic reprogramming to generate iCMs for cell-based therapy or other potential theranostic applications (Chen et al. 2017; Ghiroldi et al. 2017).

Ultrasound-Mediated Localized Direct Reprogramming via Lentivirus Delivery for Cardiac Repair With the recent developments in cardiac regenerative medicine, especially where stem cell therapy is being combined with gene delivery for a combinatorial approach, gene transfer has become a valuable tool in therapeutic research (Wu and Li 2017). The cells are genetically modulated to overexpress the gene/s of interest for delivery to the heart. The established delivery systems, including intramyocardial injection and coronary perfusion, are based on invasive procedures, which hinder their further clinical translation (Ni et al. 2016; Suzuki et al. 2011; Ebrahimi 2017). The UTMD strategy is safe, noninvasive, and ensures high rate of organ-specific gene delivery. Several researchers have demonstrated the suitability of UTMD combined with in vivo viral vector transduction after intravenous administration in experimental animal models (Robertson 2016). By loading adenoviral vectors onto the surface of albumin microbubbles, investigators have proven the targeted gene expression in the heart facilitated by UTMD (Qian et al. 2018, Liao et al. 2014, Ma et al. 2015, Sun et al. 2013). Although these studies suggest that MCAs could be used in synergy with viral vectors to enhance a virus-based gene delivery to the myocardium, they did not show significant therapeutic effects of this approach. On the contrary, the abovementioned experiments successfully used CMBs loadedGMT lentiviral vectors capable of directly reprogramming the CFs. With ultrasound at the resonance frequency (1.5 MHz), destruction of the microbubbles is induced in the heart, thereby ensuring targeted delivery of viral vectors. The newly formed cardiomyocytes were visualized by immunofluorescence specific for PHH3 in the infarcted area in all the treatment groups of animals (Fig. 4). After 4 weeks of followup, the treated groups showed a significant decrease in the total area of fibrosis and increased vascular density in the peri-infarct region (Fig. 5). Cardiac MRI revealed significantly higher mean change in LVEF for each rat from the time of lentivirus administration until 4 weeks in the treated groups (Fig. 7). From the in vivo experiment, it can be concluded that the novel strategy of UTMD can target direct-reprogramming lentiviral vectors into the rat heart. In comparison with direct intramyocardial injection or in situ microbubble destruction, the therapeutic effect of intravenous administration of viral vectors by UTMD as a noninvasive technique was not significantly different. The microbubble–virus hybrids seem to be an efficient platform for direct reprogramming and may be suitable for translation into a potential clinical strategy for patients with end-stage heart disease.

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Although the lentiviral vectors are useful tools for long-term gene transfer into the target cells and are extensively used in cell reprogramming research, the critical problem of this gene delivery strategy is its toxicity to unrelated organs, especially due to hepatic uptake (McCarron et al. 2016; Sirsi and Borden 2012). MCAs, however, can increase the therapeutic index and thus may reduce the toxicity of viral vectors to some extent. Histological examination of lungs, the liver, kidneys, and spleen was performed after H&E staining, which detected no damage or inflammation in these organs, thus revealing the safety of organ-specific technique to avoid damage to the unrelated/nontarget organs (Fig. 6).

Conclusion and Future Perspective Direct reprogramming possesses several theoretical advantages, resolving the main challenges and issues associated with cell therapies. The approach has come a long way to define specific factors and cues needed to help somatic cells cross lineage restrictions and undergo transdifferentiation to adopt the phenotype of interest (Wang et al. 2021). The most significant feature of direct reprogramming is that the transdifferentiating somatic cells are guided to avoid intermediary pluripotency states and new phenotype. Because the lentiviral-based gene delivery is gaining popularity and acceptability for clinical applications due to of their superior characteristics (Milone and O’Doherty 2018), methods to generate integration-free iCMs are necessary and widely sought. This study implements the strategy of delivery of direct-reprogramming lentiviral vectors encoding for the triad of cardiac transcription factors-CMBs hybrid to a target infarcted myocardium by UTMD serving as a viable myocardial tissue repair therapy. Systematic in vitro and in vivo results have confirmed that administration of GMT induces post-infarct ventricular functional improvement, whereas UTMD enhances the efficacy. Based on this strategy, lentiviral CMBs are likely to be a safe and effective platform for targeted delivery of reprogramming factors for myocardial repair via neomyogenic differentiation of non-myocytes, i.e., CFs, in the infarcted myocardium. Furthermore, the strategy will not only help recover the lost myocytes during infarction episodes; it will also curtail CFs availability for cardiac fibrosis and fibrotic scar tissue formation. In summary, the combinatorial approach using lentiviral-CMBs hybrid together with UTMD may be developed as a potential therapeutic modality in the future for myocardial repair and regeneration.

A Summary of the Experimental Methods and Design Vectors and Cells Transcription factor genes Gata4 (GenBank accession No. NM_144730.1), Mef2c (GenBank accession No. XM_006231731.2), and Tbx5 (GenBank accession No. NM_001009964.1) were synthesized based on sequences available in the NCBI database and were cloned into plasmid pUC57. Amplified sequences were

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then cloned separately into plasmids pCDH-MSCV-MCS-EF1-copGFP, Plvx-Puro, and pLVX-IRES. These plasmids were used to generate lentiviral vectors by means of the Tronolab lentiviral production system (Shanghai Telebio Co., Ltd.), which consisted of pRsv-REV, pMDlg-pRRE, and pMD2G. The packaging plasmids were co-transfected into the 293 T human embryonic kidney cell line (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) to produce the lentiviral vectors encoding for respective transcription factor. The control lentivirus vector was constructed using a homologous lentiviral vector encoding an eGFP cassette and was also generated by means of the lentiviral-packaging plasmids in 293 T cells. The collected supernatants containing the virus at 48 or 72 h after transfection were pelleted by centrifugation and then filtered with a 0.45 μm pore size syringe. The generated lentiviruses were further concentrated for high titer and preserved in an 80  C freezer. Rat CFs harvested from a Sprague–Dawley adult male rat (iCells Biological Engineering Co., Ltd., China) were cultured in Dulbecco’s modified Eagle’s medium with nutrient mixture F-12 (DMEM/F12, Thermo Fisher, USA) supplemented with 15% of fetal bovine serum and 1% of a penicillin and streptomycin solution. The rats CFs were cultured in a humidified incubator under specific conditions (37  C, 5% CO2, 21% O2, and 74% N2).

Creation of CMBs and Microbubble–Virus Hybrids The CMBs were fabricated via the typical emulsification–vibration process. Briefly, DPPC, DPPE, and DOPE were dissolved in chloroform uniformly and then redissolved in saline (10 ml) after rotary evaporation. Glycerine (500 μl) was added at 70  C and emulsified for 2 h in a water bath. The obtained emulsion (1 ml) was transferred into a glass bottle (5 ml), for sulfur hexafluoride injection treatment. The emulsion and gas were integrated after strong vibration via CMBs. CMBs was characterized for concentration, size distribution, and zeta potential on a Zetasizer NANO ZS system. Based on the electrostatic adsorption of the cationic CMBs and the anionic lentivirus, immunostaining of loaded and unloaded microbubbles was performed to examine the binding efficiency. The initial mean microbubble count in this experiment was 107. The virus/microbubble ratio was maintained at 1000:1. Before injection, virus/microbubble hybrid size and concentration were measured to ensure the proper characteristics (Nikon, Germany). To confirm the successful conjugation of the lentivirus and CMBs, 100 μl of a fluorescein isothiocyanate (FITC)conjugated mouse antihuman p24 antibody (catalogue #ab20569, Abcam; dilution 1:50) was added to 200 μl of microbubbles in 1.0 ml PBS and mixed for 30 min at 4  C. After that, the upper layer was used for fluorescence microscopy. Microbubbles loaded with the lentivirus were analyzed to determine whether the microbubble surface emitted strong fluorescence to prove the attachment of the viral vector, using microbubbles without lentivirus as a control.

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To further demonstrate the efficiency of lentivirus hybridized with CMBs and mediated by UTMD, the experiment included the following groups: GFP-lentivirus alone, GFP-lentivirus with ultrasound exposure, and GFP-lentivirus loaded by CMBs with ultrasound exposure. Exposed to ultrasound at diverse power intensities, those CFs transfected by CMB–lentivirus complexes were further tested for appropriate intensity. The optimum conditions were identified via the evaluation of different settings, including the duty cycle of 1.5 W/cm2, power of 1.5 MHz, and 15, 30, or 60 s duration. The cells were then seeded in 6-well culture plates for 48-hour incubation and were analyzed for fluorescence signals using a microscope.

Direct Reprogramming of CFs In Vitro GATA4, MEF2, and Tbx5 vectors (3.05
108, 3.25
108, and 3.02
108 TU/ml, respectively) or the GFP control lentivirus vector (108 TU/ml) were added into the rat CF culture medium (DMEM/F12 plus 10% FBS8 and μg/ml polybrene) for 48-hour incubation. Under routine culture conditions, the cells were allowed to undergo transdifferentiation for 14 days after removing the medium. Later, the cells were collected for analysis.

A Western Blot Analysis Reprogrammed cells (7 or 14 days after transfection) were extracted for total protein samples with RIPA buffer containing phosphatase inhibitors and protease. Using the Bradford protein assay kit (Bio-Rad, USA) and BSA as a protein standard, protein concentrations were determined for further experiments. The protein samples were electrophoresed on SDS-PAGE in a 10% gel, blotted onto membranes, and incubated with various antibodies: anti-Gata4 (Abcam, cat. # 84593), anti-Mef2c (ImmunoWay, cat. # YT2702), anti-Tbx5 (Proteintech, cat. # 13178-1-AP), anti– cardiac troponin T (anti-cTnT; ImmunoWay, cat. # YT5362), and anti–β-actin as an internal control (Abcam, cat. # 8226). A horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (Abcam, cat. # 205718) was used as the secondary antibody. The quantitative results of bands density were calculated using ImageJ software.

Development of Experimental Rat Model of MI Male Sprague–Dawley rats (weight 200–220 g) were purchased from the Department of Laboratory Animal Science, Shanghai Medical College, Fudan University, P.R. China. All animal experiments were performed according to the policy of Fudan University of Health (including its guide for the care and use of laboratory animals).

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Figure S5 Statistics results of α-actin and PHH3 immunofluorescence for quantification of reprogrammed cells. (a) Statistics results of α-actin immunofluorescence (red) in peri-infarct area. (b) Statistics results of PHH3 immunofluorescence (green) positive rate in peri-infarct area

The rats were anesthetized with 10% chloral hydrate (0.3 ml per 100 g, administered intraperitoneally), intubated, and ventilated with an animal ventilator (Chengdu Instrument Factory, China). After ligation of left anterior descending (LAD) coronary artery with an 8/0 suture, elevated S-T segment and a Q-wave appearance on ECG confirmed the success of MI induction (Fig. S5). After the surgical procedure, 40 rats were randomly assigned into 5 experimental groups (n ¼ 8 animals/per group): (i) Sham (opened/closed chest without ligation, untreated), (ii) Saline (rat chest opened with ligation and injection with 0.9% saline in the infarcted area, untreated), (iii) GMT lentivirus alone (MI-GMT), (iv) CMB/GMT lentivirus with ultrasound (MI-CMB/GMT þ US), and (v) GMT lentivirus with CMBs injected via a tube inserted into the right internal jugular vein and ultrasound (MI-CMB/GMT þ UTMD).

Delivery of Microbubble–Lentivirus Hybrids Using UTMD In Vivo At a virus/microbubble ratio of 1000:1, lentiviral vector was incubated with CMBs at 37  C. After 30 min, the mixture was diluted with saline to 0.5/rat (Zhang et al. 2017; Muller et al. 2008; Delalande et al. 2017). Ten minutes after LAD ligation surgery, 0.5 ml CMB–GMT lentivirus mixture was injected into the right internal jugular vein in the UTMD rat groups. Simultaneously, an ultrasound probe administered by a sonoporator (Chattanooga Group) delivered transthoracic ultrasound beam at frequency of 1.5 MHz for 5 min, power 1.5 W/cm2, and probe diameter 1.2 cm.

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Histological Examination Rats were euthanized on day 28 after their respective treatment; the hearts removed for histological examination. Masson trichrome and hematoxylineosin (HE) staining were performed on 5 mm transverse sections from the entire left ventricle. The other organs removed, including kidneys, spleen, lungs, and liver, were also removed for histological examination. Immunohistochemical staining for CD31 (Abcam, cat. # 182981) and α-SMA (Abcam, cat. # 7817) was performed to evaluate angiogenesis in the peri-infarct area. Immunohistochemical staining for GATA4 (Abcam, cat# 84593), MEF2C (ImmunoWay, cat# YT2702), TBX5 (Proteintech, cat# 13178-1-AP), and cTnT (ImmunoWay, cat# YT5362) was also performed to evaluate in situ direct reprogramming in the periinfarct area. We prepared cryo-sections of the peri-infarct area for phosphohistone H3 (PHH3, Abcam, cat. # 32107) expression to identify neomyocytes using fluorescence microscope fitted with a camera (Nikon, Japan). Optical overview images were captured using “scan large image” function of the microscope. Representative sections of the large image were next enlarged and evaluated after α-actin expression (Abcam, cat. # 179467). Both channels were later merged during image analysis.

Cardiac Magnetic Resonance Imaging (cMRI) and Echocardiography Measurements were performed on a 7 T MRI system for small animals (Bruker, USA), provided by the Centre for Biomedical Imaging, Fudan University, China. According to the operating manual of Bruker Company, a series of four pilot transverse images and then a single slice (coronal and sagittal images) were acquired so that the software could get enough anatomical information for planning scans. A gradient cine sequence was employed to acquire contiguous short-axis slices from the apex to the base of the heart. Data analysis was performed in analytical software Segment v.1.8 to determine LV-functional parameters. After inhalation anesthesia with 3% isoflurane, echocardiography was performed using a Vevo 2100 Imaging System (VisualSonics, Inc., Toronto, Ontario, Canada) provided by the Shanghai Institute of Cardiovascular Disease. Unaware of grouping and treatment, an investigator captured echocardiographic images of parasternal long-axis and short-axis views at specified points. M-mode tracings provided data on LVESV and LVEDV. The change in the LVEF after GMT administration was calculated for cardiac function assessment.

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Beatrice Aramini, Valentina Masciale, Giulia Grisendi, Federico Banchelli, Roberto D’Amico, Massimo Dominici, and Khawaja Husnain Haider

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosome Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Functions of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosomes and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Pathways of Exosomal miRNA Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exosomal miRNAs in Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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B. Aramini (*) Division of Thoracic Surgery, Department of Experimental, Diagnostic and Specialty Medicine– DIMES of the Alma Mater Studiorum, University of Bologna, G.B. Morgagni–L. Pierantoni Hospital, Forlì, Italy e-mail: [email protected] V. Masciale Division of Thoracic Surgery, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected] G. Grisendi · M. Dominici Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected]; [email protected] F. Banchelli · R. D’Amico Center of Statistic, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected] K. H. Haider Department of Basic Sciences, Sulaiman AlRajhi University, Al Bukairiyah, Saudi Arabia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_28

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Tumor-Derived Exosomes and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1376 Future Perspectives of Exosomal miRNAs in the Clinics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1378 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1381

Abstract

Exosomes are nanovesicles, which were first described in 1983 as a method by which cells disposed of their metabolic waste. Since the publication of these early reports, exosomes have emerged as important regulators of various processes and mechanisms that ensure the maintenance of cellular homeostasis. In cancer cells, exosomes have added roles in immunomodulation, metastasis, tumor growth and progression, chemo- and radioresistance, cell proliferation, and everything that puts the cancer cells in a position of advantage as compared to the normal cells. More recent studies have revealed that almost every cell type can release exosomes rich in the specific payload of bioactive molecules including miRNAs which are integral to intercellular communication via the transfer of the payload from the senders to the recipient cells. This chapter highlights the newly emerging function of exosomes as miRNA carriers in lung cancer and presents new perspectives for next-generation cancer treatments and targeted personalized medicine. Keywords

Cancer-associated fibroblast · Cancer · Exosome · Lung cancer · Microvesicles · miRNA · Metastasis · Payload · Tumor Abbreviations

Alix BMP EMT ESCRT EVs HIF hnRNP HPV HUVECs MALAT1 MHC miR miRNAs MVBs nSMase PADC VEGF

Programmed cell death 6-interacting protein Bis(monoacylglycerol) phosphate Epithelial-to-mesenchymal transition Endosomal sorting complexes required for transport Extracellular vesicles Hypoxia-inducible factor Heterogeneous nuclear ribonucleoproteins Human papillomavirus Human umbilical vein endothelial cells Metastasis-associated lung adenocarcinoma transcript 1 Histocompatibility complex MicroRNA MicroRNAs Multivesicular bodies Neural sphingomyelinase 2 Pancreatic adenocarcinoma Vascular endothelial growth factor

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Introduction Exosome Composition The history of exosomes dates back to 1946, when it was discovered that plasma prevented blood clotting (Hargett and Bauer 2013). It was also demonstrated that the observed anticlotting activity was due to the presence of platelet-derived nano-sized vesicles. In 1983, researchers in the Johnstone laboratory reported an interaction between the reticulocyte transferrin receptors and nanovesicles derived from sheep reticulocytes. These multivesicular bodies (MVBs)-derived vesicles were named exosomes (Wolf 1967; Pan and Johnstone 1983; Pan et al. 1985). It was also observed that exosomes were reservoirs of RNA for transfer to the recipient cells as part of cell-to-cell communication. Since the publication of these data, various research groups have extensively studied and reported the complex composition of the exosomal payload that may include proteins, lipids, mRNA, miRNA, etc. (Mashouri et al. 2019). Given their ultramicroscopic size, they are generally viewed by electron microscopy. Earlier after their discovery, exosomes were considered as a part of the cell’s disposal system to get rid of metabolic waste. Subsequent research revealed that exosomes mirrored the MVBs in their constitution and some proteins usually associated with MVBs were also detectable in exosomes (Pettersen and Llorente 2018; Henne et al. 2013). These data also helped in the elucidation of the underlying mechanisms of their release, although some of these molecules have not been well-characterized due to the complexity of their structure. Based on their size, structural features, and payload composition, exosomes are one of the subgroups of extracellular vesicles (EVs), which are secreted by the cells. Electron microscopy and in-depth proteomic, lipidomic, and genomic analysis have been used to characterize exosomes for their structure and molecular composition (D’Asti et al. 2012). They are the intraluminally generated, and their composition shows a close similarity with the parent cells of their origin. Typical constituent proteins of exosomes include the adhesion molecules, the major histocompatibility complex (MHC) class I and class II, and the transferrin receptors. Additionally, some nonspecific exosomal proteins include Rab2, Rab7, flotillin, annexin, heat shock proteins, cytoskeleton proteins, and the programmed cell death 6-interacting protein (Alix), which mediate MVB formation (Mathivanan et al. 2010). The lipid components are cell-specific and play a major role to protect the exosomes and maintain a correct balance in the recipient cells (György et al. 2011; Azmi et al. 2013). Lipids such as lysobisphosphatidic acid in MVBs generate intraluminal vesicles, i.e., exosomes (Mashouri et al. 2019). The interaction between lysobisphosphatidic acid and Alix promotes the inward generation of the MVB membrane (D’Asti et al. 2012). Sphingomyelin, phosphatidylcholine, and bis(monoacylglycerol) phosphate (BMP) contribute to differentiate the vesicles from each other. In particular, BMP, a negatively charged protein, is specific for endosomes, and it is involved in exosome formation (Akgoc et al. 2015). Recent researches defined exosomes as

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microparticles that can influence cellular homeostasis by changing the lipid profile of the recipient cells. ExoCarta and Vesiclepedia are two of most comprehensive databases about exosomes characteristics registering thousands of entries for proteins, mRNAs, and lipids, and they are useful for exosomes’ molecular definition (Pathan et al. 2019; Mashouri et al. 2019).

Biological Functions of Exosomes Exosomes show active participation in intercellular communications which has been ascribed to several physiologic and pathologic processes (Meldolesi 2018). Their genesis is generally classified as an ESCRT-dependent or ESCRT-independent mechanism; however, the pathways might not be entirely discrete (Zhang et al. 2019). Rather, these two mechanisms work in harmony, while the resultant diverging subpopulations of exosomes originate by the different contributions from ESCRTdependent and ESCRT-independent process machinery involved therein (Valadi et al. 2007; Andre-Gregoire and Gavard 2016). Although the complete mechanism involved in the formation of MVBs proceeds in a controlled fashion but remains less well understood, it is believed that a late endosome infolding occurs to form intraluminal vesicles before becoming an MVB. It has been described that the process of exosomal membrane invagination is aided by a well-orchestrated and controlled interaction between syndecan-syntenin and Alix protein (Tkach and Thery 2016; Simons and Raposo 2009). It is suggested that subpopulations of MVBs product depend upon the cell type, while others have proposed that the various mechanisms may take place in the same cell (Pitt et al. 2016). It is pertinent to mention that the release of exosomes is an important feature of several cell types and that too in terms of their release into various body fluids carrying a specific payload composition that is influenced by the culture conditions. Despite the scarcity of information regarding the biological and therapeutic benefits from in vivo experimental animal models and limited in vitro characterization data, the actual problem is related to the purification protocols based on the ultracentrifugation technique which is an inefficient method for exosomes purification and quantification because exosome may get recaptured by the cells. The method of purification fails to ensure fool-proof complete recovery of the exosomes from the conditioned medium from the in vitro cultured cells or from the biological fluids. The inefficiency of the isolation and purification protocols creates a discrepancy between the secreted amount of exosomes and their biological activity. Exosomes also carry rich amounts of immunosuppressive molecules. For example, the placenta-derived vesicles isolated from the blood of pregnant women are rich in ligands for natural killer cells. Exosomes have also been found in mice bronchoalveolar lavage fluid which may carry tolerizing molecules from a specific allergen-tolerized animal and release of proinflammatory cytokines in the airway epithelium (Prado et al. 2008). Yang et al. have ascribed the tumorigenic potential of lavage fluid to its exosomal contents during in vitro as well as in vivo studies (Yang et al. 2019). Similarly, the release of exosomes by the eukaryotic parasites or

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pathogens significantly contributes to the host’s tolerance by quenching the immune response or promoting reactions against the pathogen. More recent studies have shown that tumor-derived exosomes are rich in immunosuppressive molecules, which help the cells to evade host immune response (Olejarz et al. 2020). In addition to the immune-regulatory properties, exosomes also participate in tissue repair. For example, mesenchymal stem cells (MSCs)-derived exosomes effectively participate in myocardial repair (Haider and Aramini 2020). They also transfer pathogenic proteins such as prions and amyloid peptides besides others with as yet less-wellcharacterized functions (Bellingham et al. 2012; Bissig et al. 2013). The normal healthy cells also secrete exosomes to transfer their payload to other recipient cells as an integral part of intercellular communication. A payload analysis of exosomes reveals that a typical exosome contains, besides other components, a panel of miRNAs, although the mechanism of miRNA selection to be part of the select miRNA panel has not been clarified yet. Moreover, it is also not known whether the members of the select panel of miRNAs cooperate, synergize, or inhibit each other to achieve a balance in their functioning. These mechanisms have been discussed in-depth in numerous previously published researches (Schwarzenbach and Gahan 2019; Urbanelli et al. 2013; Hammond 2015; Simons and Raposo 2009). The off-loading of the exosomal payload and its uptake by the recipient cells primarily occurs by endocytosis that facilitates the formation of a vesicular early endosome, which can be recycled in the cell by the cell machinery, or it is passaged into the nucleus (Tkach and Thery 2016). At this level, the function of the vesicle is similar to a lysosome.

Exosomes and Cancer Similar to the other areas of research, the application of extracellular vesicles is gaining popularity in the field of cancer research as they provide interesting novel targets for future anticancer diagnostics and therapies (Pitt et al. 2016; Keller et al. 2011). The factors released by exosomes contribute significantly toward the formation, progression, dissemination, and recurrence of tumors. Moreover, the exosomal payload also reduces their responsiveness to the medical treatments due to a specific cell-to-cell communication with the tumor microenvironment (Pitt et al. 2016; Keller et al. 2011). It is pertinent to mention that cell-to-cell communication is fundamental to various physiological and pathological processes both during embryonic development and in the postnatal life to ensure cellular homeostasis and reparability (Chargaff and West 1946; Meldolesi 2018). From among the various possible mechanisms responsible for the transfer of biomaterials between the cells, cell-tocell contact is important for the nearby cells in each other’s vicinity, while the involvement of body fluids to carry vesicular payload is important for long-distance communication (Sung et al. 2015). Luga et al. have shown that in orthotropic breast cancer model in mice, concomitant injection of breast cancer cells and fibroblasts increased the rate of metastasis, which showed dependence on Wnt-planar cell polarity in the breast cancer cells and an exosomal component of CD81 in the fibroblasts (Luga et al. 2012). The authors concluded that the exosomal activity of

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fibroblasts altered breast cancer cell behavior. Similarly, the role of tumor-associated exosomes in contributing integrin has been elucidated that enhanced tumor progression (Paolillo and Schinelli 2017). The malignant cell-derived exosomes alter the tumor microenvironment such that it becomes conducive for the development and progression of the tumor via interference with the cell adhesion mechanisms involving integrins and integrin ligands (Maia et al. 2018; Zheng et al. 2018). In particular, exosomal integrins are involved throughout, from colonization by cancer cells to the composition of a niche to facilitate metastasis (Richards et al. 2017). Exosomal miR-105 facilitates the dissemination of the tumor cells by acting on the tight junction, thus compromising the integrity-endothelial cell barrier and weakening its functioning as a barrier (Zhang et al. 2018a, b). Exosomes also interfere with the permeability of the blood vessels. In colorectal cancer cells, miR-25-3p regulates VEGF-receptor 2, tight junction protein, and claudin-5 in endothelial cells. Exosomes secreted by the tumor cells also cause differentiation of the cells constituting their microenvironment by remodeling the extracellular matrix (Syn et al. 2016). Besides miRNAs, tumor cell-derived exosomes also contain a complete panel of bioactive molecules including HIF, TGF-β, caveolin, and b-catenin which is supportive in epithelial-to-mesenchymal transition and contributes to premetastatic niche formation (Syn et al. 2016; Becker et al. 2016). Pancreatic adenocarcinoma (PADC) is rich in cancer-associated fibroblasts which are inherently resistant to gemcitabine and promote survival of cancer cells (Richards et al. 2017). While elucidating the underlying mechanism, it was observed that cancer-associated fibroblasts-derived exosomes were responsible for this cytoprotective and proproliferative activity of PADCs in the presence of gemcitabine. Molecular studies revealed that cancer-associated fibroblasts-derived exosomes were able to transfer Snail and its target miR-146a to the recipient cancer cells to impart cytoprotective and cell-cycling activity in the recipient cells. Interestingly, abrogation of the exosome release from cancer-associated fibroblasts also abrogated the cytoprotective effects on the cocultured cancer cells in the presence of gemcitabine. Cancer-associated fibroblasts-derived exosomes also have a role in glycolysis via increasing the absorption of glucose, which is considered as a contributing factor in the ongoing growth of cancer even under unfavorable reduced nutrition and hypoxic condition (Richards et al. 2017). In another study, it was observed that PADCs with high metastatic potential could release exosomes rich in migration-inhibitory factor, which supported premetastatic niche formation in the liver (Costa-Silva et al. 2015). The abrogation of emigrational inhibitory factor also abolished metastatic niche formation as well as metastasis PADCs in the liver. Angiogenesis is indispensable for tumor development, progression, and dissemination (Katoh 2013; Zuazo-Gaztelu and Oriol 2018). In this regard, tumor cellderived exosomes contribute significantly to the formation of neovasculature and increase in vascular density via activation of VEGF signaling (Nishida et al. 2006; Gluszko et al. 2019). Besides VEGF, the angiogenic activity in the tumor tissue is supported by various other proangiogenic molecules, i.e., basic fibroblast growth factors, platelet-derived growth factor, etc., which are also delivered by the tumorderived exosomes TDEs (Sharghi-Namini et al. 2014; Monteforte et al. 2017).

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Ludwig et al. have evaluated the role of tumor cell-derived exosomes in proangiogenic response during in vitro assays on HUVECs. The exosomes were derived from PCI-13 (HPV
) and UMSCC47 (HPV+) cell lines and the plasma of head and neck squamous cell carcinoma patients and were labeled before use in angiogenic assays (Ludwig et al. 2018). The exosomes were taken up by HUVECs in the culture within 4 h after treatment and stimulated HUVEC proliferation, migration, and tubulogenesis. Similar data were obtained after the treatment of experimental mice model of oral carcinoma after intravenous exosome treatment. On the same note, profiling of the glioblastoma-derived exosomes revealed high levels of miR-221, proteoglycans, glypican-1, and syndecan-4 that was ascribed to the acutely angiogenic nature of glioblastoma (Hoshino et al. 2015). Tumor cell-derived exosomes containing metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which is associated with angiogenesis and metastasis in epithelial ovarian cancer, induced proangiogenesis gene expression in HUVECs (Qiu et al. 2018). Put together, these data signify the importance of exosomes in angiogenesis and support their future use as targets for tumor cells by suppressing endothelial cell migration, their phenotype alteration, and vascular sprouting (Harding et al. 2013).

The Pathways of Exosomal miRNA Secretion The cancer cells-microenvironment interaction essentially occurs through the release of bioactive molecules which contributes significantly toward the development and progression of tumors (Ungefroren et al. 2011; Kohlhapp et al. 2015). Despite the cell-specific role of miRNAs in tumor suppression and drug resistance, the participation of miRNAs in non-cell-autonomous mechanisms remains less explored. Although knowledge about the secretory RNA has been around since long, however, only recent reports have shown that miRNAs, which control various cellular processes through the regulation of specific multiple gene targets, are secreted by cells as part of the paracrine activity of the cells in the form of exosomal payload (Yu et al. 2016; Sansone et al. 2017). These data signify the emergence of both miRNAs and exosomes as the novel constituents of cells’ paracrine activity with relevance to intercellular communication (Kalluri and LeBleu 2016). The role of neutral sphingomyelinase 2 (nSMase2) in the release mechanism of miRNAs and the functional activity of the released miRNAs in the recipient cells has been reported by Serrano-Heras et al. (2010). It is interesting to note that the tumor-suppressor miRNAs released by the normal cell potentially retain and impart gene silencing activity in the recipient tumor cells that may lead to abrogation of tumor cell growth (Huang et al. 2013). The critical role of exosomal miRNAs in intracellular communication, specifically between immune cells, endothelial cells, and cancer cells, is now becoming more obvious; however, their role in cancer metastasis remains less well established (Bhome et al. 2018). Exosomal miR-210 transfer from metastatic cancer cells to the endothelial cell alters them to favorably support the process of cancer cell migration through neovascularization, thus helping in their metastatic spread. Molecular studies

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revealed that miR-210 controlled the process of neovascularization by the suppression of its specific downstream target genes (Jung et al. 2017). The changes in the miRNA profile have been ascribed with different types of cancers wherein the expression of some miRNAs is either upregulated or downregulated (EsquelaKerscher and Slack 2006; Girard et al. 2008; Peng and Croce 2016). On the same note, changes in the expression of miRNAs either support or inhibit the development and progression of cancer (Ota et al. 2004). The data supporting the critical involvement of miRNAs in cancer pathogenesis has led to their future use as potential therapeutic targets. Moreover, the huge amount of information regarding miRNA families that are altered in cancers will be important for the development of miRNA signatures which will be helpful in the diagnosis and therapy of cancers (BerindanNeagoe et al. 2014). The involvement of Argonaute proteins in miRNA function is well established. However, some exosomal miRNAs are Ago2-free (Arroyo et al. 2011). Instead, they are identified by specific proteins (e.g., the heterogeneous nuclear ribonucleoproteins hnRNPA2B1 and hnRNPA1) that selectively allow loading of miRNAs into exosomes (Janas et al. 2015). miRNAs loading into the exosomes is a well-orchestrated and regulated process. Villarroya-Beltri et al. have shown that miRNAs are loaded into exosomes via sumoylated hnRNPA2B1 (Villarroya-Beltri et al. 2013). Alternatively, the process involves two other heterogeneous nuclear ribonucleoproteins (hnRNP family proteins), namely, hnRNPA1 and hnRNPC. These proteins bind with exosomal miRNAs for their sorting process. The second mechanism is based on neural sphingomyelinase 2 (nSMase2) pathways (Kosaka et al. 2013), whereas the third mechanism is based on the 30 ends of endogenous miRNAs. miRNAs are transported by RNA-binding proteins to the lipid raft-like region of the cytoplasmic leaflet of the MVB-limiting membrane, where miRNAs with the highest affinity are engaged (Janas et al. 2015). Once attached, the miRNA initiates a spontaneous invagination to produce intraluminal vesicles (Schwarzenbach and Gahan 2019).

Exosomal miRNAs in Cancer Progression Several miRNAs are actively involved in various cellular processes (Yamakuchi et al. 2008; Pedroza-Torres et al. 2019; Chen et al. 2019). They are also involved in cancer progression and metastasis (Dilsiz 2020). Due to their critical roles and their presence in exosomes, miRNAs are considered to be promising markers for cancer diagnosis and prognosis (Wang et al. 2019). They can be used singly or in combination for cancer diagnosis (Tang et al. 2020). Cancer cell proliferation requires the dysregulated expression of cell cycle-related proteins. During the last decade, several cancer-secreted exosomal miRNAs have been identified as key regulators of cancer cell functioning. Some of the important ones from the long list include miR-200b, miR-21, miR-6869-5p, miR-9-3p, miR-let-7a, and miR-193a which work though diverse molecules such as p27, proinflammatory cytokines like IL-6 and tumor necrosis factor-α, FGF-5, and Caprin-1 (Teng et al. 2017; Zhang et al. 2018b; Liang et al. 2018; Tang et al. 2018; Liu et al. 2019).

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Several studies have demonstrated that exosomal miRs also have a critical role in tumor cell apoptosis (Eichelser et al. 2014; Wei et al. 2016). For example, miR-128 decreases the expression of prosurvival protein Bcl-2 to enhance tumor cell survival. Eichelser et al. have reported analyses of the blood serum samples for free and exosomal miRNAs in 168 breast cancer patients. Exosomal miRNA profiling revealed significantly higher levels of miR-101, miR-372, and miR-373, while miR-373 was shown to interact with the estrogenic receptor for inhibition of the apoptosis cascade (Eichelser et al. 2014). The authors concluded that exosomal miR-373 was strongly linked with a more aggressive form of breast cancer. A recently published study has shown that conditioned medium derived from human adipose tissue-derived MSCs inhibited cell proliferation and activated cell apoptosis signaling in A2780 human ovarian cancer cell line (Reza et al. 2016). The authors attribute these actions of the conditioned medium with the presence of MSCsderived exosomes, which upregulated proapoptotic signaling molecules including BAX, Casp3, and Casp9 with a concomitant decrease in antiapoptotic Bcl2. Exosomal analysis for miRNAs revealed two miRNAs, which were commonly expressed in three samples of the conditioned medium collected during independent experiments. These findings establish a clear interaction between MSCs and cancer cells via exosomal activity. miRNAs also participate in the relapse of the tumors. Exosomal miRNAs are regulators of EMT that contribute to the downregulation of E-cadherin and β-catenin and the expression of N-cadherin and vimentin markers which are specific to the mesenchymal phenotype. In particular, exosomal miR-21 can decrease E-cadherin expression, contributing to the induction of EMT-related changes in oral squamous cell carcinoma cells. Additionally, miR-1246 suppresses N-cadherin and vimentin activities and therefore interferes with the induction of the EMT process in prostate cancer cells (Bhagirath et al. 2018). miRNAs participate in tumor-endothelial cells’ crosstalk to regulate angiogenesis and cancer progression. For example, miR-103 targets VE-cadherin and P120catenin, which are components of the endothelial cell junction to increase vascular permeability (Fang et al. 2018). Cells secreting exosomal miR-210 in hypoxic leukemia induce the endothelial cell activation to participate in the angiogenic cascade (Tadokoro et al. 2013). In miRNA profiling of chronic myeloid leukemia cell line LAMA84, 124 miRNAs were identified out of which miR-126 was highly overexpressed in the exosomes as compared with the cellular contents. Further experimentation with miR-126 revealed that it negatively regulated the motility of leukemia cells via altering CXCL12 and VCAM-1 expression levels (Taverna et al. 2014). Exosome profiling of the metastatic breast cancer cells revealed the extensive presence of proangiogenic miRNAs including hypoxamir-210, which is a known regulator of proangiogenic growth factor expression (Kosaka et al. 2013). The data was produced by in vitro culture of mouse cell lines 4 T1 and MCF7 breast cancer cell line, and the exosome isolation was carried out by ultracentrifugation and labeled with fluorescent tracking dye. The culture of HUVEC with exosomes significantly enhanced their activity during in vitro angiogenesis assays, which were more attributed to miR-210 with a significant role for neutral sphingomyelinase-2 for exosomal transfer of miRNAs. Similar observations have

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also been reported with miR-23a from nasopharyngeal carcinoma cell-derived exosomes during in vitro angiogenesis assays which act by abrogation of its downstream target gene TSGA10 expression (Bao et al. 2018). On the other hand, exosomal miR-9 abrogated nasopharyngeal carcinoma cells’ motility and angiogenesis via its downstream target gene midkine, thus interfering with Akt signaling (Lu et al. 2018). Exosomal miRNAs also participate in the metastatic spread of cancer cells. In this regard, exosomal miR-148a is reported to cause proproliferative activity in cell glioblastoma by targeting its downstream gene CADM1 via STAT3 involvement (Cai et al. 2018). The authors also reported a negative correlation between miR148 and CADM1 gene expression profile in the patient samples. These data also highlight the possible use of exosomal miR-148a as a novel target for the development of future treatment strategies. Exosomal miR-423-5p is another important regulator of the cancer cell cycle through SUFU gene expression (Yang et al. 2018). In lung cancer, exosomal miR-96 has been shown to increase cancer cell proliferation and motility by targeting its downstream target gene LIM domain-only protein 7 (LMO7) (Wu et al. 2017). Some of the other important mediators of cancer cell activities reported thus far include miR-125a, miR-126, let-7b, miR-222, and miR-6126 (Felicetti et al. 2016; Kanlikilicer et al. 2016; Zhang et al. 2018b).

Tumor-Derived Exosomes and Lung Cancer The function of tumor-derived exosomes in nonsmall cell lung cancer (NSCLC) implicates a combination of intricate mechanisms as summarized in Fig. 1. One of the primary factors in cancer growth and progression in NSCLC is the rich presence of cancer-associated fibroblasts in the tumor microenvironment. These cancer-associated fibroblasts serve as a plentiful source of exosomes containing a payload of diverse molecules including RNA, lipids, amino acids, and tricarboxylic acid cycle intermediates (Zhao et al. 2016; Zheng et al. 2018). As discussed earlier, tumor-derived exosomes are instrumental in tumor angiogenesis, which facilitate tumor growth and metastasis. The induction of angiogenesis and tumor growth involves the TGF-β1-dependent pathway and stimulates fibroblasts differentiation (Webber et al. 2015). Hypoxic lung cancer cells cultured under low oxygen conditions released exosome which contained miR-23 for increased angiogenesis and enhanced permeability of the vessels through the targeting of tight junction proteins (Hsu et al. 2017). Exosomal miR-21 is regulated by the STAT3 pathway, which enhances the level of VEGF and consequently malignant transformation of the bronchial epithelium (Liu et al. 2016). Exosomal miR-210 is a hypoxamir, which regulates the levels of tyrosine receptor kinase A3, and induces vascular density in the tumors and supports the growth of the tumor cells (Cui et al. 2015). Lung cancer cell-derived exosomes participate in lung cancer progression by interacting with the other constituent cells of the tumor microenvironment. Mesenchymal stem cells are transformed into a proinflammatory phenotype via the Tolllike receptor (TLR)-NFκB pathway by the lung cancer cell-derived exosomes. These

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Tumor growth Tumor cells miR-200b, miR-21 miR-1246, miR-128 miR-373, miR-9-3p miR-193a, miR-6889-5p

miR-105, miR-155 miR-210, miR-1247-3p miR-10b, miR-21 miR-143, miR-378a miR-320a, miR-141b miR-451 CAFs

miRNAs miR-126, miR-100 miR-103, miR-210 miR-9, miR-23a

Angiogenesis

Cytoplasm Nucleus

miR-125b, miR-222-3p miR-21, miR-29a miR-1246, miR-940 miR-24-3p, miR-212-3p

Lysosome

Donor cell Immune response

miR-365, miR-34a miR-221/222, miR-151a miR-146a-5p, miR-100-5p miR-21

miR-21, miR-1246, miR-200 miR-101, miR-148a, miR-423-5p miR-96, miR-222, miR-221, miR-301a-3p, miR-6126, miR-940 miR-490, miR-23b, miR-675,......

Metastasis

Drug resistance

Fig. 1 Tumor-derived exosomal and their secreted miRNAs. Exosomal miRNAs disseminate information between cancer cells and immune cells (macrophages, T cells, and dendritic cells) and contribute toward the microenvironment to make it conducive for the cancer cells to thrive

exosomes facilitate tumor progression by interfering with the immune cell functioning to help the tumor cells escape the immune surveillance mechanism by reprogramming the immune cells (Ridder et al. 2015; Whiteside 2016). These studies highlight the role of exosomal payload in the growth and progression of NSCLC, which needs to be investigated further in future studies. A major cause of death in lung cancer patients is the metastasis, which is multifactorial, regulated, and completed in several steps. However, only a few of the cancer cells can complete the entire process in a tissue-specific manner during which they preferentially lodge in different body tissue. For example, NSCLC cells preferentially disseminate and home into the brain, bone, and liver with a direct or indirect mechanistic role for tumor-derived exosomes (Wood et al. 2014). The main role of exosomes in the metastatic process is their contribution to the formation of lung cancer microenvironment and increasing the invasiveness of the tumor cells (Milane et al. 2015; Fujita et al. 2015). Some aspects of the tumor, including hypoxia, acidosis, and inflammation, can induce the tumor cells to release exosomes with their specific payload necessary to form the microenvironment with the

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consequent growth of the tumor and its dissemination (King et al. 2012; Wang et al. 2016a). While elucidating the role of tumor cell-derived exosomes, Wang et al. showed that exosomal TGF-β and IL10 significantly contributed toward the emigrational activity of cancer cells (NCI-H1688 and NCI-H2228) during their experiment and demonstrated that exosomes derived from metastatic small cell lung cancer cell line NCI-H1688 were more effective in promoting lung cancer cell migration as compared to NSCLC cell line NCI-H1688-derived exosomes (Wang et al. 2016a). Hoshino et al. have shown development of specialized actin structures invadopodia which serve as exosome release and docking area. Abrogation of invadopodia significantly reduced exosome secretion during in vitro culture of the tumor cells, thus revealing a direct relationship between invadopodia formation and the rate of exosome release from the tumor cells (Hoshino et al. 2013). During an interesting in vitro study, human PCA LNCap and PC3 cells were subjected to either normoxic (21% oxygen) or hypoxic culture conditions (1% oxygen) to collect their secreted exosomes separately. The exosomes collected from the hypoxic cultured cells not only were smaller average size, but they also were more effective in promoting the motility of the naïve PCA LNCap and PC3 cells. The exosomes collected under hypoxic conditions also showed higher metalloproteinase activity besides being rich in various signaling molecules including IL6, tumor necrosis factor-α, TGF-β, Akt, and β-catenin (Ramteke et al. 2015). A recently published study has reported that exosomes released by colorectal cancer cell LM1215 triggered Wnt signaling in the coculture-recipient cells (Kalra et al. 2019). The exosomal transfer of mutant β-catenin to the nucleus of the recipient cells was confirmed by proteomic analysis. Tumor-derived exosomes are also considered to be prognostic markers in NSCLC (Wang et al. 2016b). In a study involving 276 NSCLC, exosomes were isolated from the plasma samples and assessed for surface expression of various markers using an Extracellular Vesicle Array. From among the 49 antibodies which showed reactivity with surface proteins of the exosomes, although NY-ESO-1, placental alkaline phosphatase (PLAP), epidermal growth factor receptor (EGFR), Alix, and EpCam correlated well with overall survival, NY-ESO-1 was the only one which maintained a significant correlation in all the samples, thus signifying its prognostic value in NSCLC patients (Sandfeld-Paulsen et al. 2016). On the same note, Liu et al. have reported a significant association between exosomal miR-10b-5p, miR-23b-3p, and miR-21-5p with poor overall survival in NSCLC patients (Liu et al. 2017). On account of their specificity and easy to isolate from the body fluids which can be sampled noninvasively, exosomes may be novel biomarkers with diagnostic and prognostic value for tumor patients in the clinical perspective (Zhao et al. 2015).

Future Perspectives of Exosomal miRNAs in the Clinics The molecular targets and the signaling involved therein as drug targets, and transporters to regulate cellular processes, i.e., cell survival and proliferation, have been shown with an integral regulatory involvement of several miRNAs (Corcoran et al. 2012). The payload of exosomes, despite having diverging composition even from the same cells under a different set of their microenvironment, is rich in proteins,

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DNA, and RNA that is actively involved in the activation and inhibition of the pathways that are crucial during chemotherapy, radiotherapy, and targeted therapies. For example, it was recently described that miR-34a (with tumor-suppressor activity) abrogation during chemotherapy of prostate cancer treatment with paclitaxel was the primary cause of chemoresistance in the patients. Given its significant tumorsuppressor activity, miR-34a is now being considered as an established predictive biomarker of prostate cancer progression during paclitaxel-based chemotherapy via Bcl-2 gene expression regulation (Corcoran et al. 2014). Other researchers have highlighted the increase in tamoxifen sensitivity of breast cancer cells after the internalization of exosomes in tamoxifen-resistant breast cancer cells. This mechanism seems to be regulated by miR-221 or miR-222 delivered by the exosomes (Wei et al. 2014). Furthermore, in the lung cancer field, exosomal miR-21 has been investigated as a biomarker of the therapeutic outcome in NSCLC, and it was associated with acquired resistance to the treatment with EGFR and tyrosine kinase inhibitors (Li et al. 2014). Similarly, exosomal miR-21 is a crucial determinant of the radiosensitivity of tumor cells. Successful abrogation of miR-21 inhibits PI3K-Akt signaling to enhance their radiosensitivity (Ma et al. 2014). These data signify the role of miR-21 as an important predictor of response to the drug therapy and its worsening outcomes during chemotherapy (Ma et al. 2014; Schwarzenbach 2017). Hence, exosomal miRNAs could form the basis of personalized therapy on account of their potential as targets for therapy and their role in drug resistance. Another important aspect of exosomal miRs is their potential role to serve as biomarkers (Schwarzenbach 2017). Several exosomal miRs isolated from tumor patients’ plasma samples have the potential to serve as biomarkers as summarized in Fig. 2. For example, exosomal miR-21 is contained in plasma but has also been detected in fecal samples of the patients and therefore can be exploited as for colorectal cancer diagnosis (Rotelli et al. 2015). Other exosomal miRNAs that are upregulated in receptor-negative breast cancer patients have been recently reviewed and discussed in-depth (Yu et al. 2016; Stevic et al. 2018; Wu et al. 2020). The upregulated expression of miR-1290 and miR-375 has a negative prognostic value on account of its association with the poor overall survival in castration-resistant prostate cancer (Corcoran et al. 2012, 2014), while exosomal miR-19a is linked to the recurrence in colorectal cancer (Matsumura et al. 2015). Exosom-derived miRs are also the inhibitors of tumor development which changes their use as part of future novel anticancer therapies. Nevertheless, the correlation and interaction between miRNAs, TDEs, and the immune system require further clarification. For example, the release of miR-142 and miR-223 posttranscriptionally regulates the expression of various proteins in hepatocarcinoma cells (Aucher et al. 2013). Similarly, exosomal miR-29c has antiapoptotic activity due to the suppression of Bcl2 and MCl-1 expression that leads to preventing bladder cancer cells’ apoptosis (Xu et al. 2014). Moreover, exosomal miR-127 and miR-197 increase the rate of cell proliferation and promote the metastatic transfer of tumor cells. Hence, these exosomal miRNAs can reduce the effectiveness of tumor treatment by promoting resistance to therapies (Lim et al. 2011). The manipulation of these miRNAs may significantly contribute to the development of novel cancer treatments, especially in patients with metastatic disease. One

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Biomarkers Patient

Exosomes Blood Exosome -based therapies Stem cells Exosomes

Therapeutic molecules (eg. siRNA) Drug delievey

Fig. 2 Summary of the teranostic applications of exosomal miRNAs

of the most important characteristics of exosomes is that they can protect their payload due to lipid bilayered structure and their small size that allows them to remain in the circulation for a long time, thus allowing their detection over longtime periods (Syn et al. 2016). Once taken up by the recipient cells, the miR payload is successfully delivered by the exosomes to take part in the ongoing cellular processes (Barile and Vassalli 2017). Exosomes are also able to spread many pathogens, including viral proteins or viral genomes. The incorporation of pathogens into exosomes affects the immune response to infection (He et al. 2018). There are some limitations to exploit exosomes as a miR delivery system such as cellular toxicity due to extensive regulatory influence on various cellular processes and activities. The current understanding of exosomal miRNAs has highlighted the importance of these microvesicles not only for cancer but also for other diseases. For example, different miR-21-5p, miR-29a-3p, and miR-126-3p are related to diabetic kidney disease process and progression that also tips them as prospective biomarkers of the disease (Assmann et al. 2018). miR-21 and miR-29a are also integral to the NSCLC growth and dissemination, while miR-126 fosters metastatic activity in hematological malignancies (Fabbri et al. 2012; Chevillet et al. 2014). However, the impact of exosomal miRNA release on tumor development remains to be determined. Exosomal miRs offer an effective pool of circulating miRs as compared to the nonexosomal miRNAs. Future studies are therefore required to discriminate the exosomal and free miRNAs besides determining whether miR exosomal packaging and uptake of miRNAs are specific and triggered by specific cues.

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Tiziana Annese, Roberto Tamma, and Domenico Ribatti

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An Overview of Endothelial Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endothelial Progenitor Cells in Preclinical Cancer Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Side Effects and Potential Risks of EPCs Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer is the second leading cause of death worldwide after cardiovascular diseases, accounting for an estimated ten million deaths annually. Researchers are making a great effort to identify more efficient therapeutic strategies. To date, genetically modified stem cells are a potential candidate for the development of new antitumor therapies and diagnostic investigation methods. Among stem cells, endothelial progenitor cells (EPCs), a subpopulation of multipotent hematopoietic stem cells (HSCs), appear promising. In response to specific stimuli, EPCs are fundamental to tumor progression because of their role in vasculogenesis and sprouting angiogenesis. In a healthy adult individual, the process of neoangiogenesis is activated only during wound healing and in the female uterus during ovulation. Therefore, it is reasonable to use them in anticancer therapy by taking advantage of their natural tropism to the altered microenvironment. Diverse studies demonstrated that EPCs predominantly home into the tumor mass, and hence, they are useful as a cellular vehicle for site-directed drug targeting to the tumors or for the delivery of imaging probe. T. Annese · R. Tamma · D. Ribatti (*) Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_29

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This chapter explores the underlying molecular mechanisms and the potential application of stem cell therapy in cancer with special reference to EPCs application in targeted gene therapy. How they could be modified, obtained in a significant amount, and administrated to treat cancer has been discussed.

Keywords

Angiogenesis · Cancer therapy · Diagnostic imaging · Endothelial · EPCs · Progenitor cells · Vasculogenesis Abbreviations

5-FC Ac-LDL ASCs BMMCs CD CEPCs CFU-ECs DR4 DR5 ECFCs ECs EC-SPs eNOS EOCs EPCs EPO ESCs FBS FSCs GCV GMCSF GSCs HIF-1α HPCs HSCs iPSCs MAPCs MCP-1 MRI MVD NK PARP PBMCs

5-fluorouracil acetylated low-density lipoprotein adult stem cells bone marrow mononuclear cells cytosine deaminase circulating EPCs colony-forming unit-EC death receptor 4 death receptor 5 endothelial colony-forming cells endothelial cells endothelial cell-side progenitors nitric oxide synthase endothelial outgrowth cells endothelial progenitor cells erythropoietin embryonic stem cells fetal bovine serum fetal stem cells ganciclovir Granulocyte-macrophage colony-stimulating factor glioma stem-like cells hypoxia-inducible factor 1 alpha hematopoietic progenitor cells hematopoietic stem cells induced pluripotent stem cells multipotent adult progenitor cells monocyte chemotactic protein magnetic resonance imaging microvessel density natural kill cells poly ADP-ribose polymerase peripheral blood mononuclear cells

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Endothelial Progenitor Cells from Bench to Antitumor Therapy and Diagnostic. . .

PD-1 PEI2k PlGF PSCs QQc SCs SDF Sirt1 SPECT SPIO TRAIL UEA1 uPAR VEGF VEGFR VESCs VW-EPCs vWF VW-VSCs

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programmed death-1 polyethylenimine 2 kDa placental growth factor placenta stem cells quality and quantity culture stem cells stromal cell-derived factor sirtuin-1 single-photon emission computed tomography superparamagnetic iron oxide tumor necrosis factor-related apoptosis-inducing ligand Ulex europaeus agglutinin 1 urokinase-type plasminogen activator vascular endothelial growth factor vascular endothelial growth factor receptor vascular endothelial stem cells vascular wall endothelial progenitor cells von Willebrand factor vascular wall-resident vascular stem cells

Introduction Cancer is a vast group of diseases that share some characteristics. Cancer cells can develop in all tissues/organs of the body, have a high proliferation rate, and can invade the normal surrounding tissue and beyond. Metastasizing is a leading cause of death from cancer (Dillekas et al. 2019). Cancer is the second dominant death source worldwide after cardiovascular diseases, accounting for an estimated ten million deaths annually (Bray et al. 2018). Lung, breast, colorectal, prostate, stomach, liver, esophagus, cervix uteri, thyroid, and bladder cancers are, in order, those with the highest incidence (Bray et al. 2018). As a general trend, patients’ survival rate and life quality are improving thanks to early diagnosis, prevention campaigns, and improved standards of care. Despite this, patients’ physical and economic efforts and the entire health system make cancer a huge problem and a considerable challenge for researchers. Different therapeutic designs are distinguished according to the type of cancer and the stage of development. These include surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, precision, and personalized medicine (NCI 2020). Surgery for cancer treatment is called curative surgery and is usually applied when the tumor mass is well-confined to a specific body part. Before and/or after resection, the patient could be treated with radiotherapy or chemotherapy. Radiation therapy includes different approaches such as external beam radiation therapy, internal radiation therapy (brachytherapy), oral or systemic radiation therapy, and photodynamic therapy. The operating principle consists of high-energy electromagnetic waves or molecules that create DNA

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damage in proliferating tumor cells. Chemotherapy consists of using one or more drugs that act mainly against proliferating cells and thus against cancer cells to prevent or limit their growth and spread. Immunotherapy is a treatment that uses cells or molecules of the immune system, such as use of antibodies or vaccines, or T-lymphocytes, to restore or boost the patient’s immune system. Targeted therapy applies drugs designed to “target” specifically cancer cells or cells of the surrounding microenvironment without affecting normal cells, exploiting their unique expression of some genes or proteins. Hormone therapy is a systemic one in which hormones are administrated to destroy cancer that depends on them to grow, like breast and prostate cancers that depend on sex hormones. Stem cell transplant is exploited to replace the patient’s bone marrow cells treated with chemo and/or radiation therapy against such cancers as leukemia and lymphoma. Precision and personalized medicine is the newest approach and is based on the patient’s genome and epigenome characterization because there is high intra-tumoral heterogeneity. Still, it is only in clinical trials for now. Among these approaches for cancer treatment and to adopt high-performance methods in terms of improved therapeutic efficacy and fewer undesirable effects, stem cell transplant, alone or in combination with other therapies, could be the right strategy for treatment and the development of new diagnostic investigation methods due to its enhanced target on tumors.

Stem Cells In all development stages from the embryo to the adult, all organs and tissues possess undifferentiated precursor cells, mitotically active, multipotent, and capable of regenerating mature cells, called stem cells (SCs). They are a reservoir of precursor cells playing a homeostatic role essential for replacing dead or damaged cells due to trauma or diseases (Galli et al. 2003). SCs are highly undifferentiated cells that do not possess morphological, structural, molecular, and antigenic characteristics found in the tissue’s differentiated cells to which they belong. SCs can perpetuate themselves through their ability to self-renew (Weissman et al. 2001). In general, the in vivo self-renewal last for the organism’s whole life, but in vitro, it is unlimited under the appropriate experimental conditions. Two types of stem cell divisions are distinguished, symmetric cell divisions and asymmetric ones (Shahriyari and Komarova 2013). In symmetric divisions, the two daughter cells are identical to each other and to the mother cell (expansive symmetric division) or, in the alternative, identical to each other but different from the mother (differentiative symmetric division), called progenitors. In asymmetric division, a stem cell produces one differentiated cell and one stem cell. This system allows the number of stem cells to remain constant at the end of each cell generation. It offers the enormous advantage of increasing or decreasing the number of stem cells within a tissue. Another critical feature of stem cells is multipotentiality, which is the ability to give rise to a differentiated progeny comprising all types of cells of the residence tissue or, in the case of embryos, to all cells of the adult organism. Stem cells,

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according to their potential, are classified as totipotent if they are not specialized and can give rise to a new embryo, such as embryonic cells at the stage of 4–8 cells after 4–5 days from fertilization; pluripotent, if they have the potential to differentiate into all cell types that derive from the three embryonic layers (endoderm, ectoderm, and mesoderm), but they do not have the potential to give rise to an embryo, such as embryonic stem cells (ESCs) at the blastocyst stage with 20–30 cells, after 5–7 days from fertilization; multipotent, if they can differentiate in all cell types of a specific organ or tissue such as hematopoietic stem cells (HSCs); unipotent, if they can give rise to a single cell type such as keratinocytes (Łos et al. 2019). SCs can also be recruited where they are required to participate in the repair process, thanks to a controlled process called homing, and once they reach the site, they settle there (engraftment). Their well-directed migration is under the control of cytokines gradient and is used to regenerate damaged tissues. Based on the source, stem cells are classified into ESCs, such as cells isolated from human blastocysts; fetal stem cells (FSCs), such as gonadal cells from abortive fetuses; umbilical cord stem cells, such as cells isolated from cord blood umbilical of newborns; placenta-derived stem cells (PSCs), isolated from the placenta of newborns; adult stem cells (ASCs), isolated from adult tissues such as HSCs; induced pluripotent stem cells (iPSCs), obtained by dedifferentiation of mature cells to embryonic cells by genetic manipulation. iPSCs open new therapeutic opportunities, which are practically the same as those of human embryonic stem cells, but without ethical and scientific concerns. ASCs, present in small quantities in stem niches of the whole organism, remain quiescent until disease or trauma reactivate them inducing proliferation and differentiation. The niche is a tissue location where a dynamic and specialized microenvironment regulates stem cell biology (proliferation, maintenance, or differentiation). They are present in different organs and tissues: the hematopoietic system (Osawa et al. 1996), brain (Galli et al. 2000; Goritz and Frisen 2012), dermis (Toma et al. 2001), muscle (Qu-Petersen et al. 2002), and liver (Shafritz et al. 2006). Until recently, it was generally thought that ASCs could at most differentiate into all cell types of the tissue they belong to (Price et al. 2007). However, today, it has been observed that, under optimal set of conditions, they can differentiate into other cell types, in addition to those of the original tissue. For example, after bone marrow transplantation enriched with HSCs, they can differentiate in all the three germinal layers’ cells (Jackson et al. 2001; Mezey et al. 2000; Orlic et al. 2001; Theise et al. 2000). Stem cells are applied in regenerative medicine for diseases such as Parkinson’s disease (Ourednik et al. 2002), spinal cord damage (Teng et al. 2002), multiple sclerosis (Pluchino et al. 2003), amyotrophic lateral sclerosis (Clement et al. 2003), stroke (Liu et al. 2009), retinal degeneration (Li et al. 2006), Alzheimer’s disease (Barnham et al. 2004), myocardial infarction (Jackson et al. 2001), and others. The unique self-renewal and differentiation potential of stem cells are the primary reasons for their use to regenerate damaged organs and correct congenital diseases. However, a major limitation for the therapeutic use of stem cells is the risk of iatrogenic oncogenesis.

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The source of the cells for therapy could be the same patients (autologous transplantation) or a donor (allogeneic transplantation). The main attraction is for the immune-privileged autologous stem cells that express the major histocompatibility complex 1 (MHC1), but not MHC2, clusters of differentiation because these can be used in immunocompetent patients, avoiding side effects and with better therapeutic efficacy and significantly improved safety. For instance, in a preclinical study to evaluate EPCs for target gene therapy, it was shown that these cells do not express MHC-I, are resistant to lysis by non-activated natural kill cells (NK), and survive and participate in tumor blood vessel formation after intravenous injection (Wei et al. 2004).

An Overview of Endothelial Progenitor Cells EPCs are mostly unipotent stem cells capable of differentiating into endothelial cells (Khakoo and Finkel 2005). In vivo, they can differentiate from hemangioblasts, bone marrow multipotent adult progenitor cells (MAPCs), and myeloid/monocytic cells. In vitro, the early and late EPCs are distinguished (reviewed in (George et al. 2011)). Furthermore, to be present as such in bone marrow, peripheral and umbilical cord blood, EPCs can be produced by transdifferentiation of stem cells present in various tissues and organs, under the influence of adequate microenvironments for endothelial differentiation (for extensive EPCs sources, readers can consider the reviewing article (Chopra et al. 2018)). EPCs express endothelial markers such as CD133, CD31, CD34, CD146, and VEGFR2 and do not express the hematopoietic marker CD45 or mature ECs markers including VEGFR1, VE-cadherin, and Von Willebrand factor (vWF) (George et al. 2011; Medina et al. 2017). CD34+/CD133+/VEGFR2+ cells are usually, but not unambiguously considered EPCs (Medina et al. 2017). EPCs can be studied in two ways, flow cytometry or in vitro culture (Medina et al. 2012). Flow cytometry is used for studying circulating EPCs (CEPCs) in the blood samples where they are quantified as the percentage of mononuclear cells CD34+/ VEGFR2+/CD133+ (Peichev et al. 2000; Wu et al. 2007). In vitro culture methods are applied to study EPCs derived from peripheral blood mononuclear cells (PBMCs) or by direct flushing of bone marrow mononuclear cells (BMMCs) and expanded using endothelial-specific media. During in vitro culture, two different cell types can be generated, the early and late outgrowth cells being hematopoietic and endothelial, respectively (see Table 1) (Medina et al. 2010). Only the last ones are considered valid EPCs (see Table 2) (Banno and Yoder 2019). The late outgrowth cells, also called endothelial colony-forming cells (ECFCs), originate from CD45 / CD133 /CD34+ MNCs, in vitro arise after 7 days, have a highly proliferative polygonal shape, do not differentiate into hematopoietic cells, and produce vascular tube in vitro and in vivo. Moreover, ECFCs can affect neovascularization in vivo, take up acetylated LDL, bind to Ulex europaeus agglutinin 1 (UEA1), and express the surface markers CD31, vWF, CD105, CD146, VE-cadherin, and VEGFR2 (Timmermans et al. 2009; Yoder et al. 2007). Some studies have shown a synergistic

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Table 1 EPCs isolated using in vitro culture methodologies classification based on a specific phenotype and a biological function

In vitro From PBMCs or umbilical cord blood appear after Achieve peak growth at Survive up to Markers

Role in new vessel formation

Secretion/ expression of pro-angiogenic factors

Name MACs; early outgrowth EPCs; early EPCs; hematopoietic EPCs; CACs; PACs; CFU-ECs; CFU-HILL; small EPCs; Myeloid EPCs 4–10 days

ECFCs; late outgrowth EPCs; LATE EPCs; non-hematopoietic EPCs; OECs; BOECs’ EOCs; large EPCs >2 weeks

2–3 weeks

4–8 weeks

4 weeks CD45+ CD14+ CD31+; CD146 CD34

12 weeks CD31+ CD105+ CD146+ VE-cadherin+ vWF+ VEGFR2+; CD45 CD14 Became ECs and participate in new blood vessel formation or vascular repair

Do not differentiate into ECs but promote angiogenesis through paracrine factors that indirectly augmented proliferation, migration, and the tube forming capability of ECFCs VEGF; IL-8; MMP9

VEGFR2; CXCR-1; MMP2

The table shows the complex EPCs nomenclature and the two leading EPCs population features studied for their pro-angiogenic properties Abbreviations: BOECs blood outgrowth ECs, CACs circulating angiogenic cells, CFU-ECs colony-forming unit-EC, CFU-HILL colony-forming unit HILL EPC, ECFCs endothelial colonyforming cells, EOCs endothelial outgrowth cells, MACs myeloid angiogenic cells, PACs pro-angiogenic hematopoietic cells, PBMCs peripheral blood mononuclear cells, OECs outgrowth ECs

effect of early and late outgrowth cells when used together compared with one of the two cells alone as cell therapy (Pearson 2010; Yoon et al. 2005). Both early and late EPCs promote angiogenesis. Early EPCs contribute to new vessels formation by secreting a series of growth factors and cytokines, such as VEGF, stromal cell-derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and insulin-like growth factor 1 (IGF-1), which stimulate ECs proliferation and survival, and direct endogenous progenitor cell recruitment into sites of neovascularization (Urbich et al. 2005). Furthermore, early-EPCs provide relevant protective effects to themselves and differentiated EPCs from apoptosis under oxidative conditions in an auto- or paracrine manner, recruiting other cells within the peripheral blood (Yang et al. 2010). Late EPCs directly contribute to vasculogenesis by providing structural support via differentiation into mature ECs (Hur et al. 2004). They can also promote angiogenesis by the secretion of numerous cytokines (Moubarik et al. 2011).

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Table 2 How to distinguish “bona fide” EPCs? Potency Assys (capacity to form a vascular network in vitro and in vivo) Detailed identity immunophenotype

Clonogenicity capacity: (single-cell colony-forming) Proliferative capacity

MACs Only in conditioned media CD14+ CD31+ CD45+ CD34 CD146

Lack

ECFCs Intrinsic tube forming capacity CD31+ CD34+ CD105+ CD133+ CD146+ VE-cadherin+ VEGFR2+ vWF+ CD14 CD45 High

ECs Intrinsic tube forming capacity CD31+ VE-cadherin+ VEGFR1+ vWF+ CD34 CD133

Lack

Medium

High

Low

The table shows the tests that must be performed to identify bona fide EPCs unequivocally. The term EPCs should be restricted to only those cells that display vessel-forming potential, the right immunophenotype, and have high clonogenic potential and proliferation rate Abbreviations: ECFCs endothelial colony-forming cells, ECs endothelial cells, MACs myeloid angiogenic cells

Under physiological conditions, EPCs homing-in is aimed to maintain vascular integrity during repair of damaged tissues, restore organ function, and participate in postnatal angiogenesis (Asahara et al. 1997, 1999a; Urbich and Dimmeler 2004). However, EPCS’ vasculogenic potential is also exploited by tumors to facilitate their progression (Asahara et al. 1999a; Dong and Ha 2010). As shown in preclinical research, in response to endogenous and exogenous signals, VEGFR2+ EPCs can get mobilized from the bone marrow into the peripheral blood circulation and subsequently home-in to tumor neovascularization sites where they differentiate into ECs, thus contributing to angiogenesis (Nolan et al. 2007; Rafii et al. 2002). Endogenous signals released from tumor cells and their microenvironment induce hypoxiainducible factor 1-alpha (HIF1-α) overexpression, glucose reduction, and reactive oxygen species increase. These events promote the release of VEGF, SDF-1, monocyte chemotactic protein (MCP-1), and erythropoietin (EPO), which facilitate EPCs homing-in to neovascularization sites (Annese et al. 2019; Dong and Ha 2010; Ribatti 2004). More precisely, this occurs before the angiogenic switch in the avascular tumor phase (Gao et al. 2008). Once recruited, the EPCs can directly participate in new blood vessel formation or can merely release pro-angiogenic factors. The neoangiogenesis is also sustained by co-mobilization of VEGFR1+ hematopoietic progenitor cells (HPCs), which home-in to the tumor-specific pre-metastatic sites and form cellular clusters, the so-called pre-metastatic niche (Kaplan et al. 2005). There is convincing evidence from both preclinical and clinical studies that exogenous signals, such as disruptive vascular agents, chemotherapy,

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and surgery, might induce an acute release of EPCs from bone marrow, contributing to tumor growth (Bertolini et al. 2003; Furstenberger et al. 2006; Roodhart et al. 2009; Shaked et al. 2008). Of particular importance is the ability of EPCs to home-in not only into the tumor’s vasculature but also into the tumor proper.

Endothelial Progenitor Cells in Neovascularization The development of EPCs-based therapies to induce or suppress new blood vessel formation necessitates the comprehension of cellular and molecular mechanisms of neovascularization. EPCs have a role in both vasculogenesis and angiogenesis. Physiological vasculogenesis is also known as developmental vasculogenesis because it occurs during embryo development. From hemangioblast, which is a common precursor of hematopoietic and vascular systems, EPCs differentiate in the bone marrow and then extravasate into the peripheral circulation in response to VEGF/VEGFR2 stimuli. From circulation, EPCs follow the stimuli gradient and upon arrival at the site of injury, they differentiate into mature ECs and participate in the ongoing vascular development (Masuda and Asahara 2003). In the adult, these EPCs from the bone marrow could participate in physiological blood vessel formation and pathological one during the early phase of tumor neovascularization. Vasculogenesis involves the recruitment and participation of circulating cells, and de novo formation of blood vessels from these cells, while angiogenesis results from the proliferation of existing blood vessels. To be more precise, two types of angiogenesis are distinguished as sprouting and non-sprouting angiogenesis. Sprouting angiogenesis occurs when ECs migrate (tip cells) toward the VEGF gradient source and proliferate (stalk cells) to form abluminal sprouts that subsequently fuse and generate new vessels (Risau 1997; Uccelli et al. 2019). On the other hand, non-sprouting angiogenesis, or intussusceptive angiogenesis, occurs in the absence of a gradient, and all ECs respond to VEGF by assuming a stalk phenotype. During intussusception, an already existing vessel splits into two by forming intraluminal endothelial pillars, which fuse longitudinally (Risau 1997; Uccelli et al. 2019). Angiogenesis plays an essential role throughout embryonic development, besides wound healing, tissue ischemia, and tumor vasculature formation during postnatal life. Hence, it is now being exploited as a novel therapeutic target in cancer treatment. During angiogenesis, EPCs can indirectly contribute to tumor vascularization via autocrine/paracrine mechanisms (Asahara et al. 2011). In addition to the extravasation of EPCs from the bone marrow and homing-in to the site of injury, the neovascularization process is also supported by immature cells present in the vascular wall of various organs. These cells are called vascular wallresident vascular stem cells (VW-VSCs) that differentiate in smooth muscle cells and ECs (Tamma et al. 2020; Torsney and Xu 2011). The subpopulation called vascular wall EPCs (VW-EPCs) differentiate in ECs and are also known as endothelial cellside progenitors (EC-SPs) CD200+/CD157+ (Ingram et al. 2005; Wabik and Jones 2015). In hypoxia conditions, these cells are under self-renewal and differentiation to stalk cells contributing to long-term ECs proliferation and, thus, angiogenesis (Takakura 2018).

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Given the ubiquitous EPCs’ role in neovascularization, their concentration in peripheral blood can be a surrogate biomarker indicating vasculogenic/angiogenic tumor activity and therapy efficacy on tumor vasculature as currently done with microvessel density (MVD) and VEGF expression (Bianconi et al. 2020; Nico et al. 2008; Schluter et al. 2018). EPCs concentration is advantageous because it is accurately but noninvasively compared to MVD and VEGF evaluation, but it is disadvantageous because only 0.025% of the PBMCs are EPCs (Peichev et al. 2000). The small amount limits the translation of prosperous findings of EPCs from bench to practical use. EPCs amount is even less if EPCs from VESCs in the preexisting blood vessels are considered. Therefore, for EPCs-based therapy, they should be first expanded ex vivo.

Endothelial Progenitor Cells Applications EPCs contribute to tissue regeneration processes via neovascularization through paracrine mechanisms or differentiation in mature ECs (Asahara et al. 1999a; Kalka et al. 2000). The freshly isolated autologous PBMCs or BMMCs have been applied to clinical vascular regenerative therapy in patients with peripheral arterial disease, critical limb ischemia, or myocardial infarction (Deutsch et al. 2020; Koshikawa et al. 2006; Kudo et al. 2003; Lara-Hernandez et al. 2010; Li et al. 2016a; Liotta et al. 2018). These researches indicated that cell-based therapy was safe, feasible, and useful. Potential stem cell applications against cancer have been well-reviewed elsewhere (Chu et al. 2020) are for cell transplantation, post-cancer treatment, vaccine production, therapeutic carriers, or immune cells generator. Clinically, EPCs can be employed in three different manners: (i) for neovascularization; (ii) as target cells in anti-EPCs therapy against tumors; (iii) as biomarkers for disease identification and severity (Chopra et al. 2018). With the purpose of neovascularization, vascular EPCs can be exploited for their ability to release several angiocrine growth factors, or other bioactive molecules, to maintain and sustain tissues/organs’ regeneration, for example, by increasing the releasing of oxygen and nutrients through neoangiogenesis. EPCs present in preexisting blood vessels or recruited from bone marrow could be used in vascular regeneration therapy in many diseases like revascularization of ischemic tissues after heart infarction (Huang et al. 2013; Moubarik et al. 2011; Steinle et al. 2018). EPCs could be applied to non-angiogenic and angiogenic tumors to induce blood vessel formation, which will be a direct access route of the drug on the tumor cells, or induce blood vessel normalization, which will alleviate hypoxia and pro-tumor microenvironment, respectively (Collet et al. 2016). As potential therapeutic carriers, a Trojan horse, EPCs combined with targeted antiangiogenic drugs for cancer treatment act as a delivery vehicle that protects the therapeutic agents from rapid biological degradation, reduce systemic side effects, and increase local therapeutic levels due to the intrinsic tumor-targeting effect. Recent advances in cellular engineering have led to stem cell-based vector development to serve as a vehicle for angiogenesis inhibitors or genes directly into the tumor

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endothelium (Janic and Arbab 2010; Nakamura et al. 2004). These novel approaches are useful in oncology to selectively destroy cancer cells, leaving healthy cells unaffected thus alleviating the side effect of cancer therapy (Chong et al. 2016; Ruggeri et al. 2018). For instance, in Sprague-Dawley rats, EPCs isolated from PBMCs were genetically modified to induce a stable expression of antiangiogenic endostatin, reducing VEGF expression. These genetically modified EPCs were successfully tested to suppress retinal vascular leakage and could be advanced for clinical assessment because endostatin overexpression may serve as a potential therapeutic agent (Ai et al. 2018). Antitumor treatments’ efficacy is usually evaluated by imaging techniques such as X-ray, computed tomography, magnetic resonance imaging (MRI), and ultrasound. EPCs can be used for diagnosis, prognostic prediction, and follow-up. EPCs can be labeled for CD133 for tracking their in vivo fate after injection by MRI for diagnosis and follow-up. As biomarkers of tumor development and/or progression, several studies have demonstrated clinical correlations between CEPCs concentration and tumor stage (Nowak et al. 2010; Ramcharan et al. 2013; Yu et al. 2013), tumor size (Richter-Ehrenstein et al. 2007; Su et al. 2010), VEGF serum concentration (Rafat et al. 2010; Yang et al. 2012), and MVD (Li et al. 2018a; Maeda et al. 2012). During hematological malignancies’ comparison with solid tumors, many studies have demonstrated a close association between EPCs and disease activity, so much so that circulating EPCs are useful diagnostic, therapeutic, and prognostic biomarker (Ge et al. 2015; Ruggeri et al. 2018). The EPC-based therapies are much better known for cardiovascular diseases compared to oncological ones. EPCs were proposed to induce angiogenesis in ischemia (Li et al. 2018b; Zheng et al. 2014), for post-injury vascular endothelial regeneration (Abd El Aziz et al. 2015; Guo et al. 2017), and ex vivo tissue engineering (Sales et al. 2010). However, the enthusiasm for the possible applications of EPCs in clinical therapy is severely limited by the lack of in-depth characterization and understanding of early and late outgrowth EPCs.

Endothelial Progenitor Cells Sources, Ex Vivo Culturing, and Implantation Mouse, monkey or human ESCs, fetal liver, human umbilical cord blood, bone marrow, and peripheral blood might be used as the potential EPCs sources (Debatin et al. 2008; Zakrzewski et al. 2019). The use of stem/progenitor cells from embryos is advantageous and ideal because they can show unlimited and undifferentiated proliferation and evade immunological rejection as they do not express MHC-I. Still, there are ethical considerations and risk of malignant transformation that restrict their progress to the clinical setting (Wei et al. 2004; Werbowetski-Ogilvie et al. 2009). EPCs derived from the fetal liver can be easily isolated and cultured; however, the clinical applicability of these cells is limited by the challenges of creating fetal liver tissue banks and host immune incompatibility (Cherqui et al. 2006). Stem/progenitor cells present in umbilical cord blood have a higher proliferative capacity, readily available, and easy to isolate than adult bone marrow-derived cells.

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However, umbilical cord donation has yet to achieve widespread acceptance, besides the chance of immunologic graft-versus-host disease in the recipients (Ingram et al. 2005; Murohara 2001; Murohara et al. 2000; Qin et al. 2017). Also, multipotent adult progenitor cells isolated from postnatal bone marrow have extensive proliferation potential ex vivo and can differentiate into mesodermal lineage cells as EPCs (Reyes et al. 2002). Thus, they can be effectively applied in autologous therapy, thanks to potentially low-level immune recognition and destruction of these cells by the host immune system. More recently, EPCs have been isolated from human adult somatic cells, that is, fibroblasts, through transdifferentiation into iPSCs (Purwanti et al. 2014; Taura et al. 2009). Nevertheless, like ESCs, EPCs iPSCs-derived will need an in-depth characterization to exclude tumorigenic potential. An easily accessible EPCs’ source is either peripheral blood or bone marrow. Circulating autologous EPCs could be isolated from these sources using markers like CD34, CD133, or VEGFR2 (Asahara et al. 1997; Shi et al. 1998). Circulating EPCs and bone marrow-derived EPCs are among the least complicated sources to use, but the main obstacle in their use in regenerative medicine is low quality and quantity, and immune recognition (Asahara et al. 2011; Sukmawati and Tanaka 2015). Chemotactic molecules as VEGF, placental growth factor (PlGF), granulocyte-macrophage colony-stimulating factor (GMCSF), or statins are used to treat patients/donors to increase EPCs number (Asahara et al. 1999b; Dimmeler et al. 2001) (for schematic EPCs application as therapy see Fig. 1).

Fig. 1 Schematic representation of EPCs as cellular vehicles. EPCs derived from PBMCs or BMMCs are expanded and transduced in vitro to express pro-drugs or tratted for the expression of imaging probe can be systematically or in-situ replanted in the patients.

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EPCs, after isolation from PBMCs or BMMCs, are expanded in vitro. There are several optimized protocols available for ex vivo culture and expansion of EPCs. However, all of these protocols require that EPCs are plated on extracellular matrix proteins coated dishes and maintained in endothelial basal medium with added supplements and growth factors (Au et al. 2008; Kawamoto et al. 2001; Lu et al. 2014). Concerning supplements, EPCs’ expansion is expensive and time-intensive due to high concentrations and frequent supplementation of growth factors because of their short half-life at physiological temperatures (Khalil et al. 2020). Moreover, supplements, such as fetal bovine serum (FBS), could be unsafe for clinical application due to their animal origin, prone to batch-to-batch variations, xenoimmunization, and possible contamination of mycoplasma, viruses, endotoxins, and prions (Dessels et al. 2016). The development of optimal protocols to expand EPCs without growth factors is a promising approach to simplifying clinical translation. Interestingly, polyphenols benefit EPCs number and functional activity (Di Pietro et al. 2020; Huang et al. 2010). For example, an attractive agent to expand EPCs is the natural flavonoid quercetin. It increases the number and functional activity of EPCs and protects them against serious glucose-induced damage by inducing Sirtuin-1 (Sirt1)-dependent endothelial nitric oxide synthase (eNOS) upregulation (Zhao et al. 2014). To avoid the use of animal serum, several laboratories have developed novel serum-free expansion methods enriched with optimal cytokine and growth factor combinations (Hagiwara et al. 2018; Kado et al. 2018; Masuda et al. 2012). These methods are known as the quality and quantity culture (QQc) system and ensures optimizing EPCs-based therapy by augmenting their qualitative and quantitative vasculogenic properties and providing measurable regenerative capacity (Sukmawati and Tanaka 2015). To isolate and expand EPCs, it is imperative to consider that EPCs are decreased in number and functional activity related to age and cardiovascular risk factors (Huang et al. 2014; Kaur et al. 2018). Therefore, isolation and application of EPCs from patients with these backgrounds have a high chance of receiving EPCs with low therapeutic effect. Before administration, expanded EPCs are characterized for their morphology, surface markers expression by flow cytometric analyses, eNOS levels, and Ac-LDL uptake/fluorescein isothiocyanate (FITC)-lectin binding actives. Moreover, their routine functional characterization also involves assessment of angiogenic potential by in vitro tube formation assay or in vivo by chick chorioallantoic membrane (CAM) assay (Kukumberg et al. 2020; Merckx et al. 2020; Qin et al. 2018; Song et al. 2010). Furthermore, recent studies advise performing isolated cells’ efficacy and safety, for example, by transplantation in a nonhuman primate model (Qin et al. 2018). Once collected sufficient number in vitro, EPCs can be conditioned to enhance their functionality for more efficient functionality and therapeutic benefits. Several studies about the revascularization of ischemic tissues have employed various growth factors or recombinant proteins or genes using nano- or microparticles to improve tissues’ revascularization and upregulate pro-angiogenic proteins (Bhise et al. 2011; Simon-Yarza et al. 2012). Recombinant proteins are costly, and it is not

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easy to maintain adequate protein levels at the target region due to their relatively short half-lives (Gupta et al. 2009). Gene therapy with viral and nonviral delivery system was applied as an alternative strategy to express the desired pro-angiogenic proteins, and it has shown to be promising. EPCs can be genetically manipulated by stable transduction using retrovirus or lentivirus-based vectors encoding for the gene/s of interest, allowing a long-term transgene expression. Besides their high transduction efficiency, their use is convenient when EPCs are used as a vehicle due to their susceptibility to gene transduction protocols. However, since viral vectors are usually replication-defective, there will be a physiological clearance reduction of EPCs vehicle following administration. This reduction can be advantageous if a temporary rather than stable presence of the cells is the goal of cell therapy. EPCs can also be modified with non-integrating viral vectors such as adenovirus or herpes simplex virus, or plasmid vector, inducing short-term effects. Moreover, using a non-integrating method, the EPCs can be modified with synthetic mRNAs, which can express exogenous proteins without the hazard of insertional mutagenesis since the delivered mRNAs remain in the cytoplasm for translation without passing into the nucleus (Sahin et al. 2014; Steinle et al. 2018). The synthetic mRNA transfection leads to the transient production of exogenous proteins of interest in the cells, and subsequently undergoes natural degradation thus leaving no traces of the delivered mRNA. After isolation, modification, and characterization, EPCs can be implanted in patients. Generally, stem cells could be introduced in different ways, such as intravenous, intramuscular, intra-articular, and intrathecal (Saeedi et al. 2019). Intravenous is the safest and most straightforward method to deliver the EPCs throughout the body. This way is a preferred route administration as it is simple and feasible, does not require general anesthesia, and allows administration of repeated doses at different times (Haider et al. 2017). The transplanted EPCs mainly home-in to a tumor site due to attraction to the tumor vasculature by its angiogenic drive, but the efficiency is not 100%. In effect, most cells intravenously inject end up in the non-target sites, including lungs, liver, and spleen (Leibacher and Henschler 2016; Varma et al. 2013b). Several factors could explain the lack of efficiency, including tumor microenvironment composition variability, vascular network size, or angiogenic stimulus power (Li et al. 2011; Wang et al. 2016). Moreover, after intravenous administration, EPCs have to compete with the endogenous EPCs for incorporation into the target organ such as a tumor. This necessitates the suppression of endogenous EPCs. One of the strategies for improving EPCs tumor homing is to deliver these cells as an adjuvant to chemotherapy or radiation therapy because this can increase the migration and incorporation of EPCs into the tumor (Shaked et al. 2008). Furthermore, to improve EPCs delivery and homing-in capacity, they might be directly injected into the arterial circulation or infused at multiple time-points (Dudek 2010; Lin et al. 2020). To successfully translate EPCs into cell-based therapy for routine patient application, it is critical to develop a technique to monitor transplanted cells’ in vivo biodistribution after delivery. Among the in vivo cell-tracking and cell-fate determining techniques, magnetic resonance imaging (MRI) is one of the most powerful

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one because of its satisfactory resolution (Aicher et al. 2003; Hu et al. 2012; Wang et al. 2003). For MRI, the cells are labeled with MRI contrast agents that are not efficiently loaded into cells to avoid cytotoxicity (Crabbe et al. 2010; Hu et al. 2012). An alternative label is the paramagnetic agent IronQ, a complex of iron and quercetin, added to cell culture (Kantapan et al. 2017). It is not only traceable by MRI but also serve as cell proliferation inducer (Kantapan et al. 2017).

Endothelial Progenitor Cells in Preclinical Cancer Studies Searching for clinical trials involving EPCs in cancer therapy, it is possible to find only five studies, all providing information regarding the characterization and quantification of CEPCs using biomarkers (ClinicalTrials.gov Identifier: NCT00393341; NCT00753610; NCT00325871; NCT00826683; NCT00067067). These studies are based on complete evidence about the emerging role of CEPCs in tumor angiogenesis as surrogate markers of antiangiogenic therapies efficacy. It is widely known that neovascularization is a crucial cancer hallmark that facilitates cancer cells proliferation and progression. Blood vessels deliver oxygen and nutrients to cancer cells allowing them to grow further 2 mm in diameter. When tumor mass is over, in response to hypoxia and microenvironment signals, cancer cells overexpress molecules that promote vasculogenesis, angiogenesis, and evasion. As discussed earlier, resident ECs and EPCs, which line all blood vessels or are present in the peripheral circulation respectively, or are recruited from bone marrow migrate toward an angiogenic cue, proliferate and form new vessels. EPCs’ involvement in tumor vasculogenesis, contributing to the development of vascular network or vascular mural cells, and their homing-in to the site of tumor angiogenesis means that they have access to distant and, in most cases, undetectable micrometastases are the reasons behind the use of these cells in cancer therapy (Rajantie et al. 2004; Reyes et al. 2002). In an experimental mouse orthotopic hepatoma model developed using tumor liver cell line HepG2, Zhu et al. (2012) demonstrated that intravenous tail-vein injection of BMMCs-derived EPCs preferentially migrated into the site of tumor development (liver) compared to the other organs. They also demonstrated that EPCs migrated to the tumor site in response to the cytokines (VEGFR, HIF-1α, SDF1) cues secreted by the tumor cells. On the contrary, some researchers have reported failure of EPCs’ chemotaxis in all kinds of tumors in response to the chemical cues emanating from the tumor cells (Annabi et al. 2004; De Palma et al. 2005; Larrivee et al. 2005; Lyden et al. 2001; Purhonen et al. 2008). The orientated homing of EPCs in hepatomas as in other tumors enhances their possible clinical applications as delivery vehicles for suicide gene therapy, antiangiogenesis gene therapy, or tumor suppressor gene therapy. An effective EPCsbased strategy in cancer therapy is to genetically manipulate EPCs with the genes encoding for the enzymes that metabolize pro-drugs into pharmacologically active anticancer drug derivatives that would kill the surrounding cancer cells based on a spectator effect (bystander effect) (Freeman et al. 1993; Zweiri and Christmas 2020).

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The death of the donor EPCs and their ineffectiveness against rapidly growing or large tumor limit these suicide gene therapies after drug activation (Wei et al. 2007). During the last few years, the effectiveness of an exciting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) anticancer activity has been reported (Lim et al. 2015; Yuan et al. 2018). TRAIL initiates the pro-apoptotic pathway by selectively binding with its death receptors-4 and -5 (DR4, DR5), while sparing the healthy cells unaffected (Forster et al. 2013; Kichev et al. 2014). Deng et al. (2018) engineered EPCs (isolated from PBMCs of neonatal Sprague-Dawley rats) with a lentivirus encoding for TRAIL for glioma treatment. TRAIL has a short half-life and also fail to cross through the blood-brain barrier and (Guo et al. 2011; Holoch and Griffith 2009). Thus, EPCs-based TRAIL gene delivery has overcome these problems (Choi et al. 2016; Redjal et al. 2015; Wang et al. 2014). TRAIL-EPCs migrate to glioma cells SHG44 in the transwell assay and induce glioma cell apoptosis in a co-culture in vitro system by increasing the cleaved caspase-3 and -8 levels and poly ADP-ribose polymerase (PARP) (Deng et al. 2018). To solve EPCs ineffectiveness due to their small number, an indirect strategy is to target tumor vasculature cells, which are critical for tumor growth and survival, instead of the whole enormous tumor cell mass. For instance, Dudek et al. (2007) have shown that genetically engineered EPCs overexpressing the antiangiogenic molecules endostatin significantly decreased tumor vascularization and growth after tail vein injection into NOD-SCID vein mice with subcutaneously implanted Lewis lung carcinoma cells. Laurenzana et al. (2014) developed a personalized therapy against melanoma using autologous MMP12engineered EPCs to treat both tumor cells and tumor vasculature. MMP12 is a metalloelastase with a bivalent role: protective if expressed by macrophages and non-protective if expressed by tumor cells (Houghton et al. 2006; Margheri et al. 2011; Martin and Matrisian 2007). MMP12 application as an anticancer strategy is based on MMP12’s enzyme activity to cleave urokinase-type plasminogen activator (uPAR). The full-length isoform acts as a potent endothelial activator responsible for tumor progression. uPAR can be expressed by both endothelial and tumor cells (Andolfo et al. 2002). Laurenzana et al. (2014) demonstrated that EPCs transfected with a lentivirus encoding for MMP12 are recruited into melanoma mass under CXCR4/SDF1 system stimuli after intravenous delivery in experimental settings. Moreover, in vitro and in vivo, it was shown that MMP12engineered EPCs reduced melanoma progression, intra-tumoral angiogenesis, and lung metastasis in old CD-1 nude mice, degrading uPAR on tumor cells and ECs (Laurenzana et al. 2014). Noteworthy, these ex vivo MMP12-engineered EPCs lost the capacity to perform capillary morphogenesis in vitro and, at the same time, acquired the antitumor and antiangiogenetic activity. Thus they seem to show no side effects in vivo (default pro-angiogenic role) (Duda et al. 2000). EPCs-based therapy using genetically transduced cells was also applied in a preclinical study for nasopharyngeal carcinoma. Wang et al. (2018) demonstrated that EPCs genetically modified with a lentiviral encoding for the metastatic gene suppressor KAI1/CD82 successfully inhibited lung metastasis in a nude mice bearing human nasopharyngeal carcinoma xenografts. However, there was little evidence regarding their potential to suppress the tumor cell graft.

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A third EPCs-based strategy in cancer gene therapy is to accentuate the host immune system against cancer. For instance, Ojeifo et al. (2001) engineered EPCs to express IL-2 to stimulate natural killer and cytotoxic T cells in a syngeneic mouse model of melanoma lung metastases. They demonstrated that multiple intravenous injections abrogated the tumor metastases and prolonged animal survival. Muta et al. (2003) manipulated EPCs with a retrovirus vector carrying IL-12, showing that, in vivo, this gene therapy selectively delivers the protein to the tumor site in a xenograft rat model of breast cancer where its overexpression induced natural killer and cytotoxic T cells. To overcome the controversies associated with the ESCs from human embryos, recently, iPSCs are considered the primary source of autologous or allogeneic pluripotent stem cells. They were explored as a source of human EPCs suitable as a delivery system of immune-stimulatory molecules to inhibit cancer. Purwanti et al. (2014) obtained CD133+/CD34+ EPCs from human iPSCs. They demonstrated that the cells expressed EPC-specific markers (i.e., CD31. VEGFR, cadherin) did not express hematopoietic cell markers (i.e., CD45), exhibited tubulogenesis in vitro, showed tumor tropism in an orthotropic lung metastasis mouse model for breast cancer, and did not enhance tumor growth and metastasis. Moreover, when these iPSCs-EPCs, engineered with a baculovirus encoding for the immune co-stimulatory molecule CD40 (with a pivotal role in the T-cell activation), were systemically injected in breast cancer-bearing mice, the animals showed prolonged survival (Purwanti et al. 2014). Noteworthy, in this study, an insect baculovirus was used instead of the conventional animal viral vectors. Insect virus bypasses the risk of virus replication and infection in the human host cells, and there is no host immune response (Bessis et al. 2004; Strauss et al. 2007). However, they are not adapted for long-term transgene expression. A combination of suicide gene-targeting therapy with an antiangiogenic molecule was established in a human HepG2 liver cancer preclinical model to improve patients’ treatment outcomes. Zhang et al. (2020) developed a gene therapy protocol with cytosine deaminase (CD) and endostatin gene transfected in EPCs obtained from fresh heart blood of adult BALB/c nude mice. Cytosine deaminase is one of the most widely investigated suicide gene/pro-drug that converts the nontoxic antifungal agent 5-fluorocytosine into the toxic chemotherapeutic agent 5-fluorouracil (5-FC) (Lawrence et al. 1998). The abovementioned preclinical model showed a total tumor volume reduction by MRI, angiogenesis inhibition visualized by VEGF- and CD31positive immunostaining, decreased ECs, and increased tumor cell apoptosis assessed by TUNEL assay in mice transfected with CD/endostatin-EPCs plus 5-FC (intraperitoneally injected) compared to control treatment group (Zhang et al. 2020). CD/endostatin synergistic action could be translated in to clinical trials to target the hepatomas site via vein grafting. Neoangiogenesis is mostly proven via CD31/VEGF immunohistochemistry, but this method is inadequate because it requires experimental animals to be sacrificed or human biopsies taken for immunohistological studies renders follow-up is impossible. Recently, studies are focused on advanced, noninvasive, and real-time molecular imaging methods as tracking strategies to monitor transplanted EPCs-based drug

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vectors for antitumor therapy (Arbab et al. 2006). EPCs can be applied for noninvasive MRI investigation, as demonstrated by Chen et al. (2014a). They have approximately 100% of human PBMCs-derived EPCs efficiently labeled with N-alkyl–polyethylenimine 2 kDa (PEI2k)-stabilized superparamagnetic iron oxide (SPIO) nanoparticles. Moreover, functional assay outputs such as proliferation, migration, and tubulogenesis rates and incorporation into tumor neovasculature in vivo results have shown that these magnetic-labeled EPCs have the same activity as unlabeled ones. Once labeled, EPCs were intravenously or subcutaneously injected in a lung carcinoma xenograft model and were effectively detected by seven-tesla micro-MRI at the tumor site. The results showed excellent biocompatibility and magnetic resonance sensitivity even at a small alkyl-PEI2k/SPIO concentration than other contrast agents (Chen et al. 2014a). An EPC-based theranostic method has also been also proposed using the abovementioned MMP12-engineered EPCs radiolabeled with 111In 8-oxyquinoline (oxine) for all the tumors displaying uPAR-dependent cancer progression (Laurenzana et al. 2014). In glioma, the herpes simplex virus TK (HSV-TK)/ganciclovir (GCV) gene therapy is a suicide gene therapy widely used in both experimental and clinical trials thanks to its potent bystander effect (Zhang et al. 2010). A combination of HSV-TK suicide gene therapy with real-time molecular imaging has been reported for glioblastoma. Varma et al. (2013a) employed human cord blood-derived EPCs as a delivery vehicle for replication-competent adenovirus AD5 carrying both suicide genes, yeast CD (yCD) and mutant HSV-TK mutTK (SR39), and reporter gene, human sodium iodide symporter (hNIS) for I-131 (radioiodine) for diagnostic MRI imaging and single-photon emission computed tomography (SPECT). Their results indicated that AD5-yCD-mutTK-EPCs reached the glioma mass upon intra-tumor injection. Furthermore, double staining experiments demonstrated that both EPCs (hNIS+/vWf+) and tumor cells (hNIS+/EGFR+) expressed the transgenes thanks to the transfected EPCs’ ability to deliver the vectors in the surrounding tumor cells (Varma et al. 2013a). Noteworthy, this study exploited intra-tumor injection instead of the prevalent systemic injection. The intra-tumor injection is advantageous to alleviate the virus’s entry into circulation and curtail side effects (Lohr et al. 2001). Moreover, a replication-deficient virus is used as a transgenes delivery system to improve tumor cell death by their self-replication properties and infectivity of the surrounding cells in the vicinity (Barton et al. 2011; Barton et al. 2003). The reporter gene system with hNIS also overcame the short monitoring time (~7 days) with In-111Oxine labeling. hNISm allows repeated detection of the injected cells for extended periods (Barton et al. 2003; Varma et al. 2013a). EPCs have also been proposed as the best vehicle to deliver therapeutic genes and imaging probe targeting glioma stem-like cells (GSCs) (Chen et al. 2014b). Glioma is a vascular-rich tumor with high resistance to antiangiogenic therapy because tumor cells can pass from the vascular phase of growth to the nonvascular one and vice versa. The mechanisms by which glioma achieve neovascularization are vascular co-option, angiogenesis, vasculogenesis, vascular mimicry, and GSCs-ECs transdifferentiation. GSCs-ECs transdifferentiation is implicated in the resistance against anti-VEGF therapy which currently in practice (Baisiwala et al. 2019;

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Yan et al. 2017). Using in situ C6 glioma rat model, Chen et al. (2014b) showed that exogenous spleen-derived EPCs labeled with USPIO (ultra-small SPIO) integrate into the vessels containing glioma-derived ECs without inducing any promoting effect of GSCs transdifferentiation. Despite these promising results, in vivo MRI with iron-nanoparticles presents some inconveniences: (i) the signal is lost over time due to the contrast agent biodegradation or dilution following cell division; (ii) time of EPC migration to the tumor site depends on multiple factors, such as tumor location and size, and chemotaxis factors expression levels; (iii) it is challenging to monitor real-time EPCs’ migration into blood circulation; and (iv) imaging devices may lead to different results due to its sensitivity and resolution. To enhance in vitro expanded EPCs translation from preclinical studies to clinical trials, the in vivo safety issues should be addressed because adverse effects and responses caused by EPCs therapy have been reported, such as collapse, sepsis, breast cancer development, and even death (Granton et al. 2015). In this context, Lee et al. (2019) proposed EPCs transplantation in dogs as a possible safety test of deleterious effects that should be conducted before EPCs application in human clinical trials. The choice of dogs lies in their physiological similarity with humans. They performed physical and laboratory examinations of human EPCs isolated from healthy donor PBMCs and transplanted intravenously into dogs. This in vivo safety assessment could be useful to test the minimal number of EPCs for transplantation because a high number is associated with pulmonary emboli or infarctions and affect immune responses (Beggs et al. 2006; Grigg et al. 1996; Prockop and Olson 2007). Systemic delivery of EPCs gene therapy to primary tumors and metastases is the most attractive feature of using EPCs. Nevertheless, it remains to understand what factors permit EPCs persistence during hypoxia, migration, and proliferation to angiogenic sites, and if they are detained within the blood vessel wall or migrate further outside, and if they participate to vessel maturation.

Side Effects and Potential Risks of EPCs Cell Therapy Given their advantages, EPCs are anticipated to play a pivotal role in cancer theranostics, for both therapy and biomarkers in future. However, various issues relevant to their use must be resolved before routine clinical use. New optimized stem cell differentiation protocols and animal models must be explored to better understand the molecular events involved in EPCs generation and differentiation. How to isolate and unequivocally identify the phenotype and functionality of EPCs remains problematic. The standardization of cell culture conditions, doses, and administration schedules will make it easier to understand different studies’ results to interpret their data for future applications (Morales-Cruz et al. 2019). EPCs are rare in both peripheral blood (0.01%) and bone marrow (0.05%). This necessitates their in vitro expansion to get them in large number for in vivo use. However, in vitro culture may alter their immunologic characteristics and tumorigenic potential. For example, during in vitro expansion, EPCs are exposed to

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exogenous culture conditions different from physiological niches’ microenvironment wherein stem cell proliferation and differentiation are under maintained under strict control. Consequently, EPCs could change their genome and phenotype that could render them tumorigenic thus contributing to tumor initiation. On the same note, sub-culturing will reduce stemness at every passage. This means that the development of new EPCs isolation methods is required to improve their yield as well as quality. EPCs have a natural tropism for the sites of vascular injury. To avoid systemically adverse effects when used as delivery vehicles, advances in nanotechnology and tissue engineering are required to improve EPCs’ homing-in and incorporation to the site of interest. When EPCs are employed to target drugs or genes to tumor cells, they could cause drug toxicity or drug resistance. For instance, after systemic injection, a small amount of them will reach the tumor site because most of them will be trapped in the lung, liver, or lymph nodes, causing therapeutic ineffectiveness and drug resistance (Brooks et al. 2018). Another side effect could be a viral infection when viral carriers are used to genetically engineer EPCs (Goswami et al. 2019). Viral vectors currently employed in patient’s treatment are classified as non-integrating or stable host-genome integrating vectors. The former include adenovirus and adenoassociated virus vectors and are primarily used for in vivo gene delivery in patients. Adenovirus can accommodate a large cDNA but are highly immunogenic. In contrast, adenoassociated viruses can only accommodate a smaller cDNA and are less immunogenic but retained for a longer time in the non-dividing cells. The last are retroviruses and lentiviruses. Both can harbor small cDNAs such as adeno-associated viruses, but unlike these, they allow for the prolonged-expression of the therapeutic gene, although there is a risk of insertional mutagenesis. The evaluation of EPCs source for transplantation is essential. Allogeneic or autologous (via iPSCs technology) stem cell transplantation may provoke severe host immune responses or autoimmunity, respectively (Li et al. 2016b). Hematologic and lymphoid cancers are commonly treated by allogeneic HSC transplantation, but often patients incurred in Graft-versus-Host Disease (GVHD), acute or chronic, due to the induction of a complex immunological reaction of the donor’s immunocompetent cells toward the recipient’s tissues and organs. Different studies confirmed significantly improved outcomes, with a reduced incidence of chronic GVHD, but not acute one, after umbilical cord blood transplantation compared to allogeneic hematopoietic stem cell transplantation (Chen et al. 2017; Narimatsu et al. 2008). It is necessary to consider the modulation of the host microenvironment as well. Cells and molecules of the microenvironment during hypoxia or increased inflammation have adverse effects on EPCs survival. For example, given the complexity and immunosuppressive properties of the tumor microenvironment, stem cell transplant combined with other therapies, such as immune checkpoint inhibitors, may better eliminate cancer and its recurrence. For example, Hu et al. engineered the surface of HSCs with the checkpoint inhibitor programmed death-1 (PD-1) antibodies-decorated platelets for the treatment of recurrent leukemia in mice (Hu et al. 2018).

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A general recommendation is to pay attention when choosing EPCs as a therapy in oncology: it must be considered that mobilization and integration in tumor blood vessels depend on tumor type, stage, and treatment (Farnsworth et al. 2014). For example, cell therapies hold much more promise for treating diseases in which tissue can be ablated, such as bone marrow or skin cancers that can be easily removed with drugs or surgically, respectively. These procedures favor transplanted cells’ engraftment because they will not have to compete with diseased resident cells. In more morphological complex tissues such as the brain, where massive ablation of diseased tissue is impossible, engraftment of transplanted cells is lower, and consequently, therapeutic efficacy is reduced.

Conclusion and Future Perspective The chapter explores the underlying molecular mechanisms and potential applications of EPCs in cancer therapy. The chapter also discusses the protocols to obtain EPCs in a significant amount and their modification, administrated, besides the possible undesired effects and potential risks for cancer patients. Despite that cancer is one of the leading public health problems, there are still no adequate and exhaustive therapeutic and diagnostic protocols available due to the incomplete knowledge of cancer cell biology. Among the various approaches for cancer theranostics, manipulated-stem cell transplant, alone or as an adjuvant for other therapies, could be a new strategy to treat cancer patients. Stem cells reside in almost all organs and tissues in the body, with the potential for self-renewal, migration, and differentiation that justifies their use in antitumor therapy. Therefore, studying stem cells for tissue engineering and theranostic resolutions is exciting. The existing results concerning stem cell therapy for cancer are highly encouraging. ESCs and iPSCs are the most powerful ones, but the diversity in their applications is still limited due to the possible risks related to viral vectors and ethics issues. EPCs are one of the autologous stem cell types for human use as they lack MHC-I expression, resistant to NK-mediated cytolysis, and primarily involved in blood vessel formation besides ease of availability and isolated and expanded ex vivo/ in vivo, efficiently transduced to carry a therapeutic payload and home-in to the tumor and its vasculature. Hence, they are excellent cellular vehicles for systemic and local cancer therapy in general and angiogenic cancer in particular, as they primarily depend on blood vessels for growth and metastasis. It would be interesting to interfere with tumor vascularization by restoring a balance between pro- and antiangiogenic signaling and ensure direct access to drug delivery at the tumor site (Collet et al. 2016). Therefore, EPCs can be manipulated to selectively deliver the therapeutic molecules to the cancer cells while sparing the healthy cells. Given their biocompatibility and sensitivity when labeled, EPCs may serve as near-ideal vasculature tracker for diagnostic imaging. Currently, only CEPCs are in clinical trials as surrogate biomarkers of antiangiogenic therapy. However, to validate the diagnostic value of CEPCs, the selection criteria of both cancer patients and healthy controls should be stricter due to the

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Table 3 Summary of EPCs’ advantages and disadvantages EPCs versus other stem/progenitor cells in cancer therapy Advantages Disadvantages Isolation Less complicated Low amount Ex vivo Can generate enough Massive tests must be performed to identify expansion cells for therapy bona fide EPCs Transduction Efficient Tropism Home to tumor proper Only a minority of systemically administrated and tumor vasculature EPCs incorporate into tumor vessels Drug Protect the drug from Systemically exposure inactivation Immunological Autologous EPCs not tolerance affected

involvement of numerous confounding factors, that is, background cardiovascular diseases, diabetes mellitus, and lifestyles, which include smoking status and physical exercise, among others (Mayr et al. 2011). Despite success in preclinical experimental animal models and enormous possibilities yet to be explored, the cell availability in small number, low-quality preparations, poor retention, low survival rate, and engraftment after transplantation still hamper EPC’s routine clinical application (Sukmawati and Tanaka 2015; Terrovitis et al. 2010). Besides, there are no unique identifying markers for EPCs, and functional characterization of the rare putative EPC population-based on FACS phenotypes is challenging to realize for a large dataset. Hence, a consensus on the exact characterization and biology of EPCs is required to create a standardized, generally accepted methodology to develop the use of EPCs in clinical settings for regenerative approaches (Sabbah et al. 2019). Another drawback is the optimization of culturing protocols containing media without animals-derived supplements. Moreover, the establishment of stem cell-based anticancer therapies is slowed down by the lack of adequate financial support, the existence of ethical and political issues, and the easy authorization of new therapeutic protocols for which the efficacy has not been adequately tested. The current gap between public expectancy and actual progress of stem cell-based therapies in the clinical threatens regenerative medicine’s social license to operate (Cossu et al. 2018). A possible step forward is to develop a combinatorial approach on several fronts (tumor vasculature, tumor cell tumor microenvironment, immune system) to achieve a better outcome. In conclution, EPCs translation from bench to antitumor therapy and diagnostic imaging depends on a more in-depth assessment (Table 3).

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Non-cryopreserved Peripheral Stem Cell Autograft for Multiple Myeloma and Lymphoma in Countries with Low Resources

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Mohamed Amine Bekadja

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliographic Literature Search for the Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data Analysis from the Selected Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conditioning Regimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Engraftment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Posttransplant Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparative Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An In-Depth Analysis of the Published Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conservation and Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Intensification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Donor Cell Engraftment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1422 1424 1424 1425 1425 1425 1425 1427 1428 1428 1429 1431 1435 1435 1436 1436 1438 1438

Abstract

Autologous peripheral blood progenitor cell (PBPC) transplant is a standard indication in the multiple myeloma (MM) and lymphoma. The use of non-cryopreserved PBPCs is not usual despite its safety, feasibility, and efficacy. Few data exists in the literature regarding the procedures for non-cryopreserved autologous PBSCs transplant in countries with limited resources. The bibliographical research of this work was limited on sites like PubMed, Google scholar; using the following articles on the non-cryopreserved autologous PBPCs in hematological malignancies in developing M. A. Bekadja (*) Department of Hematology and Stem Cell Therapy, Etablissement Hospitalier Universitaire 1st November, Ahmed Benbella 1 University, Oran, Algeria © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_52

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countries have been selected. These papers were analyzed in terms of mobilization, apheresis, preservation and viability, conditioning regimen, engraftment, response, and finally survival. This chapter sums up experience from 17 transplant centers which carried out autografts with non-cryopreserved PBPCs in 1541 patients suffering from hematologic malignancies. The results in terms of mobilization showed a median CD34+ ¼ 3.94  106/kg (range, 0.32–24.6), a viability >90% and >75% respectively in MM and lymphomas after a conservation of 24–144 h at +4  C. The engraftment (mean neutrophils ¼ 12.38 (6–86) days, mean platelets ¼ 15.57 (7–89) days, and TRM (4.39%) were very satisfactory. In conclusion, this method is easy, efficient, and safe, and it is expected to grow in developing countries due to its low production cost and procedure simplicity. Keywords

Cryopreserved · G-CSF · Hematopoietic · Progenitor · Stem cells · Transplantation List of Abbreviations

AfBMT ASCT BEAM BM BMPCs CBV CEC DFS DLBCL DMSO EMBMTG G-CSF HPCs LK MEL/VP16 NE OS PBSCs

African Blood and Marrow Transplantation Autologous stem cell transplantation BCNU-Etoposide-Aracytine-Melphalan Bone marrow Bone marrow progenitor cells Cyclophosphamide-BCNU-Etoposide/VP16 Cyclophosphamide, Etoposide, Carboplatin Disease-free survival Diffuse large B cell lymphoma Dimethyl sulfoxide Eastern Mediterranean Bone Marrow Transplantation Group Granulocyte-colony stimulating factor Hematopoietic progenitor cells Leukapheresis Melphalan, Etoposide Neutrophil engraftment Overall survival Peripheral blood stem cells

Introduction Cryopreservation of hemopoietic stem cells was established in the early 1970s (Gorin and Duhamel 1975, 1978; Gorin et al. 1978a, b; Gorin 1986; Douay et al. 1982) on bone marrow (BM) collected from iliac spines and stored usually at 140  C or 196  C in vapor or liquid phase nitrogen. Efficient cryopreservation of stem cells opened the way to autologous stem cell transplantation (ASCT) after high-dose chemotherapy and/or

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total body irradiation. Since the early 1990s, peripheral blood stem cells (PBSCs) collected by leukapheresis (LK) have replaced bone marrow as a source of hemopoietic stem cells in almost 95% of the patients (Storb et al. 1977). Cryopreservation of stem cells requires facilities, equipment for programmed freezing, and liquid nitrogen availability for storage with temperature monitoring. It also requires quality controls at thawing to ensure good quality cell preparation (Haider 2017). All steps are laborious, time-intensive, and expensive to ensure clinical grade quality of the cell preparation (Gorin 1986). Several attempts have been made to assess alternative strategies to avoid cryopreservation, including maintaining the autograft at 4  C in a conventional refrigerator. It has been rapidly shown that the viability of marrow stem cells cannot be preserved beyond 56 h (Burnett et al. 1983). On the contrary, PBSCs are more purified, devoid of erythrocytes and neutrophils, and can be easily preserved for extended periods. Several teams have demonstrated the possibility of using non-cryopreserved PBSCs to autograft patients with multiple myeloma who received high dose Melphalan over 1 or 2 days, with a preservation duration not exceeding 3 days (Wannesson et al. 2007; Lopez-Otero et al. 2009; Kayal et al. 2014; Bekadja et al. 2017; Bittencourt et al. 2019; Bekadja et al. 2012). Patients with lymphomas autografted with PBSCs receive pretransplant regimens, such as the BEAM (BCNU-Etoposide-Aracytine-Melphalan), TEAM (as BEAM but Thiotepa instead of BCNU), or CBV (Cyclophosphamide-BCNUEtoposide/VP16) over longer periods, lasting up to 6 days. Some studies show promising results with non-cryopreserved PBSCs kept at 4  C for 3–6 days (Sierra et al. 1993; Karduss-Urueta et al. 2014). Even a few studies have claimed better results with non-cryopreserved as compared to the cryopreserved PBSCs (Sarmiento et al. 2018; Bittencourt et al. 2019). Autologous bone marrow progenitor cells (BMPCs) or PBPCs provide a supportive therapy that allows the use of high doses, intensive antitumoral chemotherapy in hematological malignancies (Kessinger et al. 1986, 1988). Many studies have shown the superiority of autologous BMPCs or PBPCs over the conventional chemotherapy in hematological malignancies, such as multiple myeloma (MM) (Fermand et al. 2005) or lymphoma (Philip et al. 1995; Andre et al. 1999; Carella et al. 2009). PBPCs are currently used in autologous stem cells transplantation in hematological malignancies only, and BMPCs are reserved to specific indications, such as haploidentical allografts, or bone marrow failure. PBPCs are typically cryopreserved in liquid nitrogen at 180  C in dimethyl sulfoxide (DMSO) and albumin (Billen 1957; Bakken 2006; Berz et al. 2007). Progenitor cells should be washed and cleaned from DMSO before use in the patient. This preservation technique requires expensive equipment. In vitro study concerning the use of non-cryopreserved hematopoietic progenitor cells (HPCs) was first published in 1957 (Billen 1957). It was then followed by studies on the conservation of PBPCs at +4  C (Ahmed et al. 1991; Sierra et al. 1993; Preti et al. 1994) and their clinical use (Hechler et al. 1996). Very few autologous stem cells transplant were performed with non-cryopreserved PBSCs, and in our opinion, there are no published randomized or controlled studies on the non-cryopreserved autologous PBPCs. Only two literature

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reviews on this topic have been published. The first study was published by Wannesson et al. in 2007 on autologous HPCs transplantation (bone marrow and peripheral blood) in hematological malignancies and solid tumors (Wannesson et al. 2007) while the second study was published by Al-Anazi in 2012, on the use of autologous PBPCs for transplantation in the patient with MM (Al-Anazi 2012). This chapter reports all the published data in the field of non-cryopreserved autologous transplantation in hematological malignancies in developing countries to show the clinical safety, feasibility, and efficacy of the non-cryopreserved autologous cells to promote this technology, especially in the countries with limited resources.

Bibliographic Literature Search for the Chapter Bibliographic research was based on PubMed and Google scholar, using the following keywords: hematopoietic, progenitor, non-cryopreservation, and autograft. All the articles on the non-cryopreserved autologous PBPCs in hematological malignancies in developing countries were searched. The selected papers were analyzed in terms of mobilization, apheresis, storage and viability, conditioning regimen, engraftment, response, and finally, survival. Data from this review were synthesized in a descriptive manner. This included the tabulation of study characteristics and outcomes. All the survival times were calculated from the date of transplant. Transplant-related mortality (TRM) was defined as any death related to a fatal complication in the absence of the underlying disease within 100 days from transplantation. Overall survival (OS) was defined as the duration from the date of transplantation until the date of death or the date of follow-up when the patient was known to be alive. Progression-free survival (PFS) was calculated from the date of transplantation to disease progression or death (regardless of the cause of death). The OS and the PFS were determined using the Kaplan–Meier estimation with 95% confidence intervals from standards errors.

Data Analysis from the Selected Studies Research on PubMed and other search engines identified several publications and abstracts. In developing countries, only 17 studies were selected, responding to the criteria concerning non-cryopreserved autologous progenitor cells transplant. Countries of origin are by alphabetical order: Algeria, Brazil, Chile, Colombia, Egypt, Greece, India, Iran, Mexico, Morocco, and Thailand. All the 17 studies published from 2000 to 2021 were retrospective of “single-center case series” type. The majority focused on the MM and lymphoma (Papadimitriou et al. 2000; RuizArguelles et al. 2003; Cuellar-Ambrosi et al. 2004; Mabed et al. 2006; LopezOtero et al. 2009; Ramzi et al. 2012a, b; Kayal et al. 2014; Bekadja et al. 2017, 2021; Sarmiento et al. 2018; Kardduss-Urueta et al. 2018; Naithani et al. 2018; Kulkarni et al. 2018; Bittencourt et al. 2019; Jennane et al. 2020; Piriyakhuntorn et al. 2020) and only one study focused on acute leukemia (Palumbo et al. 2009).

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Over 21 years, a total of 1541 autografts were performed with unfrozen stem cells. The results of these autografts are detailed according to the different stages of the autograft, which are mobilization, apheresis, storage, viability, conditioning regimen, engraftment, and posttransplantation results. A special section is reserved for comparison of the studies between frozen and unfrozen stem cells.

Cell Mobilization Most centers have performed PBPCs mobilization using granulocyte-colony stimulating factor (G-CSF) alone, while two groups have used G-CSF combined with chemotherapy. In multiple myeloma, all studies (Ruiz-Arguelles et al. 2003; Mabed et al. 2006; Palumbo et al. 2009; Bekadja et al. 2012, 2017; Ramzi et al. 2012b; Kayal et al. 2014; Bittencourt et al. 2019; Jennane et al. 2020) have conducted the mobilization with subcutaneous G-CSF alone at a dose of 15 μg/kg/day, or 5 μg/kg twice a day, for 4–5 consecutive days. In lymphomas, mobilization was performed using G-CSF (5 μg/ kg/day for 3 days) in combination with cyclophosphamide at a dose of 1.5 g/kg/day, for 3 days, in two groups (Mabed et al. 2006; Lopez-Otero et al. 2009).

Apheresis The PBPCs apheresis was performed using devices such as Haemonétics®, Cobe Spectra®, or Optia®. Leukapheresis was started as soon as the flow cytometer counting of CD34+ (cluster differentiation) PBSCs was greater than 1  106cells/μl. The mean number of leukapheresis was two in MM and three in lymphomas. The overall mean of CD34+ collected in all studies was 3.94  106/kg (range, 0.32–24.6). In MM, the overall mean of CD34+ collected was 4.26  106/kg (range, 0.32–27.8). It was 4.47  106/kg (range, 1.9–24.6) in lymphomas. There was no report of mobilization failure in the published series.

Cell Storage The PBPCs collected were saved in the refrigerator at +4  C for a period ranging from 1 day to 6 days (Sierra et al. 1993; Hechler et al. 1996; Bekadja et al. 2017, 2021) depending on the type of conditioning regimen used. In the MM, storage time ranges from 1 to 2 days, and it was of 3–6 days in lymphoma (Table 1).

Cell Viability The viability of PBPCs was calculated by the Trypan Blue technique and by flow cytometry (Fleming and Hubel 2006). The average viability was over 90% in MM and over 75% in lymphomas.

76

224

55 26 94

Naithani (2018)

Kulkarni (2018)

Jennane (2020) Piriyakhuntorn (2020) Bekadja (2021)

12–67 16–50 17–55 42–66 NA NA 22–65 35–65 22–68 54 (29–75) 39 (5–67) 59 (34–68) 34 (14–64) 50 (23–68) 37–67 55.7 29 (17–60)

Age (year) 8–69 9–67

MM MM HL/NHL

MM

MM Lymphoma

Diagnosis MM/NHL/HL MM/NHL/HL/AML/ ALL MM/NHL HL NHL MM HL MM MM MM MM/NHL/HL MM/Lymphoma

Mel140/Mel200 Mel200 CBV/BEAM/BeEAM/ EAM

Mel200

Mel200/140 BEAM

CBV/CTX-TBI/Mel 200 CTX/VP16/Carboplatin CBDA/VP16/CTX Mel 200 CEAM Mel140/Mel200 Mel 200 Mel140/Mel200 Mel 200 Mel200/BEAM/CBV

HDCT Mel140-180/Mel-VP16 Mel 200

4.5 (2–12.2) 3.8 (2.0–16.5) 4.12 (3.4–5.4)

4.87

2.56 (1.2–7.9)

1.36 (0–6.32) 6.4 (3.8–24.6) >3 7.6 (0.3–14.8) 3.4 NA 2.9 (0.9–7.67) 5.7 (1.9–10.5) 5.1 (2.5–5.6) 3.6

CD34+ reinfused 3(0.8–2.78) 4.68

24–48 h 48 h 6 days

24–48 h

48 h–6 days

72 h (48 h–6 days)

144 h 72 h 72 h 24 h NA 48 h 48 h 24 h

Storage at +4  C (hours) 24–60 h 24–72 h

Abbreviations: HDCT, high dose chemotherapy; MM, multiple myeloma; NHL, non-Hodgkin lymphoma; HL, Hodgkin lymphoma; AML, acute myeloid leukemia; ALL, acute lymphoid leukemia

47 28 32 26 45 38 92 240 42 359

Cuellar-Ambrosi (2004) Mabed (2006) Mabed (2006) Lopez-Otero (2009) Ramzi (2012) Ramzi (2012) Kayal (2014) Bekadja (2017) Sarmiento M (2018) Kardduss-Urueta et al. (2018)

Author Papadimitriou (2000) Ruiz-Arguëlles (2003)

Patients (n) 72 46

Table 1 Patient’s characteristics and results of non-cryopreserved autologous peripheral blood progenitor cell transplantation

1426 M. A. Bekadja

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Conditioning Regimen The conditioning regimen and the myeloablative therapy were dependent on the diagnosis. In MM, all of the studies have used the Melphalan at a dose of 180 (Papadimitriou et al. 2000) or 200 mg/m2 on D-1 (Lopez-Otero et al. 2009; Ramzi et al. 2012b; Kayal et al. 2014; Bekadja et al. 2017). In lymphoma, the protocols used were of a different type: MEL 200 (Melphalan 200 mg/m2), CBV (Lobo et al. 1991), BEAM (BCNU, Etoposide, Aracytin, Melphalan) (Smith and Sweetenham 1995), CEAM (Lomustine, Etoposide, Aracytin, Melphalan), EAM (Etoposide, Aracytin, Melphalan) (Bekadja et al. 2018), CEC (Cyclophosphamide, Etoposide, Carboplatin), MEL/VP16 (Melphalan, Etoposide), and their length varies from 3 to 6 days (Table 2). Table 2 Results of engraftment with non-cryopreserved autologous peripheral blood progenitor cell transplantation

Patients (n) 72

Author Papadimitriou (2000) Ruiz-Arguëlles 46 (2003) CuellarAmbrosi (2004) Mabed (2006) Mabed (2006) Lopez-Otero (2009) Ramzi (2012) Ramzi (2012) Kayal (2014) Bekadja (2015) Bekadja (2017) Sarmiento (2018) KarddussUrueta et al. (2018) Naithani (2018) Kulkarni(2018) Piriyakhuntorn (2020) Jennane (2020) Bekadja (2021)

Neutrophils >0.5G/L (median day and range) 9 (6–16)

Platelet>20G/L (median day and range) 5 (0–89)

TRM (%) 0

Graft failure patients 0

14 (0–86)

25 (0–102)

2

0

47

Diagnosis MM/NHL/ HL MM/NHL/ HL/AML/ ALL MM/NHL

11 (9–15)

16 (11–44); 15 (14–20)

12.7

0

28 32 26

HL NHL MM

13 (7–18) 12 (8–17) 27 (0–53)

15 (7–20) 14 (7–19) 37 (0–73)

– 9.37 9.6

0 0 0

45 38 92 45 240 42

HL MM MM HL MM MM/NHL/ HL MM, Lymphoma

11 11 (9–21) 10 (8–27) 11 (8–12) 10 (6–17) 9 (9–16)

14 13 (10–31) 14 (9–38) 13 (10–24) 13 (9–24) 11 (10–19)

2.2 0 3.2 3 1.3

0 0 0 0 0 0

13 (9–39)

16 (7–83)

12 (9–35)

13 (9–65)

3

0

224 26

MM, Lymphoma MM MM

12 (9–22) 12 (10–19)

17 (10–44) 14 (10–23)

3.1 3.8

1/224 0

55 94

MM HL/NHL

12 (7–19) 14 (12–32)

14 (9–32) 17 (15–28)

3.6 9

0 0

359

76

0

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M. A. Bekadja

Table 3 Survival of patients with multiple myeloma and lymphoma autografts with non-cryopreserved PBPCs

Author Mabed (2006)

Patients number 28

Diagnosis HL

Follow-up (median months) 16

Mabed (2006)

32

NHL

18

Lopez-Otero (2009) Ramzi (2012)

26

MM

NA

45

HL

27

Ramzi (2012)

38

MM

31

Bekadja (2017)

240

MM

47

Kulkarni (2018)

224

MM

NA

Piriyakhuntorn (2020) Bekadja (2021)

26

MM

37.5

94

NHL/HL

62

OS 45% at 24 months 50% at 24 months 80% at 76 months 27 months (median) 30 months (median) 79% at 5 years 62.9% at 5 years NR

PFS 42% at 24 months 43% at 24 months NA

85% at 36 months

70% at 36 months

20 months (median) 27 months (median) 59 months 36.6% at 5 years 16 months

Abbreviations: MM, multiple myeloma; HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; OS, overall survival; PFS, progression-free survival; NA, nonavailable; NR, not reached

Cell Engraftment Engraftment was defined by the rate of ANC (absolute neutrophil count) over 0.5 G/L and a platelet count greater than 20 G/L, except for one study in which the threshold was 25 G/L platelets (Ruiz-Arguelles et al. 2003). The results of engraftment in the different studies are shown in Table 3. The overall mean recovery time of ANC was 12.38 days (range, 6–86), and that of the platelets was 15.57 days (range, 7–89). This recovery time was respectively 13 and 18 days in MM and 12 and 14 days in lymphoma. There was no engraftment failure recorded among the different studies, only one study (1/224) (Kulkarni et al. 2018). The overall median rate of the transplant related mortality (TRM) was 4.39%, while it was 3.51% and 4.85% respectively in the MM and lymphoma.

Posttransplant Data Considering the heterogeneity of the studies in terms of diagnosis and intensification protocols, it is difficult to analyze them in relation to response or survival. However, the median follow-up period ranged between 31 and 37.5 months in the MM, and between 16 and 62 months, in lymphoma. The overall survival (OS) and disease-free survival (DFS) in the MM and lymphoma are reported respectively in Table 4.

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Table 4 Characteristics of patients receiving PBSC stored at 4Dg C in Oran and pair matched patients receiving cryopreserved PBSC reported to the EBMT registry PBSC Stored at 4  Ca Number of patients 94 Age at SCT Median (range) 29 (17–60) IQR 25–34 Gender (%) Female 46 (49) Male 48 (51) Karnofsky at SCT (%) Good 80 79 (84) Poor 20,000/uL. Significant faster engraftment with cryopreserved PBSC ( p ¼ 0.01)

refractory forms of Hodgkin’s diseases (Hahn et al. 2013; Isidori et al. 2013; Van Den Neste et al. 2013) or non-Hodgkin lymphomas (Milpied 2013). A recent EBMT survey published in 2021 shows 48,512 stem cell transplants in 43,581 patients, including 28,714 autografts (59%). The EBMT group had

a

0

6

EBMT ORAN p = 0.365

6

12 18 24 Months after SCT

12 18 24 Months after SCT

30

30

36

36

b

0

20

40

60

80

100

0

20

40

60

80

100

0

0

6

EBMT ORAN p = 0.287

6

EBMT ORAN p = 0.1175

24 12 18 Months after SCT

12 18 24 Months after SCT

30

30

36

36

Non-cryopreserved Peripheral Stem Cell Autograft for Multiple Myeloma and. . .

Fig. 2 Outcome of the patients autografted with PBSC stored at 4  C and patients autografted with cryopreserved PBSC: No significant difference in any parameter. (a) Non-relapse mortality; (b): relapse incidence; (c): progression-free survival; and (d) overall survival

0

20

40

60

80

100

0

EBMT ORAN p = 0.3688

d

0

20

40

60

80

100

c

Cumulative incidence of NRM, %

Progression-Free survival Probability, %

Cumulative incidence of relapse, % Overall survival Probability, %

48 1433

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700 centers in 51 countries in 2019. These European centers freeze stem cells as part of the autografts. The Eastern Mediterranean Bone Marrow Transplantation Group (EMBMT) represents the Eastern Mediterranean Region (EMRO) comprising 10 countries, including Algeria, Morocco, Tunisia, Egypt, Lebanon, Iran, Saudi Arabia, Pakistan, Jordan, and the Sultanate of Oman. The 2008–2009 report has shown an activity rate of autografts of 36.5% (n ¼ 483/1322 total first transplants) in these countries, versus 59% (n ¼ 16,591/28033 total first transplants) in the developedcountry (Mohamed et al. 2011). Most transplant centers do not use stem cell freezing during autologous transplantation. The report also highlights the limited number of transplant centers in developing countries which is 14 versus 647, i.e., 46 times greater for 2009, in developed countries (Baldomero et al. 2011). Moreover, the average number of transplant center is 14/country in the EBMT Group, which counts 48 countries in 2009 versus 1.4/country in the EMBMT group which has 10 countries. The number of centers per 10 million inhabitants is respectively 7.6 and 0.3 in developed countries versus developing countries. Moreover, the number of transplants per 10 million inhabitants is 467 and 28.7 in the EBMT and EMBMT Groups, respectively. Hence, this situation showed that new transplant centers and particularly the development of the non-cryopreserved autologous PBPCs transplant in these countries and other resource-constrained countries were necessary. The first meeting of the African Blood and Marrow Transplantation (AfBMT) was held in Casablanca from April 19, 2018, to April 21, 2018, to foster HSCT activity in Africa. Out of the 54 African countries, HSCT is available only in 6 (Algeria, Egypt, Morocco, Nigeria, South Africa, and Tunisia) (Mhamed Harif 2019). The latest published report of WBMT (Baldomero et al. 2019) showed that AFR/EBMT region represented only 3% of reported activity worldwide. In the African continent, this is even lower. The health care systems in Africa are the least developed. Hematopoietic cell transplant activity in the 28 countries comprising Latin America is poorly defined. A retrospective study conducted a voluntary survey of members of the Latin American Bone Marrow Transplantation Group regarding transplant activity 2009–2012. Annual activity increased from 2517 transplants in 2009 to 3263 in 2012 represents a whopping 30% increase. The median transplants rate (transplant per million inhabitants) in 2012 was 64 (autotransplants, median 40; allotransplants, median 24). This rate is substantially lower than that in North America and European regions (482 and 378) but higher than in the Eastern Mediterranean and the Asia Pacific regions (30 and 45). However, the Latin America transplant rate is five to eightfold lower than in America and Europe, suggesting a need to increase transplant availability. Transplant team density in Latin American (teams per million population; 1.8) is three to fourfold lower than that in North America (6.2) or Europe (7.6) (Jaimovich et al. 2017). The number of publications regarding non-cryopreserved autologous transplant is scarce because of the widespread use of cryopreserved stem cells. In this chapter,

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only 17 eligible studies were collected dealing with non-cryopreserved autologous transplant in hematological malignancies (lymphoma and MM) in some developing country, from 2000 to 2020. All these studies are single-center, retrospective, non-randomized, and uncontrolled, reflecting the care of patients in real life and working conditions of countries with limited resources. Only one study is prospective and included 26 patients with MM (Lopez-Otero et al. 2009). Currently, over 1500 patients have undergone autologous transplantation with unfrozen cells, out of which 80% are relevant to multiple myeloma while the remaining 20% are concerning to lymphoma.

Cell Mobilization The first step of autologous stem cells transplantation is PBPC mobilization. There is no absolute rule in PBPC mobilization, but multiple studies have been published regarding recommendations for improving the harvesting efficiency of PBPCs (Bezwoda et al. 1994; Demirer et al. 1996). Two main methods are used in PBPC mobilization; the first relates to the use of growth factor G-CSF alone, given subcutaneously at a dose of 10 to 15 μg/kg/day or 5 μg/kg twice a day, for 5 days (Talhi et al. 2013; Yafour et al. 2013); the second consists in the combination of G-CSF with chemotherapy (Ford et al. 2006). There have been not much difference in the performance of harvesting of CD34+, but the second method requires hospitalization to manage aplastic anemia secondary to chemotherapy, whereas in the first method, the use of G-CSF alone can be done at home, which reduces the costs of the autograft procedure. Most studies have achieved mobilization with G-CSF alone, especially in the MM and lymphoma; only two groups have used G-CSF in combination with cyclophosphamide (Mabed and Al-Kgodary 2006; Mabed and Shamaa 2006). The objective of the mobilization is to reach at least 2  106/kg CD34+ in the MM in which the conservation of CD34+ is short (24–48 h) and at least 4  106/kg in lymphomas in which the conservation of CD34+ is most extended (3–6 days). Indeed, the optimal rate of CD34+ necessary for hematopoietic reconstitution is unknown with certainty, and a minimum of 2.0 to 3.0  106 CD34+ cells/kg is typically recommended. In this work, all the studies have achieved sufficient levels of CD34+, that was 4.26  106/kg (MM) and 4.47  106/kg (lymphoma), to perform autografts, and no mobilization failure was reported.

Conservation and Cell Viability Many studies have shown the possibility to save the PBPCs at +4  C in a refrigerator, for several days, with final viability of over 80% that allows the achievement of autografts (Ahmed et al. 1991; Sierra et al. 1993; Preti et al. 1994; Hechler et al. 1996). Preti and colleagues showed no difference in terms of cell viability and engraftment rate between their maintenance at +4  C and cryopreservation of PBPCs (Preti et al. 1994). In addition, cryopreservation requires the use of a

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cryoprotectant, dimethyl sulfoxide (DMSO), which is responsible for several side effects (Hoyt et al. 2000; Windrum and Morris 2003) so that it necessitates a PBPCs washing before their use. On the other hand, it allows multiple transplantations if and when needed. In all published articles, the conservation at +4  C allowed the achievement of autografts with a satisfactory rate of the viability of PBPCs despite retention periods up to 3 days, as previously reported in the studies of CuellarAmbrosi et al. and Bekadja et al. wherein the storage time was up to 6 days (CuellarAmbrosi et al. 2004; Bekadja et al. 2021).

Therapeutic Intensification Using high-dose chemotherapy (HD), therapeutic intensification is the most crucial part of the autograft procedure, as it has a direct antitumor effect. Since the 1990s, the high-dose chemotherapy of Melphalan at 200 mg/m2 on D-1, followed by the autologous transplant of PBPCs, is considered the standard first-line treatment of MM for eligible patients to this procedure. So, the schedule consisting of administration of the HD chemotherapy (Melphalan) on D-1 enables the conservation of PBPCs at +4  C for only 24–48 h, with the viability of CD34+ cells over 95%. This is consistent with an autologous transplant of non-cryopreserved PBPCs. In lymphomas, the situation is very different; the intensification protocols used are shown in Table 2. These protocols, such as CBV (Lobo et al. 1991), BEAM (Smith and Sweetenham 1995), or EAM, include administration periods ranging from 3 to 6 days, which need a collection of 3  106 CD34+/kg for varying viability of 75% to 85% (Bekadja et al. 2018). The conservation of the PBPCs up to 7 or 8 days is then feasible, but needs to obtain a number of CD34+ cells 3  106/kg at the time of the reinfusion of the PBPCs. Hence, the significant difficulty is in the mobilization of cells, especially in patients who received multiple lines of therapy. In the consolidation phase, especially in the mantle cell lymphoma or in the diffuse large B cell lymphoma (DLBCL) (Fitoussi et al. 2011; Visani et al. 2012), the probability of obtaining CD34+ cells 3  106/kg is very high, especially in view of the low number of chemotherapy lines, and non-cryopreserved PBPCs can be used easily. Improving the autograft results in lymphomas will undoubtedly reduce the number of chemotherapy cycles, by early assessment of the PET scan response, and by the availability of new intensification protocols, more myeloablative, as consolidation phase after the first-line induction. These regimens will allow the use of non-cryopreserved PBPCs in view of the high possibilities of their mobilization.

Donor Cell Engraftment Engraftment was evidenced by the rate of the ANC and platelets count. The aplastic phase was managed either with or without growth factors in case of profound neutropenia. Globally the median length of the ANC and platelets rate was similar

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to that of autografts using cryopreserved PBPCs (Lanza et al. 2013) both in the MM (Kristinsson et al. 2014) and lymphoma. Nevertheless in the lymphoma, duration was a little longer due to the number of previous chemotherapy and the refractory nature of lymphoma (Stiff et al. 2013; Rancea et al. 2013; Cook et al. 2014). Overall, these hospitalization numbers show less than 21 days in the MM and 25 days in the lymphoma, which classifies the non-cryopreserved autologous transplant in the favorable group according to Lanza et al. (2013). No engraftment failure or complications related to the infusion of PBPCs were mentioned. In addition, among the 1541 autografts, 4.39% of patients died due to the procedure and procedural complications, particularly in the autologous transplants in lymphoma. This rate of TRM is comparable to that found in the literature, demonstrating the safety of non-cryopreserved autologous PBPCs. Thus, the technique of autologous with non-cryopreserved PBPCs is a simple, reliable, and clinically feasible method. It is also safe, effective, and less costly. Only very few clinical facilities and groups in the developing countries use this procedure, whereas care is essential, especially in hematologic malignancies. Some authors question the results obtained with unfrozen cells. Their arguments are the difficulties of comparability outside of randomized clinical studies because the characteristics of the diseases and the intensification protocols are not the same (Gale and Ruiz-Arguelles 2018). Although this argument is relevant, it should be kept in mind that carrying out such a study is very difficult and challenging, and for multiple myeloma, the comparability is valid and accurate because the intensification regimen is the same, Melphalan at 200 mg/m2. The comparability results obtained in multiple myeloma can thus be applied in lymphomas despite the different consolidation regimens. On the other hand, many centers have performed autografts with unfrozen cells and have reported less toxicity and identical efficiency to those of frozen cells (Bekadja and Bouhass 2015). Currently, more than 1500 autografts have been performed with unfrozen cells, and many studies have retrospectively compared autografts with frozen and unfrozen stem cells. All the results show the non-inferiority of unfrozen stem cells. The indication for autografting of unfrozen cells in multiple myeloma is the most important because: 1. 2. 3. 4.

The duration of unfrozen stem cells is less than 48 h. A second autograft is very rare, less than 1% in case of relapse. There are now new drugs to treat relapses effectively. The storage of grafts can pose many problems regarding storage capacities and the need to destroy the grafts after the validity period expiration.

On the other hand, in lymphomas, unfrozen stem cells can only be used as the first consolidation, given the high probability of stem cell mobilization. The length of the conservation is 6 days on average. In the forms of relapse, the possibility of mobilization is less, and it is necessary to use Plerixafor (Mozobil) for mobilization of stem cells (Fricker 2013) to have very high grafts, which can be preserved for more than 6 days. Under these particular conditions, freezing at 180  C can be

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used, which requires less expensive equipment and no liquid nitrogen. On the other hand, in Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, the probability of relapse is very low and there are also many drugs that are effective against relapses.

Conclusion and Future Perspective These analyses of the published studies included in this chapter show that performing auto-HSCT without cryopreservation is possible. Although the toxicity and directly related side effects of DMSO infusion are negligible in the study population of CRYO patients, non-cryopreserved products were associated with a lower incidence of severe mucositis and febrile neutropenia. Hence, a significantly shorter hospital stay is required without sacrificing the overall response rate and survival. These results are of utmost significance because they favor better treatment tolerance and, by being cost-effective, they give obvious relief to the increasingly overloaded and already overwhelmed public health care systems.

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Van Den Neste E, Casasnovas O, André M, Touati M, Senecal D, Edeline V, Stamatoullas A et al (2013) Classical Hodgkin’s lymphoma: the Lymphoma Study Association guidelines for relapsed and refractory adult patients eligible for transplant. Haematologica 98(8):1185–1195. https://doi.org/10.3324/haematol.2012.072090 Visani G, Picardi P, Tosi P, Gonella R, Loscocco F, Ricciardi T, Malerba L (2012) Autologous stem cell transplantation for aggressive lymphomas. Mediterr J Hematol Infect Dis 4(1):e2012075. https://doi.org/10.4084/MJHID.2012.075 Wannesson L, Panzarella T, Mikhael J, Keating A (2007) Feasibility and safety of autotransplants with noncryopreserved marrow or peripheral blood stem cells: a systematic review. Ann Oncol 18(4):623–632 Windrum P, Morris TC (2003) Severe neurotoxicity because of dimethyl sulphoxide following peripheral blood stem cell transplantation. Bone Marrow Transplant 31(4):315 Yafour N, Brahimi M, Osmani S, Arabi A, Bouhass R, Bekadja MA (2013) Biosimilar G-CSF (filgrastim) is effective for peripheral blood stem cell mobilization and non-cryopreserved autologous transplantation. Transfus Clin Biol 20(5–6):502–504. https://doi.org/10.1016/j. tracli.2013.04.109

A Multilevel Approach to the Causes of Genetic Instability in Stem Cells

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Damage Response in Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors That Influence DNA Stability in Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Senescence as a Factor Inducing Stem Cells Genomic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aging and Genomic Instability in Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Telomere Damage and Genetic Instability in Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aneuploidy as an Expression of Genome Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of Genomic Arrangement in the Nucleus on Aneuploidy . . . . . . . . . . . . . . . . . . . . . . . . . . Lamins Alterations and Aneuploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replicative Stress/Replication Timing as Causes of Genome Instability . . . . . . . . . . . . . . . . . . . . . Cell Origin as Source of Genome Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adipose Stem Cells (ASC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Reprogramming Methods on Genetic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutations Induced During Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Culture Conditions, the Passage Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Bystander Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instability Factors Related to Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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E. A. Prieto Gonzalez (*) Centre for Advanced Studies in Humanities and Health Sciences, Interamerican Open University, Buenos Aires, Argentina ISALUD University, Nutrition Career, Buenos Aires, Argentina e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. H. Haider (ed.), Handbook of Stem Cell Therapy, https://doi.org/10.1007/978-981-19-2655-6_26

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Abstract

This chapter addresses the genetic instability in stem cells, a central feature that is an important determinant for the safety and effectiveness of cell-based therapies. First, DNA damage response mechanism and the gene network that regulates the DNA integrity homeostasis are revisited, focusing on relationship between genetic integrity and stemness maintenance. Also, several factors have been included that influence genetic instability in vivo, i.e., ROS generation and inflammation, and in vitro regarding cell isolation, culture conditions, i.e., oxygen levels and the use of feeder layers and different carriers, splitting procedures, passage number, etc. Telomere shortening, replicative senescence aneuploidy, and the senescence-associated secretory phenotype are different processes involved in deterioration of stem cells abilities for homing, reparability, and paracrine modulation. Moreover, less effective DNA repair upon senescence increases the propensity of developing tumors for stem cells that is one the main concerns related to genetic instability in stem cells. Nuclear organization and their determinants linked to chromosome aberrations and aneuploidy in stem cells and those epigenetic changes affecting stability are addressed. Differences between adipose, embryonic, and induced pluripotent stem cells are analyzed regarding to their ontogeny, changes in culture, and variation in proliferative capacity and stemness. The effect of reprogramming methods in genetic instability and variation in mutagenicity according to genes utilized for pluripotency induction, i.e., Yamanaka’s factors and others, is discussed. Donor-related factors, i.e., age, smoking, and alcohol consumption, comorbidities, i.e., obesity, insulin resistance, diabetes, are mentioned for wide evaluation of genetic instability. The next years must witness consensus protocols that will contribute to control the effects of factors that alter stem cell genetic stability to increase the security and applicability of cell-based therapy. Keywords

Genetic · Instability · Reprogramming · Stem cells · Stemness · Telomere Abbreviations

ASC AT ATM ATR BATF BER BID BM-hMSC BRCA1 CASP Cdc25a

Adipose stem cells Adipose tissue Ataxia telangiectasia mutated ATM- and Rad3-related serine/threonine kinase Basic leucine zipper transcription factor – ATF-like Base excision repair BH3 interacting domain death agonist Bone marrow human mesenchymal stem cell BReast CAncer gene 1 Caspase Cycle regulator phosphatase

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CDK CENP-A Cernunnos/XLF CHK1/2 c-MYC CNVs CT DBC1 DDR DDT DNA-PKcs DPPA3 DSBs EPC ESCs Exo1 FISH FOXO hADSC hESCs HIF-1α hiPSCs hPSCs HR HSC hTR ICLR IDH2 iPSCs Klf4 LIN28A MAPK p38 MDC1 mESC MMR MN/CB MNR MRE11 MSCs mTOR MUSE NANOG NBS1 NER NF-κB

Cyclin-dependent kinase p16Ink4a Centromere histone H3 variant Nonhomologous end-joining factor 1 Checkpoint kinase 1 and 2 Transcription factor, proto-oncogene Copy number variations Chromosome territories Deleted in bladder cancer protein 1 DNA damage response DNA damage tolerance DNA-dependent protein kinase Developmental pluripotency-associated protein-3 Double-stranded breaks Endothelial progenitor cells Embryonic stem cells Exonuclease 1 Fluorescent in situ hybridization Forkhead box O1 transcription factor Human adipose-derived stem cells Human ESCs Hypoxia-inducible factor-1α Human induced pluripotent stem cells Human pluripotent stem cells Homologous recombination Hematopoietic stem cells Noncoding human telomerase RNA Interstrand cross-link repair Isocitrate dehydrogenase-2 Induced pluripotent stem cells Krüppel-like factor 4 Lin-28 homolog A, RNA-binding protein Mitogen-activated protein kinase 38 Mediator of DNA damage checkpoint 1 Mouse embryonic stem cells Mismatch repair Micronucleus with cytochalasin B Trimeric complex (Mre11, Rad50, and Nbs1) Homolog double-strand break repair nuclease Mesenchymal stem cells Mammalian target of rapamycin Multilineage-differentiating stress-enduring Nanog homeobox transcription factor Nijmegen breakage syndrome 1 Nucleotide excision repair Nuclear factor κB

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NHEJ NPM2 NSC Oct-4 p21/WAF1 p53 PARP1 PBMC Pi3KK PPR PSCs RAD17 RAD18 RAD50 RAD9 Rb ROS RS RT SASP SNP SOX-2 TAD TCR TLS TRA-1-60 USSCs XPA XPA γH2AX

E. A. Prieto Gonzalez

Nonhomologous end joining Nucleoplasmin-2 Neural stem cells Octamer-binding transcription factor 4, POU5F1 Cyclin-dependent kinase inhibitor 1A p53 tumor suppressor gene Poly-ADP ribose polymerase 1 Peripheral blood mononuclear cells Phosphatidylinositol 3-kinase-related kinase Post-replication repair Pluripotent stem cells Checkpoint clamp loader component RAD18 E3 ubiquitin protein ligase Double-strand break repair protein Cell cycle checkpoint control gene RAD9A Rb tumor suppressor gene Reactive oxygen species Replicative stress Replication-timing Senescence-associated secretory phenotype Single nucleotide polymorphism Transcription factor SRY (sex determining region Y)-box Topological associated domains Transcription coupled repair Trans-lesion synthesis Pluripotency stem cell marker Human unrestricted somatic stem cells DNA damage recognition and repair factor Histone H2AX

Introduction In stem cells, genomic instability is crucial and determines the suitability for a proper and safe use in stem cell-based therapy. One of the interpretations of genomic instability is the accumulation of changes that modify genome structure, a situation that is manifested as the sustained increase in translocations, deletions, or duplications that may result in chromosome number variation, but also when is undetectable through karyotype analysis and occurs at molecular level without evident ploidy alterations, referred as the copy number variations (CNVs) (Shen 2011). Genome stability relies on cellular processes that controls DNA metabolism, i.e., replication, transcription, damage repair, and chromatin remodeling. Those process that are implicit in DNA functioning must be tightly coordinated through checkpoints controlling passages between cell cycle stages, allowing progression and detention when is needed (Hartwell 1992). The cellular checkpoints convey signals

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that may trigger DNA repair so as to assure the fidelity of genetic information transmission between cells generations. Along cell cycle, there are different checkpoints that may slow its progression to facilitate damage repair, e.g., M phase checkpoint that guarantees the completeness of DNA replication before mitosis begins. Indeed homeostasis in cells and tissues are preserved via apoptosis induction if DNA damage is too high to be fixed by DNA repair mechanism (Lo Furno et al. 2016; Toledo 2020; Giacosa et al. 2021).

DNA Damage Response in Stem Cells DNA repair mechanisms are intended to restore the integrity of genetic message, eliminating those changes in DNA molecules that could affect the fidelity of genetic information. These include nucleotide excision repair (NER), base excision repair short and long (BER), mismatch repair (MMR), and transcription coupled repair (TCR). Also, the error free homologous recombination (HR) and those error-prone mechanisms like nonhomologous end-joining repair, interstrand cross-link repair (ICLR), and post-replication repair (PPR). Malfunction of any of these mechanisms may increase genetic instability via generation of chromosomal or sub-chromosomal aberrations or indirectly through provoking mutation affecting repair checkpoints genes or other genes involved in DDR that may affect genomic stability and result in a mutated phenotype. In fact there are many reports on the overexpression of genes responsible for most of the DDR mechanism in stem progenitor cells compared with those active in adult mature cells. DDR in stem cells have been recently reviewed (Mani et al. 2020). There are many gene families that are involved in DNA damage detection, signaling, and processing; one of them is the phosphatidylinositol 3-kinase-related kinase (Pi3KK) which includes ataxia telangiectasia mutated (ATM), DNAdependent protein kinase (DNA-PKcs), and ATM- and Rad3-related serine/threonine kinase (ATR), and those enzymes are implicated in the maintenance of genomic instability in stem cells. In mesenchymal stem cells (MSCs), there are evidences of linking ATM decline with progressive lowering of the ability to respond to replicative errors as well to genotoxic damage that arises during long-term expansion in culture (Shiloh and Ziv 2013; Beckta et al. 2017). ATM plays a principal role in repairing the highly lethal double-strand breaks (DSB). ATM responds mainly to DSB and to changes in chromatin DSB, while ATR become active after nucleotide damages, impairment in replication-fork progression and also to DSB. DNA-PKcs is linked to NHEJ. CHK1/2 favors DNA repair by reducing the cell cycle progression through modification in the state of master cell cycle regulators like CDC25 proteins (Lo Furno et al. 2016). Pi3kk triggers a phosphorylation cascade that modulates the activation of downstream genes in the repair circuit, i.e., p53, BRCA1, and CHK1. Protein products of that genes are assembled in the reparosome at DNA damage sites and provokes several relevant actions beside DNA repair, i.e., promoting the blockade of DNA replication and mitosis until DNA damage is fixed; however, in certain

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circumstances, the apoptotic response may also be induced. Phosphorylation of histone H2AX (γH2AX) that is a reliable signal of genetic instability is a consequence of ATR action, that is triggered by single-strand regions that appears if replication fork is delayed or stopped at damaged sites in DNA. ATR is also activated when double-strand breaks are present (Lo Furno et al. 2016). ATM is combined to trimeric complex MNR (Mre11, Rad50, and Nbs1) that is gathered at DSB sites, keeping the extremes of the gaps to be repaired. ATM is activated through shifting to an active monomer that begins to acts as a kinase over the substrates P53, BRCA1, CHK2, RAD17, RAD9, and NBS1 in order to build up repair foci. ATM is able to phosphorylate MDC1, Rad18, p53-binding protein 1 (53BP1), and BRCA1 to trigger DBSs repair of the two main mechanism involved in this branch of DDR, homologous recombination (HR) or nonhomologous end-joining repair (NHEJ) (Lukas et al. 2011; Nair et al. 2017). DNA damage response mechanisms are crucial to avoid the deleterious effects of DSB that are repaired mainly through NHEJ mechanism that is related to the ATM pathway. An example of the significance of ATM for DNA damage repair response is seen in MSCs from FVB/N mice strain, which expressed a significant increase in micronucleus with cytochalasin B (MN/CB) frequency, both spontaneous and related to gamma irradiation, and also in DSB. Frequency of both lesions of MN/CB and DSB showed an additive effect of aging and genotoxic stress. Correspondingly these cells diminished their ability to detect DNA damage, as expressed in less γH2AX/53BP1 DNA foci formation, less DNA repair, and increased residual DNA breakage when cultured over 8 weeks. In the first week of culture, a strong response of ATM was present, but 8 weeks later, ATM impairment resulted in reduced DDR (Hladik et al. 2019). ATM importance for genetic instability in stem cells can’t be underestimated. The p53/ATM network is involved in DNA damage recognition and the triggering of DDR but also in the initiation of apoptosis. Notably, induced pluripotent stem cells (iPSCs), that shows a reduced less ATM phosphorylation activity, exhibits an increased apoptotic response when treated with low level radiation. There are evidences that cells affected by certain mutations in p53 develops an increase in interchromosomal translocations and defective G (2)/M checkpoint. Those p53 gain of function mutations promote the association of p53 to Mre-11 nuclease therefore affecting the assembly of MNR complex (Mre11–Rad50–NBS1), and this malfunction of MNR impairs its binding to DNA DSB that results in a deficient triggering of ATM function (Song et al. 2007). There is a report on the abnormal karyotype in knockout ATM tail-tip fibroblasts that albeit exhibited a lower reprogramming efficiency, their descendant iIPS conserved their pluripotency for 20 passages. A result that emphasizes the main role of ATM in stem cells genetic stability, but also is a warning for the possibility therapeutic use of genetic unstable cells that in spite of that risky conditions may be able to proliferate and differentiate, eventually in a tumor growth (Kinoshita et al. 2011). There are reports connecting low activity in ATM with reduction in iPSCs differentiation as well as increased genomic instability (Nagaria et al. 2016). Efficiency in reprogramming cells to iPSCs is reduced in cells where ATM pathway is

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impaired and is associated with more genomic variation. When was evaluated the effect on genomic variation over the frequency of CNV in the boundaries of ATM gene, those ATM-CNV bearing iPSCs showed a higher susceptibility to genome instability than wild type cells. The greater effect of impaired DDR resultant from a higher frequency of CNV affecting the gene provoking ATM-deficiency was evident at earlier stages after retrovirus-based reprogramming (Lu et al. 2016). There is a group of effectors that participate in the choice between proliferation and differentiation in stem cells. These processes are strictly regulated so as to favor the right number of and the kind of pluripotent cells in each moment during embryogenesis and organogenesis. The coordinated generation and the proper decision in each cell population is also necessary for the repair and regeneration of damaged tissues, or in response to renovation needs in the adult individual. Among those proteins that are involved in the control of those orchestrated decisions is geminin that is a target for the cyclosome, or anaphase-promoting complex. Geminin acts as an inhibitor of pre-replication through binding to Cdt1 pre-replicative complex and avoids alterations in replication timing. Geminin expression is greater in late S phase and during G2, and this factor is also a key element in the neurogenesis (Kushwaha et al. 2016). Geminin interacts with members of the suppressive polycomb protein family as well as with the trithorax group of proteins that regulates neuronal development. The tuning in these network allows geminin to participate in the regulation of Hox genes among many others that controls the differentiation routes in progenitor cells (Kingston and Tamkun 2014; Lau and So 2015). Geminin prevents pre-replication of DNA that may lead to re-replication and genomic instability. However, it seems not to be a universal characteristic, as is evident in the fact that abrogation of geminin in a murine model for brain and hematopoietic development did not increase genomic instability nor the frequency of DNA damage in this model. The complexity of its actions does not seem to be restricted to genome protection, and even this property is controversial because geminin overexpression is present in cancer cells, a “Janus” behavior that is exemplified in the geminin-induced upregulation of 24 genes involved in transition from epithelial to triple negative breast cancer metastasis, homing and stemness in cancer stem cells. Geminin is an example of the series of factors that participate in genomic stability maintenance and could be considered as next target for genetic stability interventions (Sami et al. 2021; Montanari et al. 2006).

Factors That Influence DNA Stability in Stem Cells One of the aspects that must be addressed when considering the requirements for stem cell therapeutic applications is the genomic instability, because it is a condition that could hamper the outcome of stem cell transplantation. Microenvironment, where stem cells are introduced in order to be expanded, poses a threat to cells stability. The delicate homeostatic balance in the transplanted cell is affected by several factors like oxygen level, pH, and inflammation in donor tissues and ROS (reactive oxygen species) levels in vivo and ex vivo, among several ones

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(Aerts-Kaya 2020). Superoxide anion as well as hydrogen peroxide are generated not only in donor environment but also during handling; hydrogen peroxide is not a ROS but can be easily converted in that, through Fenton or Haber Weiss reactions, where transition metals participates. Hydrogen peroxide is also involved in intracellular signaling as well in pathogen destruction when is liberated by immune cells like neutrophils. ROS are well-known DNA damaging agents that may lead to genomic instability (Barzilai and Yamamoto 2004; Seddon et al. 2021). Among the factors that increased mutation frequency in hESCs (human ESCs), one that cannot be excluded is the composition of culture medium, the origin of feeder layer, the passaging method utilized, the freeze thawing procedure, and oxygen level, that is known to generate more instability at normoxia (21%) than at hypoxia (2%). The plate or flasks where cells are cultured influence stem cell fate, and it have been reported that expansion of ESCs in microcarriers for several months conserves pluripotency besides their cytogenetic stability and normal karyotype (Oliveira et al. 2014).

Oxidative Stress DNA can be damaged by oxidative stress generating a large array of base modifications being the commonest 8-oxoguanine, a lesion that is not only highly mutagenic but also accumulates with age. This lesion triggers base excision repair (BER) a mechanism that removes the damaged base and produces a single-strand break in the DNA before filling it with a new non-affected base. Strand breaks in one or both strands induces poly-ADP ribose polymerase 1 (PARP1), an enzyme that is more active with age, and leads to a greater frequency of DNA breaks in older subjects (Petr et al. 2020). Hematopoietic stem cells (HSCs) utilizes a preventive strategy so as to avoid ROS-provoked DNA damage based on the maintenance of a hypoxic state (Goto et al. 2014; Testa et al. 2016). This implies a substitution of cell respiration in mitochondria by glycolysis in order to produce ATP, and shifting again toward mitochondrial respiration when cells reenter cell cycle. This contraption works as an antioxidant defense mechanism, because respiration ROS generation is lowered and consequently DNA oxidative damage is reduced (Takubo et al. 2013). Cells during culture experiences high metabolic exigence that increases as culture time does and the cells experienced a greater number of passages. High metabolic rate is a cause of leakage in the electronic transport chain that leads to ROS generation and genetic damage. The margin for ROS levels in cells that undergoes reprogramming is narrow. Low levels affect reprogramming while high impairs differentiation. That poses an strict dependence of reprogramming success on the correspondence of oxygen level in culture with the cell type, its age and the specific characteristic of culture conditions (Henry et al. 2019; Zhou et al. 2016; Rönn et al. 2017). The recent demonstration of the role of NADPH oxidase, a master factor in oxidative balance, in the fate of stem cells, remarks the importance of the maintenance of oxidative homeostasis over stemness during ex vivo cells expansion (Maraldi et al. 2021).

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When DNA mutations favors an increase in ROS generation, the self-renewal capacity of HSCs is hampered. ROS generation is augmented when ATM, a checkpoint for DNA damage, is abrogated. This is a curious relationship between a component of DDR, ROS generation, and cell cycle progression in HSCs. The higher ROS production reduces self-renewal capacity through activation of cyclindependent kinase (CDK) p16Ink4a and the tumor suppressor gene Rb, but in addition, interrupts quiescent state by inducing p38 kinase (Ito et al. 2004). This effect have also been observed in neural stem cells (NSCs) (Kim and Wong 2009); it is an expression of the importance of ATM pathway in DNA damage response against different stressors and the interconnection between a DNA damaging responsive kinase like ATM and induction of oxidative stress (Kitagawa and Kastan 2005). Another link with DNA function is that of FOXO, a transcription factor that is induced in response to oxidative stress, stimulates Rb and p53 activation that reduces quiescence and self-renewal in these cells. There is a relative antagonism between quiescence and ROS generation that favors reentering cell cycle through reactivation cyclin-dependent kinase. FOXO p53 and Rb are necessary to limit oxidative stress DNA damage and genome instability (Bigarella et al. 2014; Burkhalter et al. 2015). The main HSCs functions, self-renewal and differentiation, are controlled by intrinsic mechanisms, transcriptional and epigenetic regulator hormones, and cytokines but also by the close bone marrow microenvironment. HSCs are commonly assumed to reside within the hypoxic bone marrow microenvironment. It must be remarked that there are striking differences between metabolic pathways that are actives in HSCs and adult differentiated cells. In the former, responses to hypoxia are derived by hypoxia-inducible factors, dimeric Hif-1 and Hif-2, which are oxygen-regulated alpha (Hif-1a and Hif-2a) and beta subunits (such as Hif-1b or Arnt1) that modulate gene expression during adaptation to hypoxia. There is genetic evidence supporting the assumption that deficiency in Hif-1a in HSCs lowered the expression of pyruvate dehydrogenase kinase (Pdk2) that is able to enhance the “against ROS-protective glycolysis.” Hypoxia is recognized as an important factor that regulates stem cell quiescence status and metabolic shift toward energetic metabolism based in glycolysis and pentose phosphate metabolism, instead of oxidative phosphorylation in order to lower ROS generation (Forsyth et al. 2008). Low oxygen level conditioned proliferation and differentiation in ESC, cells that possess an efficient antioxidant mechanism that counteract the effect of variation of oxygen tension. It is noteworthy that when ROS become higher, the pluripotency genes OCT4, Nanog, Tra 1-60, and Sox2 lower its expression as well as differentiation is triggered. Oxygen conditions must be finely syntonized and 2% that is below physiological conditions is recommended as secure for genetic stability and stemness (Forsyth et al. 2008). When cells become differentiated, mitochondrial respiration is resumed, a change that prompts an increase in oxidative stress and higher probabilities for DNA damage and mutations. Thus transition from quiescence and glycolysis toward respiration and ROS generation contributes to mutagenic oxidative lesions in HSCs that foster stem cell genetic instability. As an example, in acute myeloid leukemia cells, mutations in the gene of mitochondrial enzyme isocitrate

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dehydrogenase-2 (IDH2) function as an oncogenic driver (Gezer et al. 2014; Testa et al. 2016). Hypoxia should be considered as a way to limit ROS damage, but also as a condition for development of bad prognosis characteristics in tumor cells. That is in agreement with that literature pointing to activation of the hypoxic response in tumors, mediated through upregulation of hypoxia-inducible transcription factors (HIFs), that are linked to genomic instability, and increased metastatic potential, and tumor stem cell characteristics among other worse phenotypic features (Böğürcü et al. 2018; Tong et al. 2018).

Senescence as a Factor Inducing Stem Cells Genomic Instability Senescence is a stable cellular program that induces growth arrest and result in a distinguishable phenotype, involving chromatin remodeling, metabolism reprogramming, a characteristic inflammatory secretome, and high autophagy. Activation of p16INK4a/Rb and p53/p21CIP1 tumor suppressor genes are determinants for the arrest in cell proliferation so as to impede the possibility of gene transmission to next cell generation. Among the triggers for senescence can be mentioned, DNA damage, mitochondrial dysfunction, telomere shortening, pro-carcinogenic aggressions, metabolic stress, and epigenetic changes. Senescence is a process relevant to tissue homeostasis, cancer, and aging. There can be two kinds of senescence: that which is caused by external causes that is acute and the chronic senescence related to DNA damage and replicative stress. That means that senescence is related to genetic instability in dependence of the kind of unrepaired DNA damage that accumulates and eventually may deteriorate the stem cells therapeutic properties that relies upon tissue cell renewal and secretome paracrine actions (Schmitt 2016; McHugh and Gil 2018; Neri and Borzi 2020). Senescence is related to the action of regulators like mTOR, the serine/threonine kinase that controls metabolism and cell growth according to nutrients, cell energy level, or other sources of stress. mTOR contributes to regulate such processes as nutrient intake, protein and lipid synthesis, autophagy, and the emergence of the senescence-associated secretory phenotype (SASP) that participates in the autocrine and paracrine senescence induction. Senescence is also linked to MAPK p38 activation or induction of p16INK4a, in response to several disturbances already mentioned, like telomere shortening, oxidative stress, oncogene activation, and DNA damage. Another factor that promotes p53 degradation bypassing and senescence are the Sirtuins (SIRT, that will be addressed in the following sections) (McHugh and Gil 2018).

Aging and Genomic Instability in Stem Cells Accumulation of mutations in the whole genome suggest that homeostatic mechanisms worsens during aging (Garinis et al. 2008; Martincorena et al. 2015), that is considered a reason for the greater amount of IPSCs bearing mutations, some of

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those have been found in white blood and skin cells, in genes linked to cancer development (Jaiswal et al. 2014; Genovese et al. 2014). Also in mitochondrial DNA, there have also been shown a mounting number of mutations in aged cells. In iPSCs bearing mitochondrial DNA mutations, the organelle is dysfunctional and provokes further metabolic disturbances (Kang et al. 2016; Wang et al. 2011), and those alterations in the mitochondria propelled further DNA damage in nuclear DNA. Another evidence of the deleterious effect of aging on IPSCs therapeutic efficacy comes from IPSCs obtained from elder mice. These cells with age-associated greater mutation level exhibited less proliferation and reprogramming capacity (Wang et al. 2011). Increase in stem cells age and mutation burden results in deterioration of damage cell responses that affects stress tolerance, tissue and organs functions, and regeneration capacity, all of them disturbances that could lead to cancer and age-related conditions. Mutations may arise in totally stochastic way, accumulating as cell ages. However, there are evidences of reduced checkpoint activities in older cells that often results in exponential increase in mutation frequency. In that sense, aged iPSCs provides a favorable scenario for genomic instability (Burkhalter et al. 2015). Burkhalter et al. (2015) have reviewed the evidences of mutation path from aging to disease. HSCs exhibits a slower DNA repair that contributes to more prevalent mutations. These HSCs mutations arose randomly and are neutral or passenger until a driver co-mutation may increase the fitness of bearing cells. If a second mutation occurs, it may lead to a greater proportion of cells with stable and transmissible changes in their genetic information (Welch et al. 2012). Sequence of phenotype changes associated to mutations reveals that in the first stage, mutations may increase self-renewal, but in the following stages, another driver mutation may help the HSCs to become a leukemic stem cancer cell. This process has been shown also to develop in male germ cells, favoring clonal expansion and the transmission of mutations (Jan and Majeti 2013; Minussi et al. 2021; Reilly and Doulatov 2021). The higher mutation frequency and telomere shortening in older HSCs could be related to DNA repair impairment during quiescence (Beerman et al. 2014; Wang et al. 2014). In HSCs, quiescent DNA damage is not repaired, but when cells reenter cell cycle, HR begins to proceed again, however, as is widely known, if DNA damage is not reversed, the pool of genetically dysfunctional cells is cleared through induction of apoptosis or senescence. It has been observed that DDR are negatively affected during aging process. Nevertheless, there are many unknown areas regarding of DNA repair mechanism during physiological aging that are under scrutiny (Ko et al. 2010). Our knowledge on DNA repair and aging arouse mainly from the study of progeria syndromes DNA damage and cancer. This is an open research avenue that will impact in the study of the aging influence in the genomic stability of stem cells utilized in therapy (Hoeijmakers 2009; Song et al. 2021). Checkpoints are critical for an adequate stem cells function. An example to whom we have previously referred to is the DNA damage detection network that includes both ATM-Chk2 checkpoint kinase and Ataxia telangiectasia and Rad3 related (ATR). These kinases promotes p53 and Rb activity as a part of the DNA damage

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response, whose nature is affected by the damage origin and burden. There are two main results: one is transient, including self-renewal suspension or quiescence, while the other triggers senescence or apoptosis that is a more definitive solution that contribute to remove damaged cells. The amount of stem cell that is required to replenish the tissue/organ that have deleted the injured stem cells depends on the return to quiescence and self-renewal, which need the expression of growth factors. Growth factors induce cell divisions so as to recover stem cell number. Some authors have proposed that removal of damaged cells mediated by p53 must occur in order to guarantee progenitor cell driven renewal, an option that relies on undisturbed naive cells harboring a more preserved genetic endowment (Schoppy et al. 2010; Smith et al. 2010). If the checkpoints ATR or Chk1 are eliminated, stemness is much compromised and a premature aging affects HSCs, and this trouble can also be observed in cells from bone, skin, and small intestine. Absence of CHk1 and ATR allows a mounting number of DNA lesions that accumulates and trigger cell cycle incapacity to progress and provokes apoptosis (Ruzankina et al. 2007; Desmarais et al. 2012). It is evident that a precise tune of the activity of these checkpoints is indispensable. Absence of their function is deleterious while its activity may result in senescence or apoptosis depending of the magnitude of damage in DNA (Shibata et al. 2010; Lossaint et al. 2011). Stem cells aging impairs its replicative capacity, and it is notorious that, during aging, several kinds of stem cells, like satellite and HSCs, stay in quiescence that helps in attaining DNA repair (Moehrle et al. 2015). However, desrepression of p16INK4a in muscle satellite cells induces a senescence-like stage that block stem cells to resume the cycle that is exceedingly affected and delayed (Rossi et al. 2007; Sousa-Victor et al. 2014; Flach et al. 2009). Cell cycle delay results from replication stress linked to chromosome gaps or breaks. Replicative stress is related to lowered expression of a helicase M component that is necessary for adequate assembly of replicative complex and affects progression of replication. On the other hand, DNA damage tolerance (DDT) mechanism is activated which allows replicative fork to bypass DNA lesions that otherwise would impede fork progression. It is necessary to remark the importance of this tolerance alternative, because if DDT fails, replication can be stalled, leading to generation of DSB, lesions that are highly toxic and may result in stem cell genomic instability (Pilzecker et al. 2019). Genomic instability as a result of replicative stress is a fact that must be considered when planning cell harvest for expansion and further use in stem cell therapy. Stem cells suffer injuries in vivo and in vitro, from several sources, like energetic metabolism, and generation of reactive oxygen species, due to inflammation, but also to manipulation, expansion over different matrices, shearing forces among many disturbances that may affect DNA integrity, not only at the beginning of their ex vivo living but alongside their life span in culture. The status of DDR machinery in stem cells and the resulting DNA stress experienced by those cells intended for therapeutic uses is a multivariable, almost random sequence. A situation that makes mandatory not only the standardized for sample selection and cell handling but also for the genomic evaluation previous to use.

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Telomere Damage and Genetic Instability in Stem Cells Telomeres, the chromosome ends, that are abundant in highly repetitive sequences, exert an influence on the whole cell genomic stability. Shortening of telomeres is one of the main features linked to aging (Hastie et al. 1990; Jiang et al. 2007; Smith et al. 2020; Grill and Nandakumar 2020). Moreover, accumulation of DNA damage in telomeres can induce premature aging (Uppuluri et al. 2021). In a progeria condition like Werner syndrome, a helicase dysfunction that favors telomere shortening is related to premature aging. Telomeres extremes are cut of at a rate of 50–100 nucleotides per cell cycle. Those ends are covered by nucleoproteins named shelterin, in order to protect chromosomes terminus from uncontrolled degradation (Henry et al. 2019; Engin and Engin 2021). There are different shelterins: TRF1, TRF2, RAP1, POT1, TIN1, and TIN2, bound to DNA and in a complex that is called shelterin/telosome, and if this protein complex doesn’t work properly, several abnormalities appears in DNA (Liu et al. 2004; Engin and Engin 2021) like fragile sites and replication defects that leads to chromosome fusions and anaphase bridges (Wieczór et al. 2014). Shelterin actively participate in the control of telomere replication and chromosome final shielding. Additionally, DNA damage response after shelterin disruption is also evidence that inadequacy in their function causes genomic instability (Smith et al. 2020; Kabaha and Tzfati 2021). These proteins are affected by oxidative stress, and there are reports on the impairment of shelterin and its association with telomere DNA if it is damaged by oxidation (Opresko et al. 2005). Telomeric oxidative damage has also been implicated in genomic instability that have been linked to protein kinase (p38)p16(Ink4a) (p16) that participates in ROS-induced HSCs senescence (Shao et al. 2011). HSCs show active DNA repair pathways to maintain their stemness. Exonuclease 1 (Exo1) participates in HR, that fails to proceed if this enzyme is inactivated. In quiescent HCSs from mice, HR is not necessary for cell maintenance, relying on NHEJ repair instead. However, after HCSs reenter in cell cycling, the HR dependence from Exo-1 activity, for DNA repair and preservation of HCS, becomes critical (Desai et al. 2014; de Lange 2018). There are reports on exonuclease 1 (Exo1) activity that after telomere shortening as component of telomeric DNA damage response, its failure results in chromosome fusion and checkpoint induction, and this fusion causes expansion of DNA damage, chromosome instability, and probably stem cell senescence (Zhang et al. 2014). Telomerase an enzyme that is present in most of eukaryotes, shows a typical retroviral reverse transcriptase-like protein core and the noncoding human telomerase RNA (hTR). This enzyme limits telomere shortage in stem cells by synthetizing telomere DNA, which protects DNA from critical shortening until number of cell divisions is completed. However, when telomerase is less expressed, it is unable to prevent shortening and affects cell functions like self-renewal in HSCs and IPC (Flores and Blasco 2009; Roake and Artandi 2020). In human cells, telomere shortening reaches critical length that are sensed as DNA damage, a situation that

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triggers the cell machinery that copes with DNA lesions and consequently activates p53/p21-related checkpoints. This results in the interruption of cell cycle progression, triggering telomeres senescence, a response to DNA damage checkpoint activation after DNA damage within telomeres (Liu et al. 2019). Cycle arrest due to DNA damage is known as replicative senescence. Cells undergoing replicative senescence experiments morphological and biochemical changes, such as enlargement or flattening, and a higher expression p21(WAF1) and/or p16(INK4A). Checkpoints like those based in p53 and Rb are responsive to telomere shortening (Sperka et al. 2012; Morsczeck et al. 2019), p53 response may interrupt cell cycle through kinase inhibitor p21 or may trigger apoptosis via induction of Puma factor, so as to eliminate those cells bearing affected telomeres from the stem cell pool that is needed for tissue renovation (Sperka et al. 2011). On the contrary, in spite of that long telomeres can also accumulate damage, their effects are less noxious for stem cells, due to protection afforded by sheltering that avoids checkpoint induction nor triggering of DDR (Fumagalli et al. 2012; Sławińska and Krupa 2021). Human ESCs with shorted telomeres shows a reduced pluripotency (Huang et al. 2011). Telomere length conservation is necessary for stemness, unlimited selfrenewal, and chromosomal stability of stem cells. Telomerase activity is necessary but not the only mechanism to guarantee telomere length. Epigenetic changes and recombination are also relevant during reprogramming and attainment of pluripotency in ESCs and IPSCs (Liu 2017; Wang et al. 2012a, b). It is noteworthy that long telomeres as well as high levels of TRF1, a shelterin, can be considered as markers of stemness. On the other side, it is important to note that higher levels of telomerase, in spite of boosting self-renewal and proliferation, also induce a greater resistance to apoptosis and have been linked to less differentiation capacity (Flores and Blasco 2009). Senescence is a process characterized by proliferative arrest. This contingence could benefit the cell trough tumor suppression or may contribute to tissue repair. As was stated previously DNA damage can initiate senescence; eventually cells increase protein secretion and many of tis proteins are cytokines; the resulting phenotype is termed senescence-associated secretory phenotype (SASP). SASP reflects an outstanding paracrine effects that may induce senescence in other cells. This evidences that senescent cells may spread senescence in their surrounding milieu (Sławińska and Krupa 2021). Induction of differentiation consecutive to DNA damage is a mechanism that have been demonstrated in several cells like HSC when exposed to aging-associated genotoxic stress. An example refers to induction of basic leucine zipper transcription factor, ATF-like (BATF) that promotes lymphoid differentiation in cells exposed to genotoxic stress (Wang et al. 2012a, b). It is as an alternative checkpoint that allows leading with DNA damage in stem cells, like occurs in melanocyte stem cells in the hair follicle, these cells after DNA damage related to ionizing radiation differentiates to melanocytes, a process that may be further enhanced if ATM is deleted (Inomata et al. 2009).

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Aneuploidy as an Expression of Genome Instability Among the causes of aneuploidy in stem cells that compromise their therapeutic usefulness, an important one is the disturbances in telomerase function. In hESCs, the enzyme is constitutively active in order to maintain telomere length, and in the case of iPSCs, telomerase activity is restarted after reprogramming (Marión et al. 2009). Telomere fusion poses a great problem in anaphase because those connected chromosomes may generate anaphase bridges that are impeded to migrate toward opposite cell poles under the mitotic spindle tubules action. Cells remain linked through the bridge that causes aneuploidy because of the illicit gain of a chromosome in one cell whereas the other suffers a loss. Telomeric shortening is a common cause for anaphase bridges that can be broken generating cycles of breakage–fusion, a situation that exhibits proneness to genomic instability and aneuploidy (Tusell et al. 2010; Srinivas et al. 2020; Henry et al. 2019). There are several and sustained evidences that telomere damage may trigger genomic instability expressed as an outburst of chromosomal aberrations as well at the sequence variant level, when those chromosome ends are disturbed, telomeres experience damage and attrition (Henry et al. 2019; Kim et al. 2021). Stem cells centromeres does not function with the same efficiency as those in mature cells, and this characteristic is probably a cause of predisposition to aneuploidy in those cells. Chromosomal aberrations are frequently found in IPSCs in culture, and among the probable causes for those abnormalities are the composition of centromere and kinetochore in ESC and IPSCs. Kinetochore is the critical spot for chromosome attachment that relies on CENP-A, the centromere histone H3 variant, or nucleosome defined chromatin. There are reports on reduced amounts of this centromere component in stem cells that could hamper chromosomal-spindle attachment. During reprogramming, CENP-A is reduced at the beginning of dedifferentiation events leading to IPSCs (Milagre et al. 2020). This acts as an epigenetic regulator of stemness, which in turn favors aneuploidy in pluripotent stem cells. Overexpression of that critical centromeric component occurs in cells with functional p53 that are able to drive cell reprogramming toward senescence, tumorigenesis, or to new centromeres linked to aberrant chromosomes (Ling et al. 2020; Jeffery et al. 2021), which is interpreted as a demonstration that a precise tuning of centromere components exerts a decisive influence in the reprogramming results.

Influence of Genomic Arrangement in the Nucleus on Aneuploidy In the interphase nuclei of differentiated cells, there is an organized distribution of chromosomes in areas termed chromosome territories (CT). Alterations in the intranuclear organization may impact the maintenance of euploidy. Inside human cell nucleus, there is a strict array of CT with predominant gene-rich chromosomal regions placed towards the inner part of nuclei while the gene-poor regions of chromosomes are placed at the nuclear periphery. In spite of some variations, there

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is a less organized intranuclear terrain in the embryonic versus mature developed cells, and the rule based in chromosomal gene enrichment is not always followed. An example of the previous assertion are chromosomes 17 and 19 that are abundant in genes and are found in granulocytes near the nuclear center while in ESCs are placed in the periphery (Bártová et al. 2001). However, more recent reports have found differences in centromeres positioning of 12, 15, 17, and 19 between hESC and mature somatic cells. There is a repositioning process that accompanies cell differentiation; impairment in this mechanism can be invoked as an additional source of genomic instability in stem cells (Bártová et al. 2008). This has been reported to influence several features of heterochromatin, as the presence of immature centromere anchorage regions that in embryos and stem cells may result in aneuploidy due to a defective attachment to the mitotic spindle (Biancotti et al. 2010). The complex machinery for intranuclear topography maintenance also includes nuclear transmembrane proteins that are responsible for gene repositioning, during myogenesis, in order to shift the genes to be repressed to more peripheral positions (Robson et al. 2016; Ahanger et al. 2021). Those peripheral domains are mainly low gene expression regions that contain histone marks of a silenced state (Smith et al. 2021). There is an increasing number of evidences that allows a less restricted nuclear organization in pluripotent cells, hereof increasing the intranuclear plasticity regarding not only to expression and organization but also to genomic instability (Vasudevan et al. 2020; Dhegihan 2021). Aneuploidy is probably an insult to the intranuclear genome organization because of the additional space occupied by the extra chromosome altering the space relationship with the nuclear envelope or other regions. The correct location of extra chromosomes that have been found in mature somatic cells is not the rule in stem cells, where there are less or not lamins A, (that will be discussed next) with a consequential impact on lamina-associated domains, therefore may alter the intranuclear lodging and hence allowing changes in gene expression (Chovanec et al. 2021). Aneuploidy-associated shifts in genome organization and subtypes of topologically associated domains (TAD) upon differentiation is under scrutiny (PhillipsCremins 2014), and is another link in the chain of event connecting the stem cell nuclear environment and aneuploidy proneness and generation of a distressful genomic landscape (Omori et al. 2017). There are also “counter measures” in stem cells against changes in chromosome number relying on DDR and protective proteins. An example of a mechanism against polyploidy is the protein Survivin that control the spindle assembly, checkpoint and cytokinesis; that protein that is protective against aneuploidy, is highly expressed in ESC and contributes to pluripotency (Wiedemuth et al. 2014). Measurement of this protein has been suggested as control for genomic stability in stem cell intended for therapy (Mull et al. 2014).

Lamins Alterations and Aneuploidy Lamins are meshing proteins that are placed covering the inner face of the nuclear membrane, and those proteins are important in the conservation of nuclear

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morphology and chromosomal location within the nucleoplasm. Lamins B1 and B2 are expressed in stem cells as well as in somatic ones. Lamins are necessary for the conservation of nuclear structure as well as the chromosomal allocation and organization (Gruenbaum et al. 2000). Lamins alterations may lead to miss-segregation of chromosomes during cell division, due to its relationships with mitotic spindle assembly and telomere binding to the inner nuclear membrane protein SUN1. That is why lamins are among the factors that may induce aneuploidy in stem cells in the eventuality of disturbances. There is a link between lamins function impairment, telomere damage, and genomic instability (Gonzalo and Eissenberg 2016; Martins et al. 2020; Dantas et al. 2021). The absence of lamins A/C has been suggested to contribute to the ESC nuclei plasticity compared to the more rigid state of somatic cell nuclei, with hESC lacking heterochromatin at the nuclear periphery. Lamin A/C and emerin determine nuclear size and shape, and hence these are contributors to gene regulation and differentiation in the first steps of embryo development (Smith et al. 2017) and to global remodeling of the genome organization during lineage commitment (Peric-Hupkes and van Steensel 2010).

Replicative Stress/Replication Timing as Causes of Genome Instability Replicative stress (RS) is another source of genomic instability in cells undergoing divisions that is present in stem cells. RS main feature is to slow down DNA replication or its complete detention. DNA synthesis can be hampered by several factors as DNA damage, (vb gr) like the inflicted by ROS or when DNA nucleotides or proteins are scarce due to a metabolic impairment RS can also arise (Lo Furno et al. 2016). Among the main causes of RS are the unscheduled restart of cell cycle that provokes interference between DNA replication and transcription but is also frequent during reprogramming into IPSCs. As a consequence of interferences between DNA synthesis and transcription, the replicative fork may lead to under or over DNA replication and also to single- or double-strand DNA breaks. RS is a cause of instability and DDR in stem cells (Lo Furno et al. 2016; Sjakste and Riekstiņa 2021). In ESCs, there is a susceptibility to RS resulting in defective chromosome condensation and segregation, leading to aneuploidy. ESCs cell cycle contains a higher number of cells in S phase, while G1 and G2 are narrowed. The more prevalence of RS in stem cells may be explained by the fact that during DNA synthesis, chromatin is more accessible to remodeling factors (Becker et al. 2006; Lambert and Carr 2013; Lamm and Kerem 2016). There is another relevant aspect to be considered among the aneuploidy leading events, which include the checkpoints for adequate chromosomal segregation, the spindle assembly, and the decatenation checkpoint. The last checkpoint during mitosis is the p53- and Rb-dependent G1 tetraploid checkpoint. The spindle assembly checkpoint has been reported as less efficient in ESCs, a feature that may lead to extra chromosomes. This spindle checkpoint is also weak, favoring association of unrepaired chromosomes. There

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are evidences that points to a lack of spindle assessment checkpoint in stem but not in progenitor cells (Brown et al. 2017; Brown and Geiger 2018). Chromosome decatenation impairment has been found in mouse cells and also in pluripotent human cells. The consequences of bad decatenation are the entanglement of chromosomes that is attributed to the absence of methylation in chromatin silence markers (Meshorer et al. 2006). In hematopoietic stem cells, the levels of Ezh1 histone methyltransferases are progressively reduced during development, and those enzymes are thought to be implicated in methylation of silencing chromatin markers like H3K9 and H3K27 (Hidalgo et al. 2012). The more open heterochromatin in stem cells may be responsible for the greater susceptibility to DNA damage and genome instability observed both in hPSCs and hiIPSCs (Henry et al. 2019). Another emerging key factor that must be addressed regarding genomic stability is the replication timing (RT), a concerted process that control the organization of a relevant array of nuclear events in eukaryotic cells. RT is triggered at certain point during cell cycle and its development is synchronized with changes in genome organization. RT is connected to processes like 3D genome organization, transcription, epigenetic modulation and mutation, and spatial distribution, and is thought to influence replicative stress and consequently genomic stability (Briu et al. 2021). Replication process is strictly timed; in the multiple origin replication process, there are regions that begin to be copied at different moments during S phase. This chronometric control of each region for replication results in the epigenetic signature of the cell genome. This process is cell specific and spans large regions of chromatin, where histone modification and compartment changes, which defined the activation of specific replication origins. Those patterns are inherited to next cell generations. However, if there are inaccurate timing as a result of replicative stress, altered gene expression and genetic instability are among the bad results of RT impairment (Donley and Thayer 2013). In other words, replicative stress may cause genetic instability through modifications in RT. In stem cells, the evidence points to a less strict correlation between histones modification and the precise scheduled RT. However, as long as stem cells differentiate, there is a more tight dependence of RT from histone changes as occurs in mature fully developed cells (Dileep et al. 2019). There is another aspect regarding the effect of increased mutation rate in stem cells in genes involved in the regulation of epigenome as have been reported in functional normal HSCs that evolves to preleukemic and acute myeloid leukemia cells (Corces-Zimmerman et al. 2014). Variation in epigenomic patterns may trigger genomic instability and clonal selection in aged tissues. Epigenetic changes may also be a consequence of mutations, and this fact could be considered as a variant of a mutated phenotype, because the disturbances over epigenome control genes may propagate not as an increase in DNA sequence changes but of altered epigenetic imprinting that at last may lead to genomic instability in a self-propelled process. Another pathway toward aneuploidy can be related to changes in methylation. Human malignancies are related to changes in methylation patterns that include hypermethylation of CpG islands, often related to gene promoters that could silence genes that affect cell cycle, DNA repair, apoptosis, and genes that are frequently

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mutated in cancer cells. These hypermethylated genes that results permanently in on “off” state notably codes for developmental transcription factors that prolonged the stemness and increases the probability of malignant transformation. Hypomethylation linked to cancer may also be present and is known to increase genetic instability but also naïve pluripotency in ESCs (Leitch et al. 2013; Pfeifer 2018). It must be stressed the role chromosome passenger proteins, as Aurora centrosomal kinases or the already mentioned, cycle-associated molecule, survivin, proteins that when dysregulated can induce aneuploidy or tetraploidy (Nguyen and Ravid 2006). In the inner centromere, a membraneless organelle is located, the so-called chromosome passenger complex, that is needed for the organization and regulation of mitotic movement of chromosomes (Trivedi and Stukenberg 2020). There are probable driver genes that may increase the occurrence of chromosomal aberrations and the concern on neoplastic potential of therapeutic candidate stem cells (Ben-David 2015).

Cell Origin as Source of Genome Instability Cell origin for stem cells therapy, including those that are isolated, expanded, and utilized directly or after induction of pluripotency, must be considered as a differential source of instability. There are differences in genetic stability that are inherent to stem cell type, isolation, handling, and culture procedures while other arises from reprogramming factors utilized in the IPSCs obtainment. Peripheral blood mononuclear cells (PBMCs) and hematopoietic stem cells HSCs can be reprogrammed to IPC. There is a large list of sources where cells to be reprogrammed can be obtained like urine, blood, cord blood, amniotic fluid, menstrual blood, teeth pulp, keratinocytes, fibroblasts, among others. These are differential in their tendency toward genetic instability, an example are human dental stem cells that are very unstable, showing around 70% of structural and numerical aberrations like polyploidy, aneuploidy, aberrations that increases during culture (Duailibi et al. 2012; Glicksman 2018). In the next section, evidences for genetic instability in three types of cells whose relevance is out of discussion will be summarized.

Adipose Stem Cells (ASC) Adult stem cells are the most relevant source of cells so as to be used in cell and tissue regenerative therapy, and those stem cells can be obtained with minimum-risk procedures (Miana and González 2018). ASCs are a source of MSCs with therapeutic properties, isolated from the stromal vascular fraction in adipose tissue. One of the main features of ASCs is their greater genetic stability when compared with other stem cell types. Adipose stem cells have been repeatedly considered as those cells that better conserve its genetic stability. ASCs isolated from the stromal vascular fraction are

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free of chromosomal aberration through G banding analysis, after during five passages in culture (Debnath and Chelluri 2019). There are evidences of negative selection of aneuploid clones of ASC that have been in culture for 6 months. A single subclone showed alterations in telomeric and subtelomeric regions at early passage, but were absent after prolonged culture time (Meza-Zepeda et al. 2008). Other studies reinforced the notion that ASC are stable after long culture time. Evaluation of chromosome stability with FISH probes for chromosomes X and 17 yielded 97.8% of diploid cells after 35 population doublings (Grimes et al. 2009). There are also evidences that points to a stable proportion of aneuploid subclones that could be increased in ASC cultures up to 16 passages (7.1%) and further during senescence (19.%), but without acquire tumorigenic capacity as was tested in nude mice (Roemeling-van Rhijn et al. 2013). Emergence of polyploidy may enhance cancer cell proliferation as have been reported in a murine model, where the polyploid ASCs was more efficient promoting breast tumor growth and metastasis than ASCs, derived from visceral adipocytes with a normal karyotype (Fajka-Boja et al. 2020). It must be stressed that quiescence of MSCs favors the generation and accumulation of genetic alterations that may result in genetic instability, especially after genotoxic insults. Genetic instability may induce senescence, apoptosis, and functional impairment that diminish or abolish the therapeutic efficacy of MSCs (Banimohamad-Shotorbani et al. 2020). It is a reason for the development and standardization of procedures intended to limit the exposition to DNA damaging procedures during cell isolation and expansion. Genetic stability is a central aspect that must be considered when discussing therapeutic application of those cells and is one of the reasons besides their accessibility and abundance to consider these cells among the best suitable for medical uses (Miana and Prieto 2018).

Embryonic Stem Cells Human pluripotent stem cells (hPSCs) include hESCs and human induced pluripotent stem cells (hiPSCs). hESCs are prone to acquire focal genomic abnormalities in culture, changes that seems to be nonrandom in origin (Lefort et al. 2009; Yoshihara et al. 2017). When considering the genetic stability in ESC, there is a consensus on the higher prevalence in genetic abnormalities like sub-chromosomal copy number variations (Laurent et al. 2011). However, in murine models, the mutation frequency in mouse embryonic stem cells (mESCs) have been estimated in one hundred percent lower than their mature counterparts from the same source, and this could be interpreted as the consequence of a better DDR mechanism so as to compensate their intrinsic genetic instability (Lo Furno et al. 2016). Blastocyst seems to sacrifice the proofreading functions during DNA synthesis in order to attain a faster cell division. In cultured ESC, there is a shortened G phase and interphase, consequently errors during replication occur more frequently (Henry et al. 2019). The p53 pathway is affected in ESCs, where in spite of the overexpression of p21 RNAm, the P21 protein itself is not produced, hence, this partially abolished

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response permits replication errors to continue (Dolezalova et al. 2012). After DNA damage in ESCs, p53 binds to the pluripotency factor NANOG’s whose inactivation promotes the progress of ESCs to differentiation. Differentiation induction is part of a mechanism intended for the enhancement of genetic stability in ESCs (Lin et al. 2005). It has been reported that p53 knockdown provokes the downregulation of NANOG and Oct4 favoring differentiation. But in the absence of damaged DNA, p53 contributes to maintenance of NANOG expression and hence favors selfrenewal in ESCs. This is an example of the complexity of the relationship between DNA damage, DDR, and stemness in these cells (Abdelalim and Tooyama 2014). Another characteristic is the increase in genetic abnormalities at different resolution levels as the number of passages in culture become higher (Maitra et al. 2005). There is a report on the early development of sub-chromosomal and chromosomal aberrations since the passage number 5, and this result points to a strong genetic instability dependence on the number of passages (Bai et al. 2015). In an excellent study that evaluated several culture conditions on genetic instability, the frequency of genetic aberration increased significantly after 80 passages that were performed mechanically in hESCs and hIPSCs (Garitaonandia et al. 2015). In newly obtained ESC, there are earlier reports on the acquisition of deletions after reprogramming; those abnormalities duplicated its frequency as the number of passages increased (Laurent et al. 2011; Ben-Yosef et al. 2013; Garitaonandia et al. 2015). Human unrestricted somatic stem cells (USSCs) represents a group of adult somatic stem cells CD45-negative population derived from human cord blood, and these cells grow adherently and maintain pluripotency to develop into cells from three layers along the expansion period without spontaneous differentiation. USSCs cultured on chondrogenic medium showed a conserved normal karyotype after expansion to 1015 cells. Those cells have shown to sustain their genetic stability until passage 6, expressed in normal karyotypes and relatively conserved telomere length (Kögler et al. 2004), that is extremely important for pluripotency, selfrenewal, and genomic stability (Liu 2017). USSCs can be grown to make feeder layers for ESC cultures (Keshel et al. 2012). ESCs cultured on conventional feeder layer compared to those grown in feederfree cultures show more genetic stability. Feeder-free culture more often results in aneuploidy, chromosomal or sub-chromosomal mutations. In a pioneer study on ESCs karyotype stability, the modality of feeder-free culture encompassed rapid changes that involved gains in chromosomes 12 and 20. Those aberration appears after passages 17 and 21, as was demonstrated in two lines (HS181and SHEF-3) and a gain in chromosome 14 after passage 10 in the SHEF-3 line. The proportion of cells with trisomy 12 increased during culture time that was interpreted as a selective advantage. The same cells lines grown in feeder cultures remained genetically stable after 185 passages, irrespective if the passage method was mechanical or enzymatic. Surprisingly, SHEF-1 that conserved genetic stability after 185 passages in feeder layer culture did not suffered any chromosomal changes when transferred to a feeder-free culture for 30 additional passages. Different susceptibility between different cell lines must be cautionary considered for ESC as therapeutic candidates (Catalina et al. 2008; Guo et al. 2018).

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In the cell line HS181, other authors have shown that chromosomal aberrations appeared in all the cells that became adapted to growth directly on plastic surface. Chromosome X trisomy, deletions in 7q11.2 and I (12) p10 were the nonrandom aberration detected. However, the greater survival and growth in feeder-free conditions was accompanied by reduction in pluripotency (Imreh et al. 2006). Anyway, the ability exhibited of certain cells bearing trisomies to properly differentiate is a real warning issue, because this aneuploidy persistence poses a risk for tumor development if those cells able to thrive and differentiate, are inoculated as a therapy procedure (Zhang et al. 2016; Bach et al. 2019). Similar results with feeder layers points to a protective effect of culture, upon feeder layers or extracellular matrix (ECM) proteins, on karyotype stability (Escobedo-Lucea and Stojkovic 2010; Lee et al. 2012) not only for ESC but also in IPSCs (Nakagawa et al. 2014; Ghasemi-Dehkordi et al. 2015), mESCs cultured on feeder layers made of hUSSCs maintain normal karyotype (Keshel et al. 2012). Feeder layer made up of bone marrow MSCs maintained the adequate phenotype in ESCs and absence of aneuploidy (Lee et al. 2012). The use of smaller feeder layer like the microdrop culture method have been applied for the generation of bovine ESCs with favorable results regarding pluripotency and the conservation of a normal karyotype (Kim et al. 2012a, b). In murine models, mouse stem cells maintain its genomic integrity during long-term cultures on a feeder of mouse embryonic fibroblasts derived matrices (MEFDMs) but non when cultured on gelatin-coated substrates (Kim et al. 2012a, b; Sthanam et al. 2017). There is a recent report of bovine stem cells that with the introduction of a defined culture substrate (N2B27) have been able to maintain a normal genetic constitution over 35 passages without the use of mouse embryonic fibroblasts feeder layers. It is a demonstration that the use of feeder layers with its potential inconveniences can be circumvented with more refined approaches in the culture conditions that include defined medium like N2B27 (Soto et al. 2021). Feeder layers favors genetic stability but this goal can be reached even without that. The passage methods as have been already mentioned is another source of karyotype changes In ESC, the impact of mechanical procedure compared to that of enzymatic treatment remain controversial. Some authors have reported that irrespective of the techniques employed, genetic instability may be detected (International Stem Cell Initiative 2011; Tosca et al. 2015). Cell passage in culture based enzymatic detachment is related to greater genetic instability when compared with the mechanical one (Garitaonandia et al. 2015). However, the use of a mixture of proteolytic enzymes, including both trypsin and collagenase activity (Accutase), does not require inactivation, hence reducing handling, and did not induce abnormal karyotype in ESCs cultured under feeder-free conditions (Kim et al. 2012a, b). In this respect, there are contradictory reports on the beneficial effects of Accutase use in cell passages. As an example, when ESCs and IPSCs were evaluated over 100 passages, those cells cultured showed deletions involving p53 locus and less activation of p53 downstream genes. But in spite of the use of Accutase-based passage, deletions were larger and appeared earlier than that in mechanical passages. Duplications and deletion involving chromosomes 20, 12, and 17 were consistently detected with a higher frequency in ESCs and IPSCs that undergo enzymatic

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passage, and karyotype mosaicism was also detected (Holm et al. 2013; Garitaonandia et al. 2015; Amir et al. 2017). Even when compared with the less harmful Accutase, the mechanical cell detachment is the best choice against the induction of genetic instability. Even with this supposedly less harmful passage procedure, a relationship between passage number and p53 mutations is noteworthy (Holm et al. 2013; Amir et al. 2017). Involvement of p53 mutations in ESCs genetic disturbances in culture was also demonstrated when 5 cell lines resulted positive for p53 dominant negative mutations, afterward when 140 lines were analyzed, from that mutated, the allelic fraction in 3 cell lines increased its prevalence to more than 50%, signifying what was interpreted as selective advantage for p53 mutations bearing cells. The authors stressed the fact that hPCS have been used in spite of containing cancer mutations in p53 (Merkle et al. 2017). Mutations in p53 were detected in cells cultured with several media, substrates, and using a variety of passage procedures. That is interpreted as evidence in the sense that current culture conditions exert a positive pressure for p53 mutations. This gene should be in the focus for the analysis of genetic adequacy of stem cell lines for therapy especially when culture goes through any intervention involving the existence of bottle neck population period, when the mutation may affect a greater proportion of cell populations (Merkle et al. 2017). In hESCs, the function of p53 is uncertain; some evidences support the notion that p53 is responsive to DNA damage as occurs in adult cells, while others have shown the absence of downstream activation of genes like p21 that build up the DNA damage response. An alternative effect of genotoxic insult is characterized by apoptotic induction that relies on p53 upregulation and relocation in mitochondria, without involvement of typical genes from p53 networks that are activated if the damaged cells are induced toward differentiation (Tichy 2011). In mESCs, cell cycle is shorter with a predominance of cells in S phase, and those cells lack G1/S checkpoint. Chk2 that phosphorylates and stabilizes p53 also acts over the cycle regulator phosphatase Cdc25a that cannot remove phosphate from Cdk2. This phosphorylated state make cell unable to proceed into S phase. However, in mESCs, miss localization of p53 does not favor Cdc25 abrogation and damaged cell may enter S phase and continue the cycle. In mESCs, there is no G1 checkpoint. However, in human ESCs, the ability of p53 to activate a DDR response and G1/S checkpoint depends on the nature of the genotoxic stimuli, and it has been shown to respond when cells are exposed to UV but not against ionizing radiation (Tichy 2011). Human ESCs, human nuclear transfer ESCs parthenogenetic hESCs, and iPSCs comprise the hPSCs that are suitable for therapeutic applications and also show susceptibility to genetic and epigenetic instability that makes mandatory for in-depth evaluation before their use (Simonson et al. 2015). Aneuploidies in hPSCs involves chromosomes whose numerical aberrations are not only incompatible with life but also resembles that chromosomal gains and losses found in human embryonic carcinoma cells, in what can be interpreted as a hint for their tumorigenic potential (Peterson and Loring 2014). However, there are results that points to the fact that the use of cells from secured sources is an example of how the genetic instability can be

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restricted in dependence of good manufacturing practice (GMP) for the obtainment of standardized ESCs. There are experiences with ESCs that after in-depth evaluation of its genetic stability did not express variations at chromosomal or sub-chromosomal levels neither gained mutations in culture (Canham et al. 2015). Duplications and deletion involving chromosomes 20, 12, and 17 were consistently detected. Additionally, chromosome 12p duplication is known to bear the loci for the pseudogene NANOGP1 and NANOG gene, a known pluripotency marker is also a hallmark for teratocarcinoma. Chromosome 20 duplication is associated with higher expression of Bcl-xL that may drive survival advantages (Nguyen et al. 2014; Clark et al. 2004). Base excision repair is active in ESC and IPC with high activity irrespective of the parental source from which they were derived. Regarding DSB repair, both stem cells show more active high-fidelity HR, which is a requisite for the conservation of pluripotency and the ability to develop a complete organism, while upon differentiation, they begin to rely on error-prone NHEJ (Tichy 2011; Tilgner et al. 2013; Zhang et al. 2018a, b). Lest say that the accuracy of DNA repair is decreasing along with the greater differentiation stage.

Induced Pluripotent Stem Cells IPSCs were obtained, as we mentioned before, through the introduction of genes or reprogramming factors with the purpose of reversing their development program to resemble ESCs. IPSCs are known for their propensity to aneuploidy, flaw that is more evident after a long culture period. When genetic instability of IPSCs is analyzed it is often difficult to stablish a a clear separation from the instability that is inherent to ESCs. Those cells, in spite of their differences, exhibit a high degree of aneuploidy. IPSCs are originated in blastocysts where aneuploidy is frequent. There is evidence for auto-correction of aneuploidy in ESCs, but the process is still poorly understood (Bazrgar et al. 2013). Developed ESCs exhibits mainly chromosomal gains in contrast to that of blastocysts which experienced both losses and gains, those ESCs chromosomal gains seems to be related to selective advantages. There are three main causes for genetic alterations in IPSCs: progenitor cells with preexisting mutations, DNA changes that arise during reprogramming, and those mutations that appears de novo during proliferation in culture (Tichy 2011). There are another causes of aneuploidy like supernumerary centrosomes that are often found in hESCs derived from human zygotes and cleaved embryos that aroused from disturbed fertilization, characteristically, the proportion of hESCs bearing the anomalies is lower than that in their earlier progenitor (Gu et al. 2016). IPSCs are very similar to ESCs but are not identical; those are produced departing from cells treated in a way that their gene expression is altered, as well as its development route that changes the original cell into another cell type. Procedures to develop IPCs are nuclear transfer, cell fusion, or transcription-factor transduction. In the last approach, IPSCs from reprogramming is a consequence of unbalance

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stoichiometry of the transcriptional regulators present in the cell through transfection of reprogramming genes that surpassed a threshold needed for the conversion to a pluripotent cell (Amps et al. 2011; Tichy 2011; Henry et al. 2019). There is growing evidence that genetic stability and pluripotency are regulated by transcriptional changes in differentiated and undifferentiated states, and is known that iPSCs are extremely prone to genetic instability, a problem that is favored by inefficient reprogramming (Paniza et al. 2020). In other words, the fidelity of genomic reprogramming is affected due to the persistence of “epigenetic memory” that reflex the fact that the epigenetics marks, inherited from the progenitor cells (Tichy 2011; Basu and Tiwari 2021). These epigenetic pattern that extended beyond reprogramming into the life of iPSCs may affect genes responsible for the maintenance of DNA integrity and euploidy. Those epigenetics modifiers are microRNA, regulators of DNA methylation, histone chemical marks, and ATP-dependent chromatin remodelers (Simonsson and Gurdon 2004; Hassani et al. 2019). There are positive and negative feedbacks, between DNA sequences expression and those modifiers, in a way that have been compared with that of the Operon-lac, described in prokaryotes. Maintenance of pluripotency also depends on those feedback loops between epigenetic landscape of DNA sequences and pluripotency genes, like the Yamanaka factors. Persistence of epigenetic memory is considered as an important contributor to genetic and epigenetic disturbances in iPSCs (Yamanaka and Blau 2010; Tichy 2011; Di Giammartino and Apostolou 2016; Pelham-Webb et al. 2020). However, other authors have not found a relationship between methylation patterns and genetic instability in IPSCs. Their results pointed to inefficient reprogramming as a cause, leading to incorrect DNA replication and replicative stress, and the evidence reveals impaired DNA replication with less origins and a high frequency of strand breaks in iPSCs (Jekaterina et al. 2020; Paniza et al. 2020). IPSCs reprogrammed carries a mutational burden accumulated during the previous somatic cell life besides the fact the event of reprogramming causes a severe disturbances in DNA chromatin homeostasis. The proportion of cells that experienced a correct reprogramming is generally low and interestingly may be increased if p53 is inactive, and this poses a greater risk for iatrogenic induction of a tumor, if cells reprogrammed for therapy eluded the response against DNA damage and mutation (Tichy et al. 2011; Amir et al. 2017). There are evidences suggesting that iPSCs genetic stability is poor, because of a lack of generation of an adequate DDR, in spite of the elevation of expression of DNA repair genes. A dissociation between expression and production of DNA repair proteins have been invoked as was already mentioned in reference to p21. There is strong evidence that genomic stability is much lower in iPSCs than in ESCs due to less DNA repair fidelity (Zhang et al. 2018a, b). The mutation frequency exhibited by ESCs is minor than that in adult somatic cells and even lower than IPSCs, it could be a reflection of the more efficient mechanism that evolved in embryonic cells, whose correct functioning is essential for the development of the embryonal tissues and organs. The fact that mutations could be transmitted to the next generation places the mechanism for DNA damage detection, repair, or amelioration in ESCs as a safeguard of the genetic integrity of the species (Adiga et al. 2010). However, both cell types exhibited a combination of

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additional mechanisms against genotoxic insults, a hyperactive and rapid DNA repair, elimination of cells with damaged DNA and differentiation in order to withdraw the mutated cell from the stem cell compartment. Another defense mechanism in ESC and IPSCs is based on lowering the number of mitochondria in order to limit the reactive oxygen generation (one of the main causes of endogenous DNA damage), while genes for antioxidants become overexpressed (Forsyth et al. 2008; Maraldi et al. 2021).

Effect of Reprogramming Methods on Genetic Instability Reprogramming based upon virally integration of the factors is also an event of insertional mutagenesis that elicit several abnormalities in the recipient genome; moreover, some of the reprogramming factor expressed in the IPSCs provokes genetic disturbances like CNVs, changes in the regulation of imprinted genes, point mutations, wrong methylation patterns, as well as chromosomal aberrations (Kang et al. 2015). Those are enough reasons to sustain and justify the search for non-integration based reprogramming procedures. Among those methods that do not rely on the integration of foreign sequences are: episomal vectors, plasmids transfection, vectors derived from Sendai virus, adenoviral vectors, synthetic mRNA, miRNA, plasmid transfection, minicircle vectors, transposon vectors, liposomal magnetofection, protein transduction, and small molecules. The non-integrating reprogramming procedures are less efficient and limited to certain types of cells like fibroblasts, but their impact on genetic instability in newly developed IPSCs is substantially minor (Goh et al. 2013; Steichen et al. 2014; Beltran et al. 2020; Schlaeger 2018; Haridhasapavalan et al. 2019). IPSCs are the basis of a strategy to obtain an unlimited source of renewable cells to be utilized in autologous transplants. These cells, however, show a high degree of genetic instability that is related to several causes; among them, the reprogramming procedure is one of undeniable importance. hiPSCs genetic stability is mostly influenced by the reprogramming method, which may be integrating vector-dependent and vector-independent methods. It is accepted that integrated methods causes more genetic instability as expressed in CNV, SNP, and mosaicism. A study of genetic aberrations in ESCs, progenitor cell lines, and IPSCs line versus IPSCs non-integrating lines through Affymetrix Cytoscan HD array resulted in CNV 20 times larger in average than those found in non-integrating IPSCs lines. CNV, SNP, and mosaicism were more abundant in integrating IPSCs than in progenitor cells, ESCs, and non-integrating iPSCs lines (Kang et al. 2015). However, there is another view point. Some researchers have found at least two CNV in progenitor cells, similar to those present in the reprogrammed iPSCs, and it is interpreted as the preexistence of low level CNV mosaicism in progenitor cells that are more often detected in iPSCs, due to their clonal expansion that lead to CNV enrichment and not necessarily new CNV linked to reprogramming (Abyzov et al. 2012).

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Mutations Induced During Reprogramming In a study that involved 22 hIPSCs reprogrammed with different procedures, 5 mutations were detected, some of them involving genes that participates in cancer development. The wide exome sequencing yielded six mutations per exome. Four new random mutations appeared as culture time was longer. Some mutations were originated during reprogramming while others, approximately the half, were preexistent in progenitor cells although at low frequencies (Gore et al. 2011). On the contrary, some potentially lethal mutations were only present during limited periods that are related to differentiation process (Fischer et al. 2012). It have been demonstrated that in early cultures of both hESCs and hiPSCs, deletions and loss of heterozygosity (LOH) are generated with a high frequency while duplications are more susceptible to occur during long-term culture. The type of genetic changes seems to be related to derivation process (Ben-Yosef et al. 2014). There are several studies that have proven that reprogramming is in itself a procedure that causes mutations and chromosomal aberrations in the iPSCs (Sugiura et al. 2014; Liu et al. 2020). Different strategies were adopted in order to dissect the precise origin of those mutations. Most of mutations arose early during reprogramming and were not clonal from a parental origin. The effect of reprogramming procedures, especially those that are integrative, is recognized as risky and poses a severe hurdle on the therapeutic used of iPSCs. Those facts have fueled the research to establish protocols that minimize the reprogramming related mutations in IPSCs (Yamanaka and Blau 2010; Haridhasapavalan et al. 2019; Yoshihara et al. 2019; Schaefer et al. 2020). In spite of the fact that there are mutations that exist prior to reprogramming in the starting progenitor cells, there are others that appear during reprogramming process and afterwards in culture. These mutations can be selected positively or negatively. The allegedly function of those mutations is that they could be involved in reprogramming process itself but unfortunately those changes also could be related to cancers, with which mutation pattern are similar to reprogramming related mutations (Gore et al. 2011). The fact that insertional based reprogramming procedures often induce de novo mutations, that not necessarily are negatively selected in culture, has stimulated the search for alternative reprogramming methods, such as those based on expression plasmids, Sendai virus vectors, and episomal plasmid vectors. An array of DNA-free reprogramming methods, including protein-based, mRNA-based methods, have also been developed (Yamanaka and Blau 2010; Basu and Tiwari 2021). The relationship between reprogramming and generation of mutations is also thought not as a consequence of the introduction of reprogramming factors, but to the process of integration in the genome of receptor cell. There is a growing number of reports on that line. The nuclear transfer procedure in mouse embryonic and tailtip fibroblasts yielded fewer mutations than those that resulted after retroviral transduction. Nuclear transfer generated 80% less mutations than retroviral transduction (Araki et al. 2017). Integrative methods induces more mutations but there are differences in their mutagenicity, like that based in Sendai versus Lentivirus, the

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alternatives within integrative methods is also opened (Sobol et al. 2015). Moreover, there are voices in dissidence that considers that reprogramming method does not affect gene expression in iPSCs (Trevisan et al. 2017). Besides the circumstance that reprogramming methods exerts an influence over the genetic and epigenetic stability in iPSCs, there is also another particular issue that must be addressed: the reprogramming factors or genes itself. Reprogramming factors, currently in use like OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 are involved in several malignancies as drivers of tumorigenesis and biomarkers of cancer severity and evolution. Reprogramming factors are at the same time oncogenes, inducers of replicative stress and genomic instability in resultant iPSCs, and this is particularly relevant when c-Myc is included as a reprogramming factor (Pasi et al. 2011; Jeter et al. 2015; Kuo et al. 2016; Lamm et al. 2016; van Schaijik et al. 2018). Shimada et al. (2019) exposed to ionizing radiation IPSCs to characterize transcriptional changes leading to pluripotency and genome stability maintenance under genotoxic pressure. The endpoints were DNA DSB repair, cell cycle checkpoints, and apoptosis in fibroblasts, iPSCs, and neural progenitor cells (NPCs) derived from iPSCs. DSB were repaired more efficiently in fibroblasts, followed by neural progenitor, while in IPCs remained a higher percentage of DSB, as was demonstrated through detection γ-H2AX foci. In IPSCs, apoptosis-related genes like p53, CASP, and BID were overexpressed as well as apoptosis markers increased. Those results points to a relevant role of apoptosis in IPCs, erasing DNA damaged cells, contributing in that way to genomic maintenance of undifferentiated cell populations exposed to genotoxic insults. Not only those genes that constitutes reprogramming factors have exerted different degree of influence in the resulting IPCs. Genes involved in remodeling of nucleosomes through deacetylation like Mbd3 are determinant in reprogramming efficiency (Jaffer et al. 2018). Another is CHK1 whose overexpression increases the efficiency of the procedure and the oocyte factor Zscan 4 that contributes to maintaining genomic stability, limiting sister chromatid exchanges and promoting telomere elongation in mESCs. Genes like developmental pluripotency-associated protein-3 (DPPA3) and (NPM2) nucleoplasmin-2 that are involved in Zscan4 regulation and stemness maintenance cooperate when co-transfected with Yamanaka factors in the genetic stabilization role in murine embryo fibroblasts (Jiang et al. 2013). Another relevant group of genes that are determinant in the success of reprogramming belongs to DNA repair mechanism, like XPA from NER, Brca1, Brca2, and Rad51 (González et al. 2013), that can be interpreted as an evidence of the need of an intact HR mechanism during reprogramming. Deficiencies in DSB NHEJ repair causes both a reduction in reprogramming efficiency and increases genomic instability. Functional impairment of ligase IV (LIG4) and Cernunnos/XLF) that participate in NHEJ also sustains the role of DDR in the reprogramming (Tilgner et al. 2013; Turinetto et al. 2017). Sirtuins (SIRT), a group of type III histone deacetylases that change chromatin conformation in specific sites, have been invoked as participants in DSB repair.

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There is a link between SIRT function and poly(ADP-ribosylation) in cells with damaged DNA. There are reports on the effect of SIRT inhibition that results in a very profound reduction in DNA-PK-independent nonhomologous end joining, a backup pathway (B-NHEJ). NHEJ is active in stem cells and the fact that SIRT inhibitions reduces this activity is in concordance with those reports about SIRT expression as relevant for the genetic stability in stem cells (Wojewódzka et al. 2007; Chen et al. 2017; Fang et al. 2019; Jeske et al. 2021). In line with the previous assertion, SIRT1 participates in the regulation of mitochondria activity in ESC (Ou et al. 2014), and consequently in their ROS production and ability to damage DNA. NAD+ levels are reduced in aging mice that in turns reduces SIRT1 function and decreased NAD+. The lowered levels of NAD+ inhibits PARP1 because a protein DBC1 that must combine with NAD+, but if NAD+ it is reduced, DBC1 is favored to combine with PARP1, that diminish its activity resulting in more DNA damage. In this circumstances, both NHEJ and HR are also reduced. The so-called NAD+/ PARP1/SIRT1 axis explains a connection between NAD+ levels and DNA damage. These links, that are relevant to explain age-related changes in the ability to respond to genotoxic insults, bring another two molecules that are involved in genetic stability in stem cells, NAD+ and SIRT family (Mendelsohn and Larrick 2017). In human MSCs, mitochondrial NAD+ restoration delays aging and replicative senescence in culture (Son et al. 2016). Moreover, SIRT have been demonstrated as a factor that alters IPSCs reprogramming through metabolic changes in the cells. This is a gene family that may be amenable for modulation in order to improve reprogramming (Shin et al. 2018). Another family of enzymes that is crucially involved in stem cell renewal, proliferation, and differentiation is that of NADPH oxidases, which mediate several basic cell processes trough modulation of redox status in embryo cell in their natural environment and also in culture expansion for therapy purposes. Oxidative stress and antioxidant capacities are antagonists in the delicate oxidative homeostasis; NOX enzymes may modulate protein activities through ROS signaling that changes stem cell main characteristics that are relevant for their clinical applications, i.e., stemness, homing, and genetic stability (Maraldi et al. 2021). NADPH oxidases, like type NOX4, are present in mitochondrial membrane, the endoplasmic reticulum, and nuclear membrane (nNOX), the last modify the expression of stemness factors Oct4, SSEA-4, and Sox2, and modulates DNA damage (Maraldi et al. 2021).

Culture Conditions, the Passage Number Stem cells are not abundant enough to be utilized straightforward in therapy, so they need to be expanded in culture. Culture comprises an array of steps and conditions that make each one a probable source of heterogeneity and further variation. Donor’s individual characteristics, cell origin, culture medium and supplements, oxygen pressure and different protocols for cell detachment, passages, and environmental radiation levels, are factors that could be summarized as a need for greater

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standardization of the whole process from cells isolation to transplantation. This shortage should be expected in a growing science field, where the development of consensus protocols is urgently needed in order to limit variability and contribute to achieve more homogeneous therapeutic cells. Culture conditions have been invoked consistently as a main contributor to genetic instability. Different types of stem cells are prone to exhibits and increase in chromosomal and sub-chromosomal abnormalities (CNV, indels), aneuploidies, and de novo mutations. Culture as a necessary procedure for cell expansion involves critical steps that may affect genetic stability, among them there are the culture medium composition, including the option of defined mediums, the normoxic or hypoxic conditions, the freeze-thaw and the passage techniques, the addition of substances that may affect DNA repair capacity, the presence or not of feeder layers, and the passage number among the more relevant (Mitalipova et al. 2005; Narva et al. 2010; Di Stefano et al. 2018; Henry 2019). Passage procedure involving singlecell or small-clump passaging is considered as relevant source of genetic changes (Bai et al. 2015). Karyotype abnormalities and copy number variations does not only occur during long-term cultures. Those genomic changes may appear early within a number as low as five passages in hESCs. Those alterations are detected after enzymatic treatment for cells detachment, as was already mentioned; enzymatic digestion induces genomic instability in hPSCs, even at the first passages. Sub-chromosomal abnormalities arise earlier than those revealed in the karyotype and were associated with higher frequency of DSB (Bai et al. 2015; Assou et al. 2020). Amniotic fluid-derived MSCs are less prone to genetic damage than BM-hMSCs, and one of the reason is the better repair response to DNA damage in the former after the same genotoxic challenge (Alessio et al. 2018). A report on genetic instability in human ASC under normoxic (21% oxygen) and hypoxic conditions (1% oxygen) points to a greater susceptibility of ASC versus BM-hMSCs to impaired DNA repair as evaluated by γH2AX signaling, the authors recommended hypoxic as the normalized culture conditions for BM-hMSCs (Bigot et al. 2015). Oxygen conditions in culture should be maintained hypoxic, but the best choice must be to match the gas pressure to the cell type. Stability in the oxygen flow must be secured in order to limit those fluctuations that is accompanied by ROS generation and DNA damage (Goto et al. 2014; Oliveira et al. 2014; Testa et al. 2016). There are proposals for enhancement of culture conditions through development of standardized platforms based in more efficient bioreactors at a greater production scale that should result in lower number of passages. Development of defined culture media that overcome the troubles associated to chromosomes abnormalities that are more often found when serum free or poorly defined media is utilized. The presence of genotoxicants during culture is been addressed (Talib and Shepard 2020). Along the chapter we have mentioned those aspects relating culture conditions and genetic instability, and hence in this section, we will focus on the proposed solutions that addressed the enhancement of genetic instability trough standardization of culture, media components, manipulation, and even selection of progenitor original cells in order to favor the homogeneity as well as stability.

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There have been proposed that cells to be used for therapy must be restrained to the first passage number, according to specific cell type, and culture media. The proposed passage number that should guarantee greater safety regarding genetic stability ranged from two to six for hESC (Merkle et al. 2017), to four passages in BM-hMSCs (Binato et al. 2013), and also four passages in MS cultured in DMEM (Yang et al. 2018). Another group found the limit for BM-hMSCs in passage three (Zhang et al. 2007). Greater stability in long-term culture have been reported for BM and adiposederived stem cells that remained genetically stable up to passage 20 in α-MEM/20% FBS (Izadpanah et al. 2008). There is another report on the suitability for BM-hMSCs that until ten passages conserved karyotype stability as well as their secretory ability, needed for their paracrine effects (Choi et al. 2010). There is a lack of consistence between different groups in finding a passage threshold of security, for instance in another work with the same cell type resulted only in two passages secure interval (Borgonovo et al. 2014). Similar results have been reported by von Bahr et al. (2012), regarding also the therapeutic efficiency declining after two passages for BM-hMSCs. ASCs yield from adipose tissue are about 500-fold times greater than from bone marrow, and this allows less expansion culture time for adipose-derived stem cells, which is an important advantage (Jeske et al. 2021). Additionally, ASC can be maintained in culture during greater time periods with less risks of decay in therapeutic properties (Danisovic et al. 2017; Jeske et al. 2021). ASC cultured during periods of 6 months maintained their genome stability evaluated through in deep high-resolution techniques, and interestingly the authors remarks that in a negligible subpopulation, certain aberrations arose in early culture, but were further eliminated, even reducing the already low probable risk (Meza-Zepeda et al. 2008). Moreover, in the search of the secure number of passages for therapy use, there are reports of adipose tissue-derived mesenchymal stem cells that shows chromosomal stability, even after scrutinized through high-resolution karyotyping, until passage five, when cultured in low-glucose Dulbecco’s modified eagle medium (L-DMEM) (Debnath et al. 2019). ASC respond with less senescence during culture, a feature that is considered as a result of higher sensitivity in NAD+ –SIRT axis (Jeske et al. 2021). Adipose-derived stem cells remain genetically stable over long periods in culture as well as keeping their pluripotency, differentiation capacities, and immunomodulatory properties; characteristics that make ASC a central protagonist in cell-based therapy (Grimes et al. 2009; Blázquez-Prunera et al. 2017; Patrikoski et al. 2019). Human umbilical cord derived MSC (hUC-MSCs) experienced changes in expression involving genes related to chromosome stability and segregation altogether with senescence morphology after passage number 15 in DMEN 10% FCS, those changes were paralleled with decreasing differentiation and regeneration abilities (Zhuang et al. 2015). Other authors report that (hUC-MSCs) are massively selected since the beginning of culture expansion, a process that results in a more homogeneous population (Selich et al. 2016), exhibiting a greater fitness. However, there is a consistency in the results indicating that mesenchymal stem cells isolated

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from adipose tissue results in more genetically stable cells than their BM counterparts. In a murine model, among the molecular features that could explain the differences between adipose and bone marrow derived cells, is significant the reduction in H-19 noncoding RNA in BM-derived cells compared with ASCs. This difference is considered as determinant to the proneness to polyploidy exhibited by BM-derived mesenchymal stem cells. In ASCs, there is also an involvement of p53 protein that is less active in BM-derived stem cells in keeping ASC diploidy (Ravid et al. 2014). Given the intrinsic nature of variations in progenitor cells and during culture, the choice should be aimed toward a cultured cell, whose amount and nature of genetic changes does not compromise their therapeutic safety, and not to the total absence of genetic changes. This is a risky statement due to the stochastic nature of the previous changes that may lead to oncogene activation or LOH as well as to mutations compromising the differentiation and proliferation cell fate. A parallel, more active, approach includes the use of mitogen-activated protein kinase (MAPK/ERK) inhibitors in stem cell culture. Inactivation of MAPK pathway with the MEK1/2 inhibitor PD0325901 often results in genetic instability in mouse cells. However, titration of this inhibitor from 1 μM to 0.3–0.4 μM or replacing it with another one may restore the epigenetic and genomic stability of mouse ESC, as well as their differentiation capacities. MAPK inhibitors are another alternative to be considered in the search for genome stability in stem cell culture (Di Stefano et al. 2018).

The Bystander Effect The paracrine effect of an aneuploid cell over the surrounding cells through exosome is known as bystander effect. One relevant example regarding culture procedure is the influence over basement membrane of feeder cells, exerted by mitomycin C. This phenomenon is considered as a contributor to the induction of chromosome aberration in untreated neighboring cells. The bystander effect is under scrutiny so as to establish the contribution to aneuploidy induction by contiguity of stem versus feeder cells in different culture conditions. Exposition to exosomes have been correlated to telomere shortening in nonirradiated human epithelial cancer cells, when exposed to irradiated conditioned medium (ICM) from irradiated ones, a phenomenon that increased with the passage number. In the progeny of non-gamma irradiated HSCs, from CBA/Ca mice, exposed to ICM, chromosomal instability similar to that of irradiated cells have been induced due to bystander effect (Lorimore et al. 2005). Exosomes from irradiated cells are proven responsible for bystander telomere effects. Proteins and RNA contained in exosomes have been found responsible for telomere-induced instability in nearby cell (Jella et al. 2014; Al-Mayah et al. 2017). Microvesicles have been previously identified as vectors for bystander effect between irradiated and nonirradiated human keratinocytes. Both exosomes (30–100 nm) and microvesicles (>100 nm) are involved in the transport of such molecules that induces

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genetic instability in non-treated cells (Jella et al. 2014). Intercellular communication junction are also proven as passage ways for instability factors between cells like products resultant from oxidation (Klammer et al. 2015; Henry et al. 2019). There is a consensus respect to bystander effect as a factor that is relevant to maintenance of genetic stability in cells that are cultured in the presence of other that have been exposed to genotoxicants (Henry et al. 2019).

Instability Factors Related to Donors Obesity In MSCs obtained from adipose deposits in obese mice under oxidative stress produces an increase in DNA damage that may cause telomere shortening and activates genotoxic checkpoints. Oxidative stress linked to adipose inflammation compromise the expression of stemness genes and provokes more senescence and apoptosis. In ASCs obtained from old obese subjects, there is more DNA damage, less renewal, and therapeutic potential due to secretome impairment (Alessio et al. 2020). Similar results were obtained when obese diabetic subjects from India and nonobese control served as donors of hADSCs so as to evaluate the on ASDC functions. Proliferation was reduced, expression of pluripotency genes was altered as well as diminishing osteogenesis (Rawal et al. 2020). Resistin, an adipokine that is increased in obesity, known for favoring insulin resistance, have been recently linked with lowering of the stemness of in cultured hADSCs from a healthy subject. Exposition to resistin obstacles differentiation to adipogenesis and reduced expression of several genes including CCAAT/enhancerbinding proteins (C/EBP)α and adiponectin in adipocytes and affect SIRT levels in derived in osteocytes (Rawal et al. 2021). Under hypoxic conditions, there have been observed a decrease in expression of DNA repair genes and consequently microsatellite instability. A reduction in the number of mitochondria accompanied with less production of ATP, simultaneously, a decrease in ATP that is independent from of O2 concentration or number of cell passages. Several point mutations, including some in a wide range of tumors, were observed. This is one in many reports on the negative effect of hypoxia in stem cell genomic instability (Zhang et al. 2018a, b; Kaplan and Glazer 2020). Donor Age Senescence is a cell response to aging that is characterized by exhaustion of stem cells and chronic inflammation. However, there are considered coordinated processes that guarantee protein homeostasis. This processes include: synthesis, folding, ubiquitination, and proteasome degradation, and a disruption in this network results in toxic damaged proteins. Also a senescence hallmark is the alterations in nutrient signaling and sensitivity to mitogens that is crucial in cell growth regulation. Cell growth is controlled by two opposite pathways, one that is anabolic called target of rapamycin complex I (mTORC1) and the alternative is the autophagy pathway that is catabolic.

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mTORC1 reacts to several types of stimuli that integrates growth as a response for a multiplicity of metabolic situations, and among those effectors are growth factors, adenosine triphosphate, oncogenes, amino acids, and ROS. Senescence includes the breakdown of the control of genome, protein homeostasis, and nutrient sensing related growth (López-Otín et al. 2013; Carroll and Korolchuk 2018). Age-associated genome instability in stem cells is linked to mutations that accumulate in a stochastic manner or tends to increase in exponential dynamics of mutations. Emergence of driven mutations may lead to senescence or cancer in the context of reduction in several DNA repair pathways (Burkhalter et al. 2015). Aging is expressed as a complex and gradual loss of functions that result in tissue and organ deterioration as well as the organism as a whole. Stem cells that are responsible for tissue homeostasis guarantee renewal and proliferation, experienced aging, and consequently accumulate damages that leads to senescence affecting their quality and usefulness to be utilized in cellular therapy (Kollman et al. 2001). In this regard, age of donor subjects is important in the success of cell expansion. A series of reports support the negative effect of aging in stem cell suitability for therapy. IPSCs reprogramming was inversely correlated to donors age (Trokovic et al. 2015; Meng et al. 2020), as well as the yield of adipose MSC (Yamauchi et al. 2017). The negative impact of cells aging, affects differentiation efficiency, and paracrine immunomodulatory properties have been reported (Jin et al. 2017; Park et al. 2021). In a mixed model of murine recipients and human cells transplantation, the analysis of peri-infarct cortex treated with hMSCs from aged donors significantly reduced the efficacy relative to neurogenesis, vessel development, and antiinflammatory secretory profile (Yamaguchi et al. 2018). A recent review that evaluated information on the links between vascular senescence, endocrine pro-senescence factors NSC responsiveness to vascular and blood factors, lead to the conclusion that aged stem cells declined in key processes like neurogenesis. These facts poses a warning signal against the use of elders as donors of NSC for therapy (Rojas-Vázquez et al. 2021). Studies on the relationship between donor age and IPSCs genetic stability have demonstrated that cells from elder donors contained a higher number of genetic alterations than the younger. In a work IPSCs were obtained from blood samples from 16 subjects (age 21–100 years). Clonal expansion via reprogramming allowed exomic analysis that revealed a linear relationship between mutations and donor’s age, and those mutation includes several related to cancer (Lo Sardo et al. 2017). There is a consensus on the greater genetic alterations and less suitability for stem cells derived from aged subjects (Kenyon et al. 2012; McNeely et al. 2020; De et al. 2021). This brings and implicit limitation for those autologous treatments in old patients that are being the subject of intense scrutiny that is continuously fueled by controversial results. Alternatively, some author reported that adipose stem cells conserved the secretome without changes neither showed senescence or reduction in cells yield irrespective of donors age (Dufrane 2017). In this direction, there are reports in reversing age signature and fulfillment of successful reprogramming IPSCs from elder even centenarians subjects. Those studies that reports cell reversion of aged

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phenotype in different kind of cells, like MSCs, NSCs, HSCs, and reduction of genomic instability hallmark, and remarkably epigenetic rejuvenation obliges to keep the possibilities opened to the acceptance of elder cells donors as the research on the continues (Lapasset et al. 2011; Rohani et al. 2014; Singh and Newman 2018; Li et al. 2020; Yu et al. 2020).

Toxic Habits and Diseases in the Donors Smoking and alcoholism are habits that may harm donor cells, preconditioning then in such a way that affects the abilities for its use in therapy. Basal stem cells from airways of smoker subjects acquires gene signature of lung adenocarcinoma (Shaykhiev et al. 2013). Long-term exposition (20 weeks) to cigarette smoke in mice have shown an increase in stem cells features in pancreatic cells, which include among others the RNA polymerase II-associated factor promoter (PASF1) that is found overexpressed in pancreatic tumors (Nimmakayala et al. 2018). Also in a murine model, long-term cigarette smoke exposure in vivo lowered MSCs and HSCs, while increased gene expression related to proliferation, in what is another warning for the use of stem cells from smokers (Siggins et al. 2014). Smoke is capable of induce stem cells in the route to lung cancer development (Lu et al. 2020). Chronic nicotine exposition modulates epigenome through ROS induction and favors stemness in Hk-2 human kidney epithelial cells, during nicotine-related kidney carcinogenesis (Chang and Singh 2019). Cigarette smoke extract (CSE) impairs stem cell development through induction of pro-inflammatory cytokines, affecting MSCs quality in mice (Cyprus et al. 2018). In human ASCs, in vitro exposition to CSE even at lower doses impaired the chondrogenic and osteogenic differentiation albeit adipogenesis was not affected (Wahl et al. 2016). Even in healthy donor’s peripheral blood, hematopoietic progenitor cells response to granulocyte colony stimulation factors is reduced in human heavy smokers but is reversed after smoking cessation (Zhen et al. 2020). Smoking is one of the conditions that should be considered when selecting stem cells donor from different compartments, so as to guarantee that cells quality should not be compromised. Pro-inflammation, epigenetic changes, overexpression of proliferating cells, changes in stemness, and even the development of cancer stem cells could cause a derangement in the potential recipients of those cells. Alcohol intake have also been reported as detrimental over certain types of stem cells (Dhanabalan et al. 2018), but the literature is more scarce on the subject that reflects the obviousness of avoids selection of alcoholic subjects as cells donors. Neurogenesis is affected by alcohol intake, through depletion of stem cells Sox-2 progenitors (Le Maître et al. 2018). On the opposite side, a moderate red wine intake have been reported to favor the increase of endothelial progenitor cells (EPCs) limited tumor necrosis factor-alpha-induced EPC senescence in what is attributed to an increase in nitric oxide availability (Huang et al. 2010). Resveratrol increases the number of EPCs and angiogenesis (Lu et al. 2019). Different conditions have shown to be detrimental for stem cell performance, i.e., hyperglycemia (Chen et al. 2007; Yin et al. 2021), that reduce proliferative capacity (Wang et al. 2018). In stem cells where metabolism is high, in concordance with the

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increased proliferation requirements, hyperglycemia and O-GlcNAcylation induce genetic instability and trigger DDR in different stem cell types (Na et al. 2020). However, there are differences in stem cell susceptibility to hyperglycemia and ischemia, and one example is the maintenance of genetic stability in MSCs derived from umbilical cord and placenta (Sharma and Bhonde 2015). Human mesenchymal stromal from umbilical cord from newborns from diabetic mothers shows impaired proliferation as well as high levels of p16 of p53, mitochondrial dysfunction, and premature senescence (Kim et al. 2015). In another study in human umbilical cord cells, less proliferation, telomerase function, lower antioxidant enzymes, reduced stemness, less expression in genes related to mitochondrial activity, and impaired differentiation capacity have been observed when pregnancy was affected by gestational diabetes (Kong et al. 2019). Developing IPSCs from diabetic patient’s cells have been proposed to circumvent the problem posed by the heavy mutation burden in islets cells from diabetic donors and open new roads for autologous transplant for diabetic patients (Maxwell and Millman 2021). Hyperinsulinemia observed in telomerase immortalized MSCs (ASC52telo) is accompanied by a reduction in their capacity to differentiate into adipocytes (Kulebyakin et al. 2021). Insulin resistance-induced oxidative stress have been related with damages that are severe enough so as to consider this relevant metabolic disturbance as a warning condition when evaluating possible stem cells donors (Kulebyakin et al. 2021).

Conclusion Genome, genetic, or chromosome instability in stem cells from different species are terms that have been in use as synonyms, and those terms describe an increase in sequence variations at molecular level, the presence of aneuploidy, changes in epigenetic patterns, as well as dysregulation of those gene circuits that control DNA damage response and its interconnection with cell cycle control and progression. These changes are the result of a large array of causes operating at different levels, from the oxidative species generated in metabolism or inflammation to those aroused from external challenges to the cell in vivo or during isolation, culture, and reprogramming. Stem cells stemness shows an outstanding dependence from DNA stability and intranuclear deployment and chromatin organization. Those properties that make stem cells the core of the cellular therapy, like stemness, proliferation capacity, and paracrine effectiveness, are linked to genetic stability. Induced pluripotent stem cell reprogramming is paradoxically the “spring of youth” for inducible cells and the source of undesirable mutations on the other side. Consequently, cell dependence on the procedures integrative or not is an issue that will be in the center of stem cell basic and therapeutic-related research. Genetic stability, of those cells intended for the treatment of many diseases that otherwise would remain without effective therapy, is a must as is the comprehension of those issues regarding to their genetic stability like, isolation, culture conditions

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(oxygen levels, type of medium, supplemented, or defined, feeder layers carriers, passage number, handling, and conservation, as well as the adequate selection of donors, even for autologous treatments. The pursue of genetic stability both for standardized cell pools to be commercialized like therapeutic drugs and for individualized medicine will be the consequence of the search for the best combinations between the multifaceted characteristics that will be responsible for the attainable genetically stable low risk therapeutic stem cells.

Cross-References ▶ Effects of 3D Cell Culture on the Cell Fate Decisions of Mesenchymal Stromal/ Stem Cells ▶ Induced Pluripotent Stem Cells ▶ Mesenchymal Stem Cell Secretome: A Potential Biopharmaceutical Component to Regenerative Medicine ▶ Response of the Bone Marrow Stem Cells and the Microenvironment to Stress ▶ The Stem Cell Continuum Model and Implications in Cancer

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Index

A Abnormal mitochondria, 679 Acetaminophen (APAP), 227 Acute kidney injury, 986 Acute lung injury (ALI), 167, 986 Acute myeloid leukemia (AML), 1264 Acute myocardial infarction (AMI), 452–460, 518, 809, 810, 1027 BMMC dosage, 253 BONAMI trial, 253 bone marrow cells, 251 BOOST trial, 254 cell treatment, 252 clinical trials, 249–250 contractility, 243 FINCELL trial, 252 HEBE trial, 252 intracoronary infusion, 252, 255 LateTIME trial, 253 LEUVINE-AMI trial, 254 LVEF, 253 REGENT trial, 252 REPAIR-AMI trial, 254 stem cell therapy, 253 Acute on chronic liver failure (ACLF), 228 Acute respiratory distress syndrome (ARDS), 167 cytokine storm, 168 hyperactivation of T lymphocytes, 177 lung repair and treatment, 180 MSCSs, 185 Adaptive immune system, 230 Adherens junctions (AJs), 832 Adipocyte(s), 1181, 1184, 1186, 1200, 1205, 1207, 1209–1214 lipolysis, 1209 metabolism, 1213

Adipogenic differentiation, 574–575 Adipose-derived stem cells (ASCs), 353, 1463–1464 applications, 698 autologous and allogeneic treatments, 698 bone reconstruction, 714 components and functions of exosomes, 700 culture-expanded, 698 description, 696 isolation of, 696 in plastic and cosmetic surgery, 704–714 recontouring depressed facial lesions, 709–712 regulatory considerations for the production and clinical application of, 700–701 safety, cell therapy, 702–704 secretome, 699 soft tissue reconstruction, 704–709 in wound healing, 712–713 Adipose-derived stromal cells, 257 Adipose tissue, 108, 131–132, 1283 and environmental stressors, 1213–1214 Adipose tissue-derived mesenchymal stem cells (Ad-MSCs), 28–29, 327–330, 332, 342, 362, 366–367 advantages, 29 clinical applications, 31 isolation, 30–31 limitations, 29–30 Adrenergic signalling, 243 Adult stem cells (ASCs), 476, 516, 693, 1393 heart failure, 517–522 Adult tissue-derived stem cells, 733–734 Advanced glycation end products (AGEs), 78 Advanced therapies, 54 Advanced therapy medicinal products (ATMPs), 80

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1500 Aesthetic surgery, 693 AT-MSCs cell-based clinical studies in, 705–708 cell-based therapies in, 700 clinical application of SVF/ASCs in, 704–714 AF-derived MSCs (AF-MSCs), 419 African Blood and Marrow Transplantation (AfBMT), 1434 Age-related macular degeneration (AMD), 333, 608, 901, 908 Aging, 242–245 stem cell genomic instability, 1454–1456 Akt phosphorylation, 1285 Alagille syndrome, 225 Alcohol intake, 1479 Alcoholism, 1479 Aldehyde dehydrogenase, 1311–1312 Aldosterone antagonists, 246 Alkaline phosphatase (ALP), 447 Allogeneic BM-MSCs, 211 Allogeneic MSCs advantages and limitations, 12, 14, 15 adverse reactions, 14 allogeneic adipose tissue-derived MSCs, 8, 9 allogeneic stem cell transplantation, 9 vs. autologous cells, 15 cell-based therapy, 14 clinical application, 14 immunological profiling, 14 immunomodulation, 14 safety and efficacy, 14 Allogeneic myoblast transplantation (AMT), 651–652, 655, 661, 662 allogeneic human myoblasts, manufacture of, 654 case selection, 653 clinical research procedures, 655 clinical trials, 653 cyclosporine immunosuppression, 654 donors, 654 efficacy, 659 MI patients, 652 muscle biopsy, 654 patient selection, 654 preparation of myoblasts, 654 regulatory, 653 Allograft, 630 Alveolar bone proper-derived stem/progenitor cells (ABMSCs), 1040 Alzheimer’s disease (AD), 405–406, 905, 1048 allogeneic MSCs, 426 amyloid β protein, 422, 424 AT-MSC-Exos, 424

Index blood-brain-barrier, 426 BM-MSC-Exos, 423 exosomes, 422 MSC-derived miRNA-21, 425 MSC-Exo-based neuroprotection, 423 MSC-Exo-treated microglia, 425 neurological disorder, 421 neuropathological characteristics, 422 pathological neurons, 422 UC-MSC-Exos, 424, 425 American College of Cardiology/American Heart Association heart failure staging system, 475 AMICI study, 141 Aminosalicylates, 196 Amyotrophic lateral sclerosis (ALS), 416, 1051, 1242 Aneuploidy, 1459 and lamins alterations, 1460–1461 Angiogenesis, 248, 458, 459, 1044, 1129, 1132, 1136, 1137, 1142, 1372, 1376, 1395–1399, 1403–1406 Angiogenic factors, 1052 Angiogenic signaling proteins, 958 Angiomyogenesis, 664 Angiomyogenic repair, 444 Angiopoietin 1 (Ang-1), 1131, 1132 Angiopoietins, 1189 Angiotensin-converting enzyme 2 (ACE2-), 180 Angiotensin-converting enzyme (ACE) inhibitors, 246, 475 Angiotensin receptor blockers (ARBs), 246 Ankle plantar flexors, 645 Anomalies, 833–835 Antiangiogenic therapies, 1313 Anti-apoptosis, 1044 Anti-apoptotic activity, 1132 Antiarrhythmic drug efficacy, 886 Antibiotics, 553 Antigen-presenting cells, 199 Anti-inflammatory effects, 198 Anti-inflammatory factors, 248 Anti-inflammatory markers, 1056 Anti-inflammatory properties, probiotics, 555 Antimicrobial metabolites, 552 Apheresis, 1425 ApoBDs, 1133 Apolipoprotein E (APOE), 853 Apoptosis, 551 Aquaporin-4 (AQP4), 856 Argonaute proteins, 1374 Arrhythmia, 878, 880–887 Articular cartilage defects, 353–355, 1137 Aryl hydrocarbon receptor (AhR), 1198–1199

Index Ascl1, 387 Astrocytes physiological roles of, 852–853 reactive gliosis, 854 Astrogliosis, 859 AT-derived MSC-Exos (AT-MSC-Exos), 424 Atherosclerosis, 772 Atorvastatin, 450 Atrial and ventricular cardiomyocytes, 729–730 Atrial fibrillation (AF), 242, 878, 880, 887 Atrial natriuretic peptide (ANP), 478, 481 Autism, 429–431 Autism spectrum disorders (ASD), 430 Autografts, 630, 714 Autologous bone marrow cells (ABMCs), 518 Autologous chondrocyte implantation (ACI), 357–358 Autologous limbal cell transplantation, 327 Autologous MSCs advantages and limitations, 12 autologous bone marrow-derived MSCs, 8, 9 in elderly patients, 13, 14 from sick patients, 12, 13 treatment, 12 Autologous stem cell transplantation (ASCT), 1422, 1430 Autologous transplantation, 928 Autonomic nervous system, 1181 Autonomous robotic cell injection catheter system, 665–666 Autophagy, 1192, 1193 Autoradiography, 824 5-Azacytidine, 779 B Base excision repair (BER), 1452 Basic fibroblast growth factor (bFGF), 636, 1131 Basic-helix-loop-helix (bHLH), 831 BCNU-etoposide-aracytine-melphalan (BEAM), 1423, 1427, 1431 Becker muscular dystrophy (BMD), 1106 Benzalkonium chloride (BAC), 329 Benzene, 1196–1197 Beta-blockers, 246 Beta-coronavirus, 176 Bioabsorbable matrix, 209 Bioactive melatonin, 363 Bio-banking, 903–904 Bioengineering, 491, 497–499, 532, 1337 Bioethicists, 1168 Biogenesis, 1133 Bioinks, for heart tissue, 789–790 Biologic skin, 675

1501 Biomarkers, 806 EVs (see Extracellular vesicles (EVs)) liquid biopsy, 1270 TEVs, 1268 theranostic applications, 1266 Biomaterials, 260 Bioreactors, 370 ® BioSeed -C, 358 Blood-brain barrier (BBB), 825, 853, 854 Blood cell test, 655 Blood coagulation indexes, 657 Body mass index (BMI), 514 Body sculpture, 675 BONAMI trial, 253 Bone marrow, 129–130 Bone marrow and stress response, 1191 cell and microenvironment response to stressors, 1191–1193 emergency hematopoiesis, 1195–1196 environmental hematopoietic stressors, 1196–1199 hematopoietic stress response, 1193–1196 Bone marrow-derived mesenchymal stem cells (BMSCs), 27–28, 130–131, 251, 254–256, 258, 362–364, 419, 694, 1129, 1232, 1288 advantages, 28 limitations, 28 Bone marrow-derived stem cells (BMDSCs), 256, 353 heart failure, 481–482, 518–519 Bone marrow microenvironment (BMME), 1200 components, 1184–1185 as contributor/driver of leukemia, 1206–1209 dynamic interactions, 1187–1191 secondary myeloid malignancy, development of, 1201–1206 stem cell niches and spatial position, 1185–1187 Bone marrow mononuclear cells (BMMNCs), 251–253, 255, 481, 1394 Bone marrow progenitor cells (BMPCs), 1423 See also Peripheral blood progenitor cells (PBPCs) Bone marrow stromal cells (BMSCs), 328–330, 334 Bone marrow transplants, 475 Bone morphogenetic proteins, 736 Bone morphogenic protein-2 (BMP-2), 447 Bone reconstruction, 714 Bone regeneration, MSCs’ secretome, 1135–1137 BOOST-2 trials, 254

1502 BOOST trial, 254 Bradford protein assay kit, 1357 Brain-derived growth factor (BDGF), 419 Brain-derived neurotrophic factor (BDNF), 856, 1042 Brain natriuretic peptide (BNP), 478, 481, 484 Brain pathologies, 833–835 Brava™ system, 709 Breast cancer EVs (see Extracellular vesicles (EVs)) TEVs (see Tumor-derived EVs (TEVs), breast cancer) Brentuximab vedotin, 1265 Bullous keratopathy (BK), 612 C Cancer breast (see Breast cancer) development and proliferation, 1264 EVs (see Extracellular vesicles (EVs)) exosomal miRNAs, 1374–1376 and exosome, 1371–1373 lung, 1376–1378 stem cells, 1262–1263 Cancer stem cells (CSCs), 1262–1263 aldehyde dehydrogenase as drug-detoxify enzymes against, 1311–1312 biological characterization of, 1314 drug-efflux pumps, 1312 Hedgehog signaling pathway, 1309–1310 markers in, 1306–1309 niche and quiescent state, 1312–1313 Notch signaling pathway, 1310 oncolytic adenovirus, 1315–1316 oncolytic adenoviruses and pancreatic clinical trials, 1317–1318 oncolytic herpes simplex virus, 1316 oncolytic herpes simplex viruses and pancreatic clinical trials, 1318–1319 oncolytic virus and effects in clinical trials, 1317 role in tumor, 1305–1306 stem cell-derived exosomes, 1319–1320 target therapies against, 1313–1318 Wnt signaling pathway, 1310–1311 Cancer therapy, 1399, 1403, 1409, 1410 Cancer treatment, MTT, 668–669 metastasis, tumor growth and cancer apoptosis, 669–672 myoblasts fuse with cancer stem cell, 670–671 plausible sequence of events, 671–672 polygenic defects, identification of, 669

Index Canine model for myocardial repair, 302 Cardiac aging, 243 Cardiac fibroblasts, 1335, 1354 Cardiac function, 658, 1348 Cardiac magnetic resonance imaging, 1359 Cardiac progenitor cells (CPCs), 482 Cardiac regenerative medicine, 499 bioengineering, 497–499 cell-free strategies, 495–497 Cardiac remodeling, 474 Cardiac stem/progenitor cells (CSCs/CPCs), 476, 516, 1009 heart failure, 485, 517–518 Cardiac stem cell therapy appropriate timing of administration, 533 challenges, 533–535 ethical considerations, 535–536 optimal cell type, 529–531 optimal method of delivery, 531 patient characteristics and sourcing, 533 Cardiac tissue engineering, 446, 447 Cardiology, 1238–1240 Cardiomyocyte maturation-specific gene expression analysis, 750 Cardiomyocytes (CMs), 474, 476, 478–480, 488–490, 493, 495, 496, 498, 500, 776, 777, 785, 877, 882, 884, 885, 887 action potential, 750 aging, 243 atrial and ventricular, 729–730 bone morphogenetic proteins, 736 cell death, 244 commitment and differentiation, 734–738 description, 728 endogenous regeneration, 516–517 fibroblast growth factor signaling pathway, 737–738 GATA family, 738–739 HAND transcription factors, 740 heart injury, 248 irreversible damage, 244 limitations of 2D culture, 750–752 molecular profiling of, 734 MSCs, 246 myocyte enhancer factor-2 family, 739 necrosis, 243 Nkx-2.5, 739 population, 243 regulation of lineage differentiation in stem cells, 743 serum response factor and myocardin, 740 size and number, 243 status of iPSC-derived, 741

Index structure of, 728–729 technical limitations, 742 transplanted cells, 242 Wnt signaling pathway, 736–737 Cardiomyogenesis, 734 Cardiomyopathy, 244, 811 Cardiopulmonary injuries, 1045 Cardiotoxic drugs, 245 Cardiovascular diseases (CVDs), 223, 224, 473, 513, 985 acute and chronic impact, 772 endothelial progenitor cell dysfunction in, 805–806 prevalence, 771 Cardiovascular mortality, 242 Cardiovascular system, 1012 Cargo, 229–232, 1260 Cartilage articular, 351 articular cartilage defect treatment, 353–355 bioreactors, 370 chondrocytes, 351–352 collagen, 352 conducive microenvironment mimicking, 367 fluid, 352 lacunae, 351 natural polymers, 368 rotating wall vessels, 371 spinner flasks, 370 synthetic materials, 370 tissue engineering, 371 Cartilage Regeneration System (CaReS), 358 Cartilage tissue engineering natural scaffolds, 368 synthetic hybrid scaffolds, 369–370 Cataract, 332 Cationic microbubbles (CMBs), 1338 design and characterisation of, 1339, 1340 destruction–mediated localised delivery, 1339 UTMD-mediated localised delivery of hybrids, 1343–1348 C-CURE trial, 258, 484 CD133+ bone marrow progenitor cells, 255 CD146, 137–138 CD26, 1189 CD271, 136–137 CD34, 929 CD45 expression, 925 CD45RA-memory, 182 Cell-assisted lipotransfer (CAL), 704 Cell-based regenerative therapy, 773

1503 Cell-based therapy, 54 experimental studies, 226–227 liver diseases, 225–228 preclinical and clinical levels, 232 Cell-based therapy, heart failure cell type, 489–490 dosage, 486–489 engraftment, survival and rejection, 485–486 ethical issues, in regenerative medicine, 494–495 Hippo-YAP pathway, 492–494 mechanism of action, 527–528 route of administration, 490–492 Cell culture, 1148 Cell cycle, 1258 Cell-free strategies, 359, 495–497 Cell-free therapy, 146, 147, 1127, 1128, 1135 animal heart models, 1013–1019 approaches, 229–231 challenges and future perspective, 1027 diagnostic applications of exosomes, 1011–1013 EPCs, 1009 experimental animal models, 1009 GMP-certified preparation, 1009 heart, 1010 stem-cell therapy approach, 1009 translational experimental studies, 1019–1023 Cell fusion, 635–637 Cell membrane defect, 639–640 Cell orientation, 780 Cell physiology methods, 754 Cell replacement technologies, 829 Cell Research and Application (CiRA), 909 Cell-scaffold construct ® BioSeed -C, 358 hyalograft, 358 MACI, 358 NeoCart, 359 Cell suspension, 226 Cell therapy, 79, 83, 86, 184, 197, 274, 278, 300, 303, 306, 307, 630, 698, 702–704 NK cell, 182 T-cell, 181 Cell Therapy Research Foundation (CTRF), 644 Cell transplantation, 229, 398–399 Cellular administration, 83 Cellular competition, 83 Cellular differentiation, 825 Cellular senescence, 572–573, 1193

1504 Cellular therapy minimal criteria, 932 Cellular transformation, 69 Cellular transplantation, 1040 Cell-wave therapy approach, 445 Central nervous system (CNS) development, 832 fetal human, 830 in vitro, 825 lifetime duration, 830 and medullary tube, 824 tissues, 823, 831 Central nervous system (CNS) disorders, 851 cell-based therapies for, 866–868 CNS autoimmune conditions, treatments for, 862–864 neurodegenerative disorders, treatments for, 864–866 stroke and CNS trauma, treatments for, 861–862 Central nervous system (CNS) pathologies, 986 CEP-41750 trial, 140 Ceramide-1 phosphate, 1190 CHART-1 trial, 140 CHART-2 trial, 140 Chemical-based reprogramming approach, 744 Chemokines, 1042, 1044, 1046 Chemosensitive cells, 1265 Chemotaxis, 1044 Chemotherapy, 1305, 1312, 1313, 1392 Childhood hematolymphoid diseases, 924 Chimeras, 1167 Chimeric antigen receptor T-cell (CAR-T cells), 168, 181 Cholinergic cells, 406 Chondrocyte implantation, 362 Chondrocytes, 351–352 Chondrogenic differentiation, 575 Chondroitin-sulfate proteoglycans (CSPGs), 833, 855 Chondrosphere, 360 Chorioallantoic membrane (CAM) assay, 1401 Choroid neovascularization (CNV), 333 Chromosome territories (CT), 1459 Chronic cardiomyopathy, 522, 530 Chronic exposure, 1197 Chronic heart failure, 513 Chronic inflammation, 416, 1194 Chronic kidney disease (CKD), 986 Chronic myocardial infarction (MI) model, 299 Chronic myocardial ischemia model, 300 Cigarette smoke extract (CSE), 1479 Ciliary body, 593

Index CIRCULATE trial, 141 Circulating EPCs (CEPCs), 1394 c-JUN/N-terminal kinase (c-JUN/JNK), 1053 Clinical studies, 1023–1027 Clinical trials, 900 allogenic cells, 902 eye disorders, 909 HLA-homozygous iPSC-RPE cells, 902 iPSC-based cell therapy, 907–910 safety and efficacy testing, 906 transplantation, 901 Clinical trials on MSC therapy of IBD, 201–216 Coding and non-coding RNAs, 981–982 Cofactors, 632 Cognitive and behavioral dysfunctions, 416 Cognitive impairment adiponectin, 428 genetic modification of MSCs, 427 hippocampus injury, 426, 429 miRNA-21, 428 MSC-Exos, 427, 428 neurotrophins, 429 pro-angiogenic and trophic factors, 429 TBI, 426 Coleman’s method, 30 Collagen, 352 scaffolds, 777–779 Colony forming unit–fibroblast (CFU-F), 276, 693, 930, 1231 Comparative genome-wide gene expression analyses, 388 CRISPR activation, 390–391 random trial and error screening of libraries, 388–390 single-cell RNAseq and computational analysis, 391 Comprehensive in vitro Proarrhythmia Assay (CiPA), 887 CONCERT-HF trial, 485, 518 Concomitant administration of cells, 445 Conditioned medium (CM), 37 DPCSs, 1048–1052 GMSCs, 1052–1055 PDLSCs, 1055–1057 SHED-CM (see Secretome/conditioned medium (SHED-CM)) Conjunctiva, 592 Conventional stem cell purification, 1257 Conventional therapies, 245–246 Copy number variations (CNVs), 900, 1166, 1447 Cord blood, 365

Index Cornea cellular layers, 609 endothelium, 590–592, 611–612 epithelium, 589–590, 609–610 stroma, 590, 610 trabecular meshwork, 613 Corneal damage, 330 Corneal diseases, models of, 325–331 Corneal endothelium, 590–592, 611–612 Corneal epithelial cells, 325 Corneal epithelium, 589–590, 609–610 Corneal hydration, 611 Corneal stroma, 590, 610 Corneal transplantation, 327, 611 Coronary artery bypass grafting (CABG), 305, 475, 479, 482, 484, 491, 497, 498, 517, 1019 Coronary artery disease (CAD), 256 Corona virus disease-2019 (COVID-19) ALI, 167 cells-based innovation, 181–182 clinical feature, 165 global health emergency, 165 infection and immune response, 176–178 MSCs clinical use, 178 MSCs (see Mesenchymal stem/stromal cells (MSCs)) pathology, 167 treatment options, 167 vaccine, 167 WHO declaration, 165 Cortex-derived astrocytes, 396 COVID-19, see Corona virus disease-2019 (COVID-19) Cranial bone (CB) MSCs, 548 Crigler-Najjar syndrome, 225 CRISPR activation-based screening, 390–391 CRISPR‑Cas9 technology, 85 Critical limb ischemia (CLI), 713 Crohn’s disease (CD), 142–143, 195, 201, 210, 213, 1027, 1237–1238 BM-MSCs therapy, 211 inflammatory (luminal), 211 perianal fistulas, 207, 208 Cryopreservation of stem cells, 1423 Culture-expanded adipose-derived stem cells, 702–704 Cuprizone, 404 Cutaneous wounds, 108 C-X-C motif chemokine ligand 12 (CXCL12), 69, 703 Cyclin dependent kinase (CDK), 1453 Cyclooxygenase-2 (COX-2), 1289

1505 Cyclosporine, 645 immunosuppression, 638, 654 Cyclosporine-A (CsA), 642 Cytochalasin B induced membrane vesicles, 1086, 1087 Cytochalasin B-induced MVs (CIMVs), 1077 Cytochrome P450 (CYP), 1198 Cytokine release syndrome (CRS), 181 Cytokines, 632, 1042, 1046 Cytokine storm, 168, 177, 178, 180, 183 Cytoprotection, 444 Cytotoxic CD8+T cells (CTLs), 417 Cytotoxicity screenings, 907 D Damage/Danger-associated molecular patterns (DAMPs), 1199, 1282 Decellularization, 1038 Deferoxamine (DFO), 1142 Defibrotide, 183 Dendritic cells (DCs), 181 De novo urea synthesis, 228 Dental follicle (DF) MSCs, 548 Dental follicle stem/progenitor cells (DFSCs), 1040, 1057–1058 Dental pulp MSCs, 548 odontoblasts, 549 tissue, 547 Dental pulp stem cells (DPSCs), 866, 868, 1040 angiogenic factors, 1049 autoimmune diseases, 1052 immunomodulatory properties, 1049 liver disorders, 1049 MSCs markers, 1049 neurological diseases, 1051–1052 osteogenic potential, 1049 Dental stem/progenitor cells (DMSCs) ectomesenchymal neural crest cells, 1040 multi-differentiation, 1040 Dentin sialophosphoprotein (DSPP), 1050 Derived Multiple Allogeneic Proteins Paracrine Signaling (d-MAPPS), 419 Dermal sheath stem cells, 107 Dermal signaling, 107 Detoxification response and signaling pathways aryl hydrocarbon receptor, 1198–1199 drug metabolizing enzymes cytochrome P450 and glutathione S-transferases, 1198 TLR signaling, 1199 Diabetes mellitus, 244

1506 Diabetic retinopathy (DR), 333 Differentiated adult somatic cells, 116 Differentiation, 1281–1284, 1289–1291, 1293 Diffuse large B-cell lymphoma (DLBCL), 1272, 1436 Digoxin, 246 Dilated cardiomyopathy (DCM), 887 Dimethyloxaloylglycine (DMOG), 1142 Dimethyl sulfoxide (DMSO), 1436 Dioxins, 1197 Direct neural reprogramming strategies, 386 Alzheimer’s disease, 405–406 autologous cell transplantation, 398–399 CNS disorder treatment with neural cells, 399–401 comparative genome-wide gene expression analyses, 388–391 fibroblasts, 393 hepatocytes, 394–395 implications of cell source, 391–396 ischemic stroke, 403–404 miRNAs, 387 multiple sclerosis, 404 neural cell subtype generation, 396–398 Parkinson’s disease, 405 pericytes, 395 primary-derived cell sources, 392 reprogramming factors, 386–388 reprogramming of astrocytes, 393–394 small molecules, 387–388 spinal cord injury, 399–402 transcription factors, 386–387 traumatic brain injury, 402–403 urine epithelial cells, 395–396 Direct cardiac reprogramming advantages of lentiviral vectors for, 1352 cardiomyocyte generation, 1337–1339 CFs in vitro, 1357 future research, 1355 improvement of reprogramming factors in, 1353–1354 ultrasound-mediated, 1354–1355 Disease modeling, 897, 907 Dishevelled (Dsh) protein, 737 Diuretics, 246 DNA damage response (DDR) signaling pathway, 1192 DNA damage tolerance (DDT), 1456 DNA stability, 1451–1454 Donor cell survival, 443–444 Donors’ right, 1170–1172 Dosage strategies, 184 Double-blind randomized trials, 810

Index Double strand breaks (DSB), 1449 Double-stranded RNA (dsRNA), 1284 Drop-on-demand bioprinting technique, 788 Drug discovery, 905–907 Drug-efflux pumps, 1312 Dual-isotope simultaneous acquisition (DISA), 302 Duchenne muscular dystrophy (DMD), 1103 genetic mosaicism, induction of, 641–642 LBT, 645–646 MTT in, 642–650 natural history, 640 SMT, 643–644 strategizing DMD treatment, 641 WBT, 646–650 Dulbecco’s modified Eagle medium, low glucose (DMEM-LG), 5 Dysbiosis, 555 Dysfunction, 324 Dystrophin, 1103 gene, 632, 633 E Eastern Mediterranean Bone Marrow Transplantation Group (EMBMT) report, 1434 conservation and PBPC viability, 1435 donor cell engraftment, 1436–1438 PBPC mobilization, 1435 therapeutic intensification, 1436 Echocardiography, 1359 Ectodomain, 1047 Ectosomes, 1107 Electrical phenotypes, 886 Electrolyte levels, 656 Electroretinography (ERG), 339 Embryonic stem cell-derived MSCs (ESC-MSCs), 1147 Embryonic stem cells (ESCs), 111–112, 228, 325, 336–338, 342, 440, 515, 546, 731–732, 1009, 1231, 1232, 1239, 1393, 1464–1468 differentiation, 927 heart failure, 515 hESCs, 588 Emergency hematopoiesis, 1195–1196 Encephalomyelitis (EAE), 1056 End-diastolic volume (EDV), 478, 485, 518 Endogenous cardiac progenitor cells, 482 Endogenous cardiac regeneration, 476–478 Endogenous stem cells, 106 Endogenous vascular reparation, 801

Index Endothelial cells (ECs), 1011, 1189 Endothelial cell-side progenitors (EC-SPs), 1397 Endothelial colony-forming progenitor cells (ECFCs), 808–810, 1394 Endothelial nitric oxide synthase (eNOS), 251, 1401 Endothelial progenitor cells (EPCs), 801, 812, 1009, 1181, 1184, 1394–1396, 1479 advantages and disadvantages, 1410 animal studies for vascular repair, 806, 808–810 applications, 1398–1399 cardiovascular diseases, endothelial progenitor cell dysfunction in, 805–806 clinical studies for applications, 810–812 definition and biological function, 802–804 and early outgrowth, in revascularization and vascular reparation, 806–812 in vitro culture methodologies classification, 1395 molecular maker antigens, 804 molecular mechanisms, 803 in neovascularization, 1397–1398 physiological conditions, 1396 preclinical applications of early and late EPCs, 807 in pre-clinical cancer studies, 1403–1407 side effects and potential risks, 1407–1409 sources, ex vivo culturing and implantation, 1399–1403 Endothelial-to-mesenchymal transition, 118 End-stage HF patients, AMT, see Allogeneic myoblast transplantation (AMT) End-systolic volume (ESV), 478, 481, 485, 518 Engraftment window, 924 Enhanced green fluorescence protein (EGFP), 279, 339 Environmental hematopoietic stressors detoxification response and signaling pathways, 1198–1199 environmental pollutants, 1196–1198 Environmental pollutants, 1196 benzene, 1196–1197 dioxins, 1197 heavy metal exposure, 1198 nanoparticles and particulate matter, 1197 polycyclic aromatic hydrocarbons, 1197–1198 Enzyme-linked immunosorbent assay (ELISA), 1131 Epidermal growth factor (EGF), 1042

1507 Epigenetics, 743, 938 Episcleral veins, 341 Epithelial and oral mucosal stem cells (EOM-SCs), 548 Epithelial cells (ECs), 553 Epithelial stem cells, 114–115 ERK-SMAD pathway, 86 Erythropoietin (EPO), 1396 ESCRT-associated proteins, 957 ESTIMATION trial, 140 Ethics advantages, 1164 hiPSCs, 1163 issues, 1163 EVpedia, 1011 Excessive wound healing, 109–111 Excitation contraction coupling, 635 ExoBCD, 1011 ExoCarta, 1011 Exogenous, 826 Exonuclease 1 (Exo1), 1457 exoRBAse, 1011 Exosomal lncRNA sequencing, 459 Exosomes, 200–201, 230, 259, 452, 1043, 1104, 1107, 1109, 1133, 1138, 1192, 1258 advantages, 146 in Alzheimer’s disease, 421 biological functions, 1370–1371 and cancer, 1371–1373 cell-free therapy (see Cell-free therapy) challenges and future perspective, 1027 in clinical studies, 1023–1027 composition, 1369–1370 diagnostic applications, 1011 in experimental animal heart models, 1013–1019 future perspectives, 1378–1380 for heart, 149–150 for IBD, 147–148 miRNAs, 1374–1376 MSCs-derived and acute graft-versus-host disease, 146–147 multiple sclerosis, 140–150 and MVs, 1043–1044 pathways of exosomal miRNA secretion, 1373–1374 rheumatoid arthritis and knee arthritis, 148–149 in translational experimental studies, 1019–1023 tumor-derived, 1376–1378 Experimental animal studies, 226–227

1508 Experimental autoimmune encephalomyelitis, 72 Experimental autoimmune encephalopathy (EAE), 340 exRNA Atlas, 1011 Extensor digitorum brevis (EDB), 643, 644 Extracellular acidification rate (ECAR), 884 Extra-cellular matrix (ECM), 447, 497, 498, 568, 776–777, 832–833, 862, 1044, 1106, 1129, 1184, 1466 Extracellular matrix metalloproteinase inhibitor (EMMPRIN/CD147), 177 Extracellular vesicles (EVs), 146, 980–981, 1043, 1105, 1133–1135, 1148, 1184, 1239 advantages, 949–951 aminoethyl anisamide-PEG incorporation, 963 anti-apoptotic miRNAs, 962 anti-inflammatory properties, 962 apoptotic bodies, 1075 applications, 952 biodistribution, 960 biofluid origin, 1075 biogenesis mechanisms, 962 biological activities, 1073 biological pathways, 1259 bioprocessing, 965 biosafety, 1072 in blood, 232 cargo, 1260 cell-based therapeutics, 1073 cell-derived nanovesicles, 965 cell-free therapy, 1072 cellular paracrine secretion, 229 characteristics, 1075 circular membrane fragments, 949 classification, 949, 1075 clinical-scale production, 964 clinical studies, 954, 955 CNS lymphomas, 1269–1271 composition, 1074 continuous medium perfusion, 963 CSCs, 1262–1263 degree of recovery, 959 development of, 1074 diagnostic and therapeutic role, 231–232 dosing strategies, 959 endosomal origin, 1075 EV-based therapeutics, 951 EV mediated mitochondria donation, 1083, 1085, 1086 exosomes, 949

Index first-pass effect, 962 functions of, 1073, 1258–1261 GelMA hydrogels, 964 gene expression profiles, 965 guidelines/recommendations, 955, 956 heterogeneous, 1259 hollow fiber bioreactor technology, 963 immune regulatory function, 963 immunomodulatory activity, 1081, 1082 in-depth analysis, 1074 inflammatory response and subsequent activation of nearby cells, 1110–1112 injured muscle environment, 1110 intravenous autologous MSCs’ transplantation, 954 intrinsic ability, 964 iPSCs-derived MSCs, 231 isolation methods, 959–961 large-scale production of vesicles, 1085–1088 in leukemia, 1263–1265 lipids, 959 in liver diseases (see Liver diseases) in lymphoma, 1265 mammalian body, 1258 manufacturing approach, 965 medicinal products, 961 microRNAs, 957 microvesicles, 949, 1075 MSC-derived EVs, 949, 1074–1078, 1080 from MSCs, 230 MSC-specific membrane proteins, 950 normal stem cells, 1261–1262 optimal time and mode, 959 physiological and pathological processes, 1073 physiological features, 963 pleiotropy, 1261 post-transplantation, 230 preclinical evidence, 951 preclinical studies, 954 production of, 229, 960 protein-based mechanism of action, 957, 958 proteins and lipids, 1107–1108 quality control and potency markers, 956, 957 regenerative effects, 1074 regenerative potential, 1261 restorative properties, 961 RNA cargo, 1108 scaled-up culturing, 963 secreted by liver cells, 230

Index shear stress, 963 stem cells, 229 structures, 1074 surface-modified EVs, 963 therapeutics, 231, 961 3D-biomaterial scaffolds, 965 3D-culture platforms, 964 thrombin-preconditioning of MSCs, 962 transplantation, 230, 231 2D-culture condition, 964 type-one disease, 232 types of, 1075 uptake of vesicles, target cells, 1109–1110 Eye ciliary body, 593 conjunctiva, 592 cornea, 609–614 (see Cornea) human, 588 iris, 592–593 lens, 589, 594–595 ocular lens, 613–614 retina, 614–616 (see also Retina) stem cell-based therapies, 608 trabecular meshwork, 593–594

F Fanconi anemia (FA), 1203–1205 Fat transplantation, 703 Fatty acid-binding protein, 1209 Fatty acid translocase, 1209 Fatty acid transport protein, 1209 Fecal incontinence (FI), 676 Fetal bovine serum (FBS), 84, 1401 Fetal development of tolerance, 923 Fetal healing process, 109 Fetal sheep model, 932 Fetal stem cells (FSCs), 1393 Fibro-adipogenic progenitors (FAPs), 1102 Fibroblast growth factor 2/basic fibroblast growth factor (FGF-2/bFGF), 1042 Fibroblast growth factors (FGFs), 496, 831 Fibroblast growth factor signaling pathway, 737–738 Fibroblasts, 393 FINCELL trial, 252 Fluid, 352 Fluorescence-activated cell sorting (FACS), 388, 1165 Fluorescent in-situ hybridization (FISH), 930 FOCUS-CCTRN trial, 255

1509 Foreign dystrophin gene, 633 Free fatty acids (FFAs), 882 Freeform reversible embedding of suspended hydrogels (FRESH), 788 Fried egg method, 595 Functional maturation analysis, 749–750

G Ganglion cell layer (GCL), 340 Gap junction, 729 GATA-4, 738–739 Gelatin/polycaprolactone–polyethylene glycol (Gel/PCEC-TGF1), 370 Geminin, 1451 Gene defects, 629 Gene-editing technology, 902, 1168 Gene-targeting therapy, 1405 Gene therapy, 38–39, 259, 633 Genetically encoded calcium indicators (GECIs), 881 Genetically encoded voltage indicators (GEVIs), 881 Genetic alterations, 900 Genetic diseases >80% of human death, 629 MTT in, 668 Genetic edition, 1145 Genetic instability, in stem cells adipose stem cells, 1463–1464 and aging, 1454–1456 aneuploidy, 1459–1461 cell origin, 1463–1470 DNA damage response, 1449–1451 DNA stability, 1451–1454 donor age, 1477–1479 embryonic stem cells, 1464–1468 induced pluripotent stem cells, 1468–1470 obesity, 1477 replicative stress/replication timing, 1461–1463 reprogramming methods, 1470–1477 senescence, 1454 telomere damage, 1456–1458 toxic habits and diseases, in donors, 1479–1480 Genetic mosaicism, 632–633 mesenchymal cell transplant, 641–642 newborn muscle transplant, 641 normal and dystrophic minced muscle mixes, 641 primary myoblast culture, 642

1510 Genetic stability, 879 Genome editing, 880–881 Genome-wide association studies (GWAS), 877, 880 Genomic instability, 703, 900 Genotoxic stress, 1192 Genotypic analysis, 880 Gingival mesenchymal stem/progenitor cells (GMSCs), 1040, 1282 neural disorders, 1053–1054 osteogenic potential, 1054–1055 secretome, 1053 skin injuries, 1054 Glaucoma models, 340–342 Glial cell line-derived neurotrophic factor (GDNF), 1048 Glial cells, 851 in CNS auto-immune conditions, 856–859 in neurodegenerative disorders, 859–861 stroke, CNS trauma and impact, 854–856 Glial-derived neurotrophic factor (GDNF), 862 Glial scar, 393 Gliogenesis, 831 Glioma stem-like cells (GSCs), 1406 Gliosis, 403 Glucose phosphate isomerases (GPI), 642 Glutamate excitotoxicity, 337, 860 Glutathione S-transferases (GSTs), 1198 Glycine residues, 777 Glycogen phosphorylase (GP), 1012 Glycogen synthase kinase-3 (GSK-3) inhibitor, 877 Glycosaminoglycans, 833 Glycosyl transferase-programmed stereo substitution (GPS), 84 Good manufacturing practice, 6, 77, 80, 83, 960, 1468 Graft tolerance, 923 Graft versus host disease (GvHD), 141–142 MSC-based therapies, 1235–1237 Graft-versus-leukaemia (GVL) effect, 74 Granulocyte-colony stimulating factor (G-CSF), 1043, 1188–1191, 1207, 1208, 1215, 1395, 1425, 1430, 1435 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 1043, 1140 Granulocyte-macrophage progenitors (GMPs), 1186, 1195 Growth differentiation factor, 114 Growth factors, 1042 G-secretase inhibitors, 1310 Gut microbiota, 551–553

Index H Haemopoietic stem cells (HSCs), 440 Hair follicle anatomy of, 107 bulge cells, 115 formation, 115 HAND transcription factors, 740 Harvey-Bradshaw index (HBI), 210 Heart, 222, 441, 443, 445, 447, 450–452, 460 bone marrow-derived MSCs, 1014–1016 cell-free therapy, 1010 cell therapy, 279 cytoprotective effects, 1013 diagnostic applications, 1011–1013 exosomes (see Exosomes) functions, 1009 genetic manipulation, 1017 and liver, 223–225 miR-125b, 1017 miR-210, 1017 MSCs-derived exosomes, 1013, 1017 myocardial injury, 1013 pharmacological manipulation, 1018 pre-clinical trials, 1014–1016 in translational experimental studies, 1019–1023 Heart diseases aetiologies, 244 aging, 242–244 clinical syndrome, 244 clinical trials, 249–250 conventional therapies, 245–246 infarcted tissue, 244 limitations, 257–258 stem cells, 242 treatment, 242 types, 244 Heart failure, 242, 255–256, 303, 304, 306, 307, 473, 514, 801 BMSCs, 258 bone marrow-derived stem cells, 481–482 cardiac stem cells, 485 cell-based therapy, factors, 485–495 clinical syndrome, 244 and death, 254 and heart disease, 255 mesenchymal stem cells, 482–485 MI, 244 pathophysiology, 513–514 pluripotent stem cells, 478–480 safety parameters, in human clinical trials, 486

Index skeletal myoblasts, 480–481 symptoms, 475 UC-MSCs, 256 Heart failure with preserved ejection fraction (HFpEF), 474, 513, 514 Heart failure with reduced ejection fraction (HFrEF), 474, 513, 514, 811 Heart function, 246–248 Heart-liver inter-organ connection, 225 Heart muscle degeneration, 650 adverse reaction assessment, 655–656 AMT, 651–655 objective evaluation, 656–657 perspectives, 663–664 safety assessment, 655–659 severe myocardial infarction, 651 statistical analyses, 655 study outcomes, 659–663 subjective evaluation, 657–659 Heart transplant, 246 Heat shock proteins (HSPs), 1192 Heavy metal exposure, 1198 HEBE trial, 252 Hedgehog signaling pathway, 1309–1310 Hematopoiesis, 1184 Hematopoietic cell transplant activity, 1434 Hematopoietic progenitor cells (HPCs), 1396, 1423 Hematopoietic stem cells (HSCs), 132, 928–930, 1181, 1184–1191, 1193–1197, 1199–1201, 1204–1208, 1231, 1257, 1393, 1452, 1453, 1455–1458, 1462, 1463, 1476, 1479 in the bone marrow niche, 933 differentiation of, 931 Hematopoietic stress response, 1193–1196 Hematoxylin-eosin (HE) staining, 1355, 1359 Hemeagglutinin esterase (HE), 177 Heparin-binding epidermal growth factor (HEGF), 1042 Hepatic artery, 223 brain level, 224 congestion, 224 HSCs, 230 lineage specification, 223 mononuclear cells, 230 parenchyma, 226 veins, 223 Hepatic growth factor (HGF), 419 Hepatic stellate cells (HSCs), 230 Hepatitis C virus (HCV), 1272

1511 Hepatocyte growth factor (HGF), 1042, 1101, 1131 Hepatocytes, 394–395 Hepatocyte transplantation, 227 Hepatocytic activity of highly purified marrow HSC, 930 HER2osome, 1268 Hereditary degenerative organs, 630 Heterogeneity, 898, 904 Heterokaryotic cardiomyocytes, 637 Heterotopic ossification (HO), 1241 High-throughput screening, 390 Histone deacetylase (HDAC) inhibitors, 1336 Hodgkin’s lymphoma (HL), 1273 Homeostasis, 241, 598 Homologous recombination (HR), 1449 Hormone therapy, 1392 Human adipose tissue-derived MSCs (hAMSCs) secretome, 982 Human corneal stromal stem cells (hCSSCs), 590 Human embryonic stem cells (hESCs), 335, 336, 440, 877 corneal endothelial cells, 612 donors’ right, 1170 informed consent, 1170–1172 patentability, 1169 reproduction, 1166–1168 research, 1162 safety, 1164–1166 scientists and ethicists, 1163 stem cell technology, 1162 substitutes, 1163 virus-dependent delivery methods, 1163 Human Fertilization and Embryology Authorities (HFEA), 1168 Human gene therapy, 642, 644 Human hair follicle stem cells-CM (HFSCs-CM), 1046 Human hepatocyte generation, 932 Human immunodeficiency virus (HIV), 1272 Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) contractility and force generation, 885 differentiation and definition of homogenous cardiomyocyte subpopulations, 881–882 electrophysiological measurements and arrhythmia assessment, 885–886 maturation, 882–883 metabolic considerations, 884 morphology, 884–885 phenotypic assays, 883–886

1512 Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (cont.) proarrhythmic drug assessment, 886–887 transcriptomic and proteomic analysis, 883–884 Human induced pluripotent stem cells (hiPSC), 228, 231, 877, 1464, 1465, 1470, 1471 donors’ right, 1170–1172 genome editing applications, 880–881 informed consent, 1170–1172 patentability, 1168–1170 quality control and genotyping, 879–880 reproduction, 1166–1168 reprogramming, 878–879 safety, 1164–1166 Human lens, 332 Human mesenchymal stem cells (hMSCs), 71 Human pluripotent stem cells (hPSCs), 1464, 1467, 1474 Human proteins, 937 Human retinal progenitor cells (hRPCs), 334, 335 Human shed deciduous teeth (SHED), 1041 DMSCs, 1040 Human telomerase RNA (hTR), 1457 Human T-lymphotropic virus-1 (HTLV-1), 1272 Human umbilical cord derived MSC (hUC-MSCs), 365, 1475 Human umbilical vein endothelial cells (HUVEC), 452, 1017, 1047 Huntington’s disease (HD), 416, 859 glutamate excitotoxicity in, 860 Hyalograft, 358 Hydrogel based therapy, 781–783 Hydrogel patches, 1023 Hydrogels, 826 Hypercoagulability, 180 Hypertension, 244 Hypertrophic cardiomyopathy (HCM), 884, 887 Hypoimmunogenic mouse, 903 Hypoxia, 1454 Hypoxia-induced apoptosis, 248 Hypoxia-inducible factor 1α (HIF-1 α), 1131 Hypoxia induction, 1140–1141 I Iliac bone marrow MSCs (iBM-MSCs), 548 Immune cell migration, 1288 Immune system, 196 Immunogenicity, 901–903

Index Immunomodulation, 36–37, 56, 73, 168, 553, 1044, 1129–1131, 1288, 1289, 1291, 1293 Immunomodulatory properties of MSCs, 70–72 Immunostaining, 425 Immunosuppressive effects, 198 Impaired wound healing, 108–109 Indoleamine 2,3-dioxygenase (IDO), 1129, 1130, 1142 Induced cardiomyocyte-like cells (iCMs), 1337, 1341 Induced pluripotent stem cells (iPSCs), 112–113, 325, 336, 440, 476, 515–516, 732–733, 1106, 1163, 1231, 1239, 1336, 1393, 1450, 1455, 1468–1470 biobanking, 903–904 cell-based therapies, 588 cell therapy-ongoing clinical trials, 907–910 corneal endothelial cells, 591 corneal epithelial cells, 608 corneal epithelium-like cells, 610 disease modelling, 905–907 donors’ right, 1170–1172 drug discovery, 905–907 economic issues, 903–904 ESCs, 896, 897 heart failure, 521–522 and hESCs, 588, 1163 heterogeneity, 904 hiPSCs, 896 immunogenicity, 901–903 informed consent, 1170–1172 lentoid bodies, 595 low-efficiency reprogramming, 898–899 patentability, 1168–1170 reproduction, 1166–1168 reprogramming of skin fibroblasts, 896 retinal pigment epithelial cells, 597 safety, 1164–1166 toxicity studies, 905–907 trabecular meshwork cells, 613 transcription factor, 897–898 tumorigenicity, 899–901 Inducible nitric oxide synthase (iNOS), 72, 1046 Infarcted myocardium, 773 Infarction, 301 acute myocardial, 271 chronic myocardial, 299 exosome-based treatment strategy, 1017 myocardial, 1017, 1019

Index Inflammation, 56, 70, 83, 168, 178, 179, 181 Inflammatory bowel disease (IBD), 195 clinical trials on MSC therapy of, 201–216 diagnosis of, 196 MSCs-derived exosomes, 200–201 pathogenesis, 199 preclinical studies, 199–200 therapy with MSCs, 197–198 Inflammatory loop, 417 Inflammatory (luminal) CD, 211 Inflammatory priming, 1141–1142 Informed consent, 1162, 1164, 1167, 1170–1172 Inherited arrhythmias, 878 Inner cell mass (ICM), 440 In situ hybridization, 932 Instant blood-mediated inflammatory reaction (IBMIR), 80 Insulin-like growth factor-1 (IGF-1), 482, 527, 636, 808, 1042, 1395 Insulin-like growth factor II (IGF-II), 1042 Intensive care units (ICU), 178 Interferon-gamma (IFNγ), 1045 Interfollicular stem cells, 114 Interleukin 6 (IL-6), 1131 Internal tandem duplication (ITD), 1203 International Bone Marrow Adiposity Society, 1210 International clinical trials registry platform (ICTRP), 907 International Society for Cellular Therapy (ISCT), 5, 25, 246, 362, 546, 1125, 1186, 1232 International Society for Extracellular Vesicles (ISEV), 1075, 1133 Interstrand crosslink repair (ICLR), 1449 Intracameral injection, 611 Intracoronary infusion, 491, 532 Intraluminal vesicles (ILV), 1104 Intramyocardial injection, 490, 532 Intraocular pressure (IOP), 338, 342 Intravenous approach, 532 In utero transplantation, 938–939 experimental tool to study stem cell biology, 928–934 hematopoietic SC, 928–930 long-term transplantation tolerance following, 923–924 mesenchymal stem cells, 930–934 Investigational New Drug (IND), 1023 In vitro and in vivo cardiac applications, exosomes, 749

1513 In vitro differentiation of BM-derived MSCs, 272–274 In vitro experimental model of traumatic brainlike injury, 987 In vitro fertilization (IVF), 1162 In vitro reprogramming, 1341 efficiency of astrocytes, 397, 398 In vivo heart function assessment, 1351 IPSCs-derived retinal pigment epithelial cells (iPSC-RPE), 901 Iris, 592–593 Iris pigment epithelial (IPE), 616 Iron oxide nanoparticle-loaded MSCs (IONPMSCs), 1023 Irradiated conditioned medium (ICM), 1476 Ischaemic heart disease, 1335, 1339 Ischemia, 299, 307 Ischemia-reperfusion-based treatment protocol, 444 Ischemia-reperfusion injury, 1012 Ischemic cardiomyopathy, 631, 650–652, 663, 664 Ischemic heart disease (IHD), 306, 440 Ischemic stroke (IS), 403–404 conditions, 990 treatments, 861 Isocitrate dehydrogenase-2 (IDH2), 1454 J Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, 451 K Kaposi sarcoma-associated herpesvirus (KSHV), 1272 Kaposi sarcoma mouse model, 75 Kawasaki disease, 809 Keratinocyte growth factor/fibroblast growth factor-7 (KGF/FGF-7), 1042 Keratoconjunctivitis, 331 Keratocytes, 590 Knee extensors, 645 Knee flexors, 645 Knee osteoarthritis, 145 L Lactobaillus rhamnosus, 552, 554–556 Laminin-rich fractions, 833 Large animal experimentation, 928

1514 Large animal models, 280–303 Large chondroitin-6-sulfate proteoglycan (LC6SP), 636 Laser trabeculoplasty, 613 LateTIME trial, 253 Leber’s hereditary optic neuropathy (LHON), 340 Left ventricular assist devices (LVAD), 246 Left ventricular ejection fraction (LVEF), 243, 447, 449, 451, 459, 460, 474, 478–485, 495, 498, 500, 513, 658 Left ventricular end-diastolic volume (LVEDV), 252 Left ventricular end-systolic volume (LVESV), 252 Lens diseases, models of, 332–333 fiber-cells, 594 hESCs, 595 iPSCs, 595 lentoid bodies, 595 progenitor cells identification, 594 transparent tissue, 594 Lens progenitor cells (LEC), 333 Leukemia, 1181, 1196–1208, 1210, 1213–1215 bone marrow microenvironment, 1263–1264 predictors of response and promoters, 1264–1265 LEUVINE-AMI trial, 254 Limbal stem cells, 609 Lineage-restricted assays, 936 Lineage-tracing studies, 276 Lipid metabolism, 1190 Lipid raft-associated proteins, 1107 Lipolysis, 1209 Lipopolysaccharide (LPS), 1141 Liver, 222–223 cell-free therapy approaches, 229–231 diseases (see Liver diseases) and heart, 223–225 metabolism, 227 Liver diseases cell-based therapy, 225–228 cell suspension, 226 clinical evaluation, 228 clinical studies, 227–228 donor shortage, 225 EVs (see Extracellular vesicles (EVs)) experimental animal studies, 226–227 in situ, 226 non-alcoholic fatty, 224

Index portal vein system, 225 surgical animal models, 226 therapeutic approach, 224 Liver failure (LF), 1045 Liver function indexes, 656 Locomotor activity, 222 Long-term HSCs (LT-HSCs), 1185, 1187 Long-term persistence, human hematopoiesis, 929 Low-density lipoprotein (LDL) cholesterol, 446 Lower body treatment (LBT), DMD, 644–646 L-type calcium channels (LTCC), 884 Lung cancer, 1376–1378 Lymphoblastoid cell lines, 897 Lymphoma, 1265 biomarker, 1269–1270 DLBCL, 1272 HL, 1273 and lymphomagenesis, 1270–1271 patient outcomes and prognosis, 1271 therapy, 1271 virally-mediated, 1272 Lymphomagenesis, 1270–1271 Lysophosphatidylcholine activation genes, 1046 M Macrophages, 1102, 1130 Magnetic-activated cell sorting (MACS), 391, 1165 Magnetic field bioreactors (MFB), 372 Magnetic resonance imaging (MRI), 1402 MaioRegen scaffold, 359 Major histocompatibility complex (MHC), 417, 1369 class-I, 631 class II, 36 Malar deformity, 710, 712 Malfunction, 1449 Mandibular bone marrow MSCs, 548 Marrow adipose tissue (MAT), 1210–1213 Massive rotator cuff tears (MRCTs) lesions, 984 Matrix-induced autologous chondrocyte implantation (MACI), 358 Matrix-metalloproteinase-9 (MMP-9), 853 Mature cells, 360–362 Medicinal products, 69, 80, 86 Megakaryocyte (MK) generation, 1195 Melanocytic stem cells, 107 Melanoma mouse model, 76 Mel-CAM, 137 Membranous bodies (MB), 679

Index Mer tyrosine kinase proto-oncogene (MERTK), 596 MESEMS study, 144 Mesenchymal progenitor cells (MPCs), 1241 Mesenchymal stem cell-based therapies, 1235, 1236 cardiology, 1238–1240 Crohn’s disease, 1237–1238 graft versus host disease, 1235–1237 neurology, 1241–1243 orthopedics, 1240–1241 potential risks, 1243–1244 Mesenchymal stem cells based therapies, 1125–1128 Mesenchymal stem/stromal cells (MSCs), 25, 55, 113–114, 129–130, 246, 330, 362, 693, 926, 1181, 1184, 1186–1189, 1191, 1200, 1201, 1205–1208, 1210, 1212, 1214, 1231–1232, 1281–1283, 1449 ACE-2, 180 acute and chronic liver defects, 228 adipose tissue-derived, 28–31, 131–132, 179, 1013 administration, 180 adult and fetus-associated tissue sources of, 275 advantages for clinical application, 66 allogeneic adipose tissue-derived MSCs, 9 allogeneic MSCs (see Allogeneic MSCs) allogeneic stem cell transplantation, 10 animal studies, 420 anti-inflammatory effects, 179 anti-inflammatory factors, 248 anti-tumor activity of, 75–76 for autoimmune diseases, 73–74 autologous MSCs (see Autologous MSCs) biological properties, 977–978 bone marrow-derived, 4, 27–28, 130–131, 271–277, 419, 1014–1016 for cancer treatment, 74–77 in cardiac regenerative therapy, 774 CD146, 137–138 CD271, 136–137 cell-based therapy, 6, 461 cell-free therapy, 461 cell manufacturing for clinical use, 80–82 cell migration toward damaged tissues, 69–70 cell types, 1009 as cellular medicament, 55 cellular regulators, 179 cellular transformation, 69

1515 cellular transplantation, 1040 characterization, 56 characterization before culture, 135–138 in clinical perspective, 138–145, 303–307 clinical studies, 420 clinical therapies, 547 clinical trials, 8, 201–216 clinical use, 178 cognitive impairment, 426–429 complications, 11 considerations for cellular medicament, 82–86 considerations for clinical applications, 78–82 in COVID-19 pneumonia treatment, 168 Crohn’s disease, 142–143 definition, 55 differentiation capacity, 68–69 differentiation potential of, 277–278 from different tissue sources for various disease conditions., 139–150 dosage strategies, 184 drawbacks, 978 Dulbecco’s modified Eagle medium, low glucose, 5 encapsulated formations, 421 and endothelial cells, 57 engineered therapeutic MSCs, 6, 7 exosomes, 200–201 (see also Exosomes) exosomes based therapeutic intervention, 145–150 extracellular vesicles, 37–38 ex vivo expansion, 5 functions, 56 future research, 86–87, 304–307 gene therapy, 38–39 good manufacturing practices, 6 graft-versus-host disease, therapy for, 141–142 growth, 418 guardians of inflammation, 56 healing of osteonecrosis, 11 healing of ulcus cruris, 9 heart failure, 482–485, 519–520 immunomodulation, 36–37 immunomodulatory characters of, 279 immunomodulatory properties of, 70–72 immunoregulatory and angiomodulatory effects, 421 immunoregulatory cells, 419 impact of statins, 446–447 imprint of disease on, 77–78 infection and immune response, 176–178

1516 Mesenchymal stem/stromal cells (MSCs) (cont.) intracoronary MSCs transplants, 11 intravenous allogenic non-ischemic cardiomyopathy, 307 intravenous infusion, 180 IONP-MSCs, 1023 isolation and characterization of, 275–277 isolation from sources, 131–135 large experimental animal studies, 280–298 left ventricular assist device, 5 local administration, 201–210 locations, 1040 mechanism of action, 69–72, 197–198 MSCs-based cell therapy, 9 multiple sclerosis, 143–144 myocardial angiogenesis, 248 in myocardial repair and regeneration, 442 nebulized administration, 180 oromaxillofacial tissue-derived, 548 paracrine effects, 81, 248 pharmacological approaches, 547 pharmacological effects, 168 phenotypic profile, 65–67 placenta, 134–135 POSEIDON trial, 303–305 postnatal development, 547 preclinical applications, 72 pre-clinical studies with, 279–307 probiotic interaction, 553–556 proliferative activity of, 68 PROMETHEUS clinical trial, 305 properties, 197, 419 pro-tumor activity of, 76–77 public registry list of phase III/IV clinical trials, 58–65 quality control, 16, 17 rheumatoid arthritis and osteoarthritis, 144–145 roots, 975–977 route of administration, 184 safety concerns, 81 secretome derived from (see Mesenchymal stem cells secretome) secretory factors, 168 side-effects, 182–183 small animal experimental models, 279–292, 1009 source of cells, 183–184 sources, 26–34, 1233–1234 SSEA-4, 137 statin treatment, 453 STRO-1, 135–136

Index stromal cell for ischemic heart disease, 306 study design, 183 systemic administration, 330 systemic (intravenous) administration, 210–213 TAC-HTF trial, 306 therapeutic effects, 57, 421–426 therapeutic potential, 421, 1234–1235 therapeutic strategies, 34–39 therapeutic outcomes of cellular medicament, 83–86 thrombopenia, 7, 8 therapy for cardiovascular pathologies, 138–141 in tissue engineering, 1040 tissue regeneration, 34–36 tissue repair, 70 trophic functions of, 278–279 umbilical cord, 132 umbilical cord blood, 132–133 umbilical cord-derived, 31–34, 228 in utero transplantation, 930–934 Wharton’s jelly, 133–134 Mesenchymal stem cells secretome benefits and barriers, 993–994 cardiovascular diseases, 985 central nervous system pathologies, 986 clinical approaches based on, 991–993 coding and non-coding RNAs, 981–982 composition and characterization of, 979–982 extracellular vesicles, 980–981 improvements needed for application, 994–995 ischemic stroke, 990 kidney and lung injuries, 986 mechanisms of action and principal effects, 980–982 neurodegenerative disorders, 990–991 pre-clinical approaches, 982–991 soluble fraction, 979–980 spinal cord injury, 988–990 therapeutic benefits of, 983 traumatic brain injury, 987–988 wound healing, cartilage repair and regeneration, 982–984 Mesoangioblasts (MABs), 1101 Messenger RNAs (mRNAs), 496, 1292 Metastasis, 1372–1374 Metastatic breast cancer (MBC), 1266 Metastatic human breast carcinoma cells, 76 Microarchitecture, 752 Microbubbles, 1338, 1341, 1351, 1354

Index Microbubble ultrasound contrast agents (MCAs), 1337–1338 Microbubble–virus hybrids creation of, 1356 delivery using UTMD in vivo, 1358 Microelectrode array (MEA), 885 Microenvironment, 830–831, 1392 Microfracture, 355 Microglial cells, 855 and physiological functions, 851–852 Microglial polarization, 855 Microglial secretions, 855 Micronucleus with cytochalasin B (MN/CB), 1450 Micropatterning, 754 MicroRNAs (miRNAs), 387, 1103–1106, 1108–1112, 1132, 1292–1293, 1319 in cancer progression, 1374–1376 future perspectives, 1378–1380 secretion pathways, 1373–1374 selection, 1371 Microthrombotic particles (MTPs), 450 Microvesicles, 1380 Microvesicles (MVs), 1133, 1380 EXs, 1043–1044 Microvessel density (MVD), 1398 Migratory ability, 852 Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018 guidelines, 949 Minnesota Living with HF Questionnaire (MLHFQ), 478, 483, 498 miRcombo, 1353 Mismatch repair (MMR), 1449 Mitochondrial stress signaling, 1192 MMP12, 1404 Molecular-based treatments, 862 Molecular profiling, 330 of cardiomyocytes, 734 Monocyte chemoattractant protein-1 (MCP-1), 1044, 1131, 1140, 1396 Mononuclear cells (MNCs), 1233 Morphological analysis, 884 Morris water maze (MWM), 423 Mouse embryonic fibroblasts, 404, 405 Mouse embryonic stem cells (mESCs), 1464, 1466, 1467, 1472 MSCs-derived exosomes (MSC-Exos), 418–421 Muller glial cells, 597 Multi-database search, 907 Multi-detector computed tomography (MDCT), 299

1517 Multi-drug-resistant proteins (MDR), 1312 Multilineage differentiation, 68 Multiple myeloma, 77, 1431 Multiple organ dysfunction syndromes (MODS), 167 Multiple sclerosis (MS), 143–144, 404, 567, 857, 858, 1048 Multipotency, 1040 Multipotent adult progenitor cells (MAPCs), 1394 Multipotent-stem cells, 594 Multivesicular bodies (MVBs), 981, 1043, 1104, 1133 Murine hematopoiesis, 926 Muscle biopsy, 654 Muscle regeneration, 633 Muscle-specific miRNAs, 1109 Muscular dystrophy, 638, 1103 etiology, 638–639 pathogenesis, 639–640 Musculoskeletal diseases, 1135–1140 Mutant Huntingtin protein, 859 Myoblasts, 631 mitotic cardiomyocytes, in vitro controlled cell fusion, 637 pertinent muscle developmental biology, 633–635 Myoblast transfer therapy (MTT), 631, 682 antiaging aesthetica, 674–676 breast and uterine cancers, 673 in cancer treatment, 668–674 cellular and molecular phases, development, 666 controlled cell fusion, 636 cyclosporine immunosuppression, 638 in DMD, 642–650 engineering genetic mosaicism with, 632–633 injection methods, cell distribution and fusion, 637 loose teeth and bone fracture repair, 676–677 mesenchymal cell transplant, 642 repairing hereditary degenerative cells, 632 replenishing dead cells, 632 stimulation therapies and fractal dynamics, 677–679 tendon repair, for injured athletes, 676 type II diabetes, gene defects in, 666–668 with primary myoblast culture, 642 Myocardial differentiation, 447

1518 Myocardial infarction (MI), 244, 474–476, 478, 482, 488, 489, 491, 495, 496, 498, 513, 801, 810, 1343 experimental rat model, 1357–1358 therapy, 447 Myocardial microenvironment donor cells, 444–445 functional mechanism of statins, 446 MSCs, impact of statins on, 446–448 Myocardial single-photon emission tomography (M-SPECT), 300 Myocarditis, 244 Myocardium, 1343, 1348, 1353 Myocyte enhancer factor-2 (MEF-2) family, 739 Myofiber(s), 634 intracellular repair, 1103–1107 repair, 1104, 1110 Myofibroblasts, 362 Myogenesis, 634 MyStromalCell Trial, 141 N Nanocomposite, 1023 Nanoparticles, 1197 Natural killer (NK) cells, 902 therapy, 182 Natural scaffolds, 775–776 collagen scaffolds, 777–779 ECM, 776–777 Neoangiogenesis, 1405 NeoCart, 359 Neomyocytes, 279, 1344 Neomyogenesis, 1343 Neoplasm inhibition rate (NIR), 673 Neovascularization, 333, 1136, 1373 endothelial progenitor cells in, 1397–1398 Nerve growth factor (NGF), 419 Neural growth factor (NGF), 1042 Neural stem cells (NSCs), 419, 866, 1453 adult mammalian brain, 823 applications and limitations, 825–830 culture strategies, 827 and disorders, 836–838 embryonic NSCs, 830–831 experimental evidence, 823 frog embryos, 824 hiPSCs, 825 historic prospectives, 823–829 iPSCs technology, 823 reprogramming of somatic cells, 823 researchers, 826

Index signalling molecules, 831–832 sources, 830 therapy, 835–838 transplantation, 835–838 Neurite extension, 1047 Neurites, 1052 Neuritogenesis, 1047 Neuroblastoma, 824 Neurocognitive disorders, 415 Neurodegeneration, 405, 416, 418 Neurodegenerative disorders, 990–991 Neuroepithelium, 831 Neurogenesis, 823, 824, 826, 831, 833–835 Neuroinflammation, 416, 1047, 1051 Neurology, 1241–1243 Neuromuscular transmission, 635 Neuromyelitis optica (NMO), 856–858, 862, 863, 866 Neuron adult mammalian brain, 823 and astrocytes, 825 doctrine, 823 growth factors, 823 microenvironment, 832 progenitors, 831, 834 Neuron-specific growth factors, 823 Neuropeptide Y, 1189 Neuroprotection, 1044 Neurotrophins, 418, 423, 1044 Neutral sphingomyelinase 2 (nSMase2), 1373 Neutrophils, 1102 New York Heart Association functional classification of heart failure, 474 Nitric oxide (NO), 419, 1101 Nkx-2.5, 739 N-methyl-D-aspartate (NMDA), 597 receptor, 337, 862–338 Non-cardiomyocytes, 882 Non-coding RNAs, 230–232, 496 Non-Hodgkin lymphomas (NHL), 1269 Nonsmall cell lung cancer (NSCLC), 1376 Non-union bone defect, 1136 Normal cardiomyocytes, 637 Normal dystrophin gene, 632 Normal wound healing, 105–108 Notch signaling, 1101 pathway, 832, 1310 Novel object recognition (NOR), 423 ® NOVOCART 3D, 359 N-terminal pro-brain natriuretic peptide (NT-proBNP), 811 Nuclear factor-kappa B (NF-κB), 859, 1286

Index Nucleotide excision repair (NER), 1449 Nutrient deprivation, 1192 New York Heart Association (NYHA) cardiac function, 660 grading, 658 O Obesity, 1477 Ocular hypertension, 342 Ocular lens, 613–614 Odontoblastic differentiation, 1051 Oligodendrocyte precursor cells (OPCs), 394 Oncolytic adenovirus, 1315–1316 Oncolytic herpes simplex virus, 1316 Open-angle glaucoma, 340 Open-chest model, 300 Ophthalmology, 326 Optical mapping, 886 Optimal combination of reprogramming factors, 389 Optomotor behavior tracking, 335 Oral disorders, 553 Organogenesis, 360 Organoids, 168, 178, 905 cerebellum, 829 cerebral, 823 hedgehog agonists, 828 new model systems, 825 technique, 826 Organ-on-a-chip (OOC) technology, 906 Oromaxillofacial tissue (OMT) biology, 548–550 identification, 548–550 isolation, 548–550 Oromaxillofacial tissue mesenchymal stem cells (OMT-MSCs), 547 co-culture, 557 isolated, 549 mechanism, 549 oral flora, 550 probiotic interaction, 554 Orthopedics, 1240–1241 Orthotropic liver transplantation (OLT), 224 Oscillatory potentials (OP), 339 Osteoarthritic-like priming, 1146 Osteoarthritis (OA), 352, 984, 1137–1139, 1240 Osteocalcin (OCN), 447 Osteochondral allograft transplantation (OCA), 356–357 Osteochondral autologous transplantation (OAT), 354, 356

1519 Osteochondral transplantation (OCT), 356 Osteoclastogenesis, 1046 Osteogenesis, 1136 Osteogenesis imperfecta (OI), 1241 Osteogenic differentiation, 573–574 Osteonectin (OCN), 574 Osteopontin (OPN), 447, 574 Outer nuclear layer (ONL), 335 Overall survival, 1424 Oxidative stress, 1452–1454, 1477 Oxygen consumption rate (OCR), 884 P Pancreatic adenocarcinoma (PADC), 1372 Paracrine, 359, 1010, 1127–1129, 1131–1133, 1138, 1139, 1144, 1145, 1147 agents, 74 hypothesis, 278 signaling, 527–528 Parkinson’s disease (PD), 405, 416, 429–431, 907, 909 Particulated articular cartilage implantation (PACI), 354 ACI, 357–358 advancement, 357 allogenic, 357 autologous, 357 treatment modality, 357 Particulate matter, 1197 Patch-based cell therapy, 783–784 Patch clamping, 886 Patentability, 1168–1170 Pathogen-associated molecular patterns (PAMPs), 177, 1199, 1282 Pathological scarring, 118 Pattern recognition receptors (PRR), 1283 Payload, 1369–1371, 1373, 1376–1378, 1380 Percutaneous coronary intervention (PCI), 475, 1027 Perfusion bioreactors, 371–372 Perianal fistulas, 207, 208 Pericytes, 395 Periodontal ligament MSCs, 548 Periodontal ligament stem/progenitor cells (PDLSCs), 1040 cytokines profile analysis, 1055 dental tissues regeneration, 1057 neural disorders, 1056 osteogenic potential, 1057 therapeutic effects, 1055 Peripheral blood mononuclear cells (PBMCs), 1394, 1463 Peripheral blood MSCs, 367

1520 Peripheral blood progenitor cells (PBPCs), 1423 apheresis, 1425 cell engraftment, 1428 cell storage, 1425–1426 conditioning regimen and myeloablative therapy, 1427 cryopreserved in liquid nitrogen, 1423 EBMT report, 1431–1438 mobilization, 1425 post-transplant data, 1428–1433 uses, 1423 viability, 1425 Peripheral blood stem cells, 1423, 1431 Persistent organic pollutants (POPs), 1197, 1213 Personalized medicine, 675 Pertinent muscle developmental biology, 633–634 excitation contraction coupling, 635 myogenesis, 634 neuromuscular transmission, 635 number of myofibers and strength, 635 Pharmaceuticals and Medical Devices Act (PMD Act), 700 Pharmacological manipulation, 441, 443, 444 Phenotypic assays, 883–886 Phenotypic screening, 906 Phosphatidylinositol 3-kinase/protein kinase B, 74 Pig models of myocardial injury, 280 Placenta, 134–135 Placenta-derived MSCs, 72 Placenta-derived stem cells (PSCs), 1393 Placental growth factor (PIGF), 1131 Placental tissue (PT), 1283 Plasminogen, 1189 Plastic adherence, 276 Plasticity, 257 Platelet-derived endothelial cell growth factor (PDEGF), 1042 Platelet-derived growth factor-α (PDGFR-α), 1102 Platelet-derived growth factor B (PDGF-B), 496 Pleiotropy, 1261 Pluripotency, 897, 898, 900, 903, 939 Pluripotent stem cells (PSCs), 476, 588, 594, 730, 1106 heart failure, 478–480, 520 Poland syndrome, 704 Poly ADP-ribose polymerase (PARP), 1404 Poly-ADP ribose polymerase 1 (PARP1), 1452

Index Polycaprolactones (PCL), 370 Polycyclic aromatic hydrocarbons (PAH), 1197–1198 Polyethyleneimine (PEI), 1054 Polygonal skin fibroblasts, 675 Polylactic acid (PLA), 370 Poly(lactide-co-glycolide) (PLGA), 370 Poly-L-ornithine, 825 Polyurethane (PU), 368 Polyvinyl alcohol (PVA), 788 POSEIDON trial, 140, 303–305 Post replication repair (PPR), 1449 Posttranslational modifications (PTMs), 883 Preclinical models, 608, 611 Preclinical research of MSC-based therapies, for ocular disease corneal diseases, models of, 325–331 EAE models, 340 glaucoma models, 340–342 hereditary diseases, optic nerve damage, 340 lens diseases, models of, 332–333 NMDA, model damage to, 337–338 PACG, animal models, 342 retinal models, 333–337 transgenic RGC loss models, 339–340 transient ischemia models, 339 Precondition(ing), 443–444, 550–551, 556 Predictors, 1264–1265 Prefrontal cortex (PFC), 431 Pre-scaffold fabrication bioprinting, 787 Primary angle-closure glaucoma (PACG), 342 Primary central nervous system lymphoma (PCNSL), 1270 Primary human corneal endothelial cell transplantation, 331 Proarrhythmic drug assessment, 886–887 Probiotics, 551–553 Progenitor cells, 934, 1423 PROGENITOR trial, 257 Programmed death ligand-1 (PD-L1), 1289 Progression-free survival (PFS), 1424 Pro-inflammatory cytokines, 199 Pro-inflammatory factors, 247 Proliferating cell nuclear antigen (PCNA), 1053 PROMETHEUS clinical trial, 140, 305 Prostaglandin E2 (PGE2), 1043 Protein-based systems, 897 Proteoglycans, 1184 Proteolytic activation, 1189 Proteomic analysis, 1044, 1107 Proton efflux rate (PER), 884 Purified exosome preparations (PEPTM), 1023

Index Q Quality and quantity culture (QQc), 1401 R Radial glial cells (RGCs), 830 Radiation therapy, 1391 Radioresistance, 1305 Randomized controlled trials (RCTs), 255 RA receptors (RAR1-3), 832 Reactive gliosis, 854 Reactive oxygen species (ROS), 243, 1128, 1185, 1194, 1451, 1452 Real time quantitative PCR (RT-qPCR), 883 Recruited leukocytes, 76 Red fluorescent protein (RPF), 442 Refractory angina (RA), 256–257 REGENERATE-AMI trial, 253 Regeneration, 1038 Regenerative effect, 200 Regenerative medicine, 54, 105, 134, 145, 532, 629, 701, 1230–1233, 1235, 1393 cardiac, 495–499 ethical issues in, 494–495 Regenerative therapy, 775 natural scaffolds, 775–780 synthetic scaffolds, 780–784 REGENT trial, 252 REGENT-VSEL trial, 257 Regulated marrow adipose tissue (rMAT), 1211, 1212 RELIEF trial, 140, 141 Renal function indexes, 656 REPAIR-AMI trial, 254 Repairing hereditary degenerative cells, 632 Replication-timing (RT), 1462 Replicative stress (RS), 1461 Reproduction, 1166–1168 Reprogramming, 596, 826, 898 of astrocytes, 393–394 Reprogramming methods, genetic instability, 1470 by-stander effect, 1476–1477 culture conditions, passage number, 1473–1476 mutations, 1471–1473 Resistin, 1477 Restoration, 1038 Retina autologous-IPE cells, 616 gene expression profiles, 615 hESCs, 615 iPSC-based autologous cell, 616

1521 iPSC-based treatments, 615 Muller cells, 597 neural retina, 596 pigment epithelial, 614, 616 pigment epithelium, 596–597 retinal photoreceptors, 614 Retinal ganglion cells (RGCs), 336–341, 608 Retinal models, 333–337 Retinal pigment epithelium (RPE), 333, 335 Retinitis pigmentosa (RP), 334, 608 Retinoic acid (RA), 832 Retinoic Acid-Receptor-Related Orphan Receptor gamma T (RORyT), 417 Retroviral transduction, 878 Rheumatoid arthritis (RA), 72, 144–145, 352 RIMECARD trial, 256, 484 Romberg disease, 704 Rosuvastatin, 450 Route of administration, 490–492 RPE-derived stem cells (RPESCs), 335 Runt-related transcription factor 2 (RUNX2), 447, 1050 Ryanodine receptors (RyRs), 884 S Safety, human-induced pluripotent stem cells, 1164–1166 Sarcomeres, 729 Sarcoplasmic reticulum (SR), 635, 884 Satellite cell (SC), 1099–1101, 1109 Scaffold-based 3D cell culture systems, 574 Scaffold-free strategies, 360 Scaffolds natural, 775–780 synthetic, 780–784 Schizophrenia, 429–431 Scotopic threshold response (STR), 339 scRNAseq analysis, 391 Secondary myeloid malignancy, 1201 leukemia-predisposing inherited BM failure syndromes, 1203–1206 therapy-related myeloid malignancy, 1201–1203 Secretome, 228, 229, 567, 575, 576, 801, 802 Secretome, MSCs, 1128–1129 angiogenesis and revascularization, 1131–1132 anti-apoptotic activity, 1132 bioactive factors, 1142–1143 bone regeneration, 1135–1137 in cartilage defects and cartilage degeneration treatment, 1137–1139

1522 Secretome, MSCs (cont.) cell-cell interactions, modulation of, 1143–1144 cell-substrate interaction, 1144 composition of EVs cargo, 1133–1135 disease-like priming, 1145–1146 extracellular vesicles, 1133–1135 genetic editing, 1144–1145 hypoxia induction, 1140–1141 immunomodulation, 1129–1131 inflammatory priming, 1141–1142 musculoskeletal disease treatment, 1135–1140 soluble factors, 1129–1132 tendon injuries, treatment of, 1139–1140 Secretome/conditioned medium (SHED-CM) cardiopulmonary injuries, 1045 dental-pulpal disorders, 1047 diabetes mellitus, 1046 hepatic disorders, 1045–1046 immunological disorders, 1046 neural injuries, 1047–1048 SEED-MSC, 141 Selective serotonin reuptake inhibitors (SSRI), 866 Self-formed ectodermal autonomous multizone (SEAM), 610 Self-inactivating (SIN) vectors, 1352 Self non-self discrimination, 923 Self-renewal ability, 977 Senescence, 1454, 1458, 1477 Senescence-associated secretory phenotype (SASP), 1454, 1458 Septovalvulogenesis, 736 Serum deprivation, 635 Serum response factor, 740 Severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), 165, 167, 176 Severe myocardial infarction, 651 Shear-thinning hydrogel (STG), 1023 Shwachman Diamond syndrome (SDS), 1205–1206 Sickle cell anemia (SCA), 907 Signalling molecules, 831–832 Signal transducer and activator of transcription (STAT)-4, 417 Simultaneous hybrid 3D bioprinting, 787 Simvastatin, 449 Single guide RNA, 390 Single muscle treatment (SMT), DMD, 643 efficacy, 643–644 safety, 643 significance, 644

Index Single nucleotide polymorphisms (SNPs), 880 Single nucleotide variation (SNV), 900, 1166 Single-photon emission computed tomography (SPECT), 257, 449 Sinusoidal obstructive syndrome (SOS), 183 Sirtuins, 1472 Skeletal muscle, 631 Skeletal muscle repair and regeneration, 1100–1101 immune response, 1102–1103 macrophages and lymphocytes, 1102–1103 muscular dystrophy, 1103 residential cells, 1101–1102 Skeletal myoblasts (SMs), 476, 529, 898, 1009 heart failure, 480–481, 520 Small animal models, 279–292 Small interfering RNA (siRNA), 865 Small molecule inhibitors, 743 and pleiotropic effects, 746–748 small molecule inhibitors in stem cell biology, 743–745 Small molecules, 387–388 Smoking, 1479 Soft tissue reconstruction, 704–709 Somatic cell nuclear transfer (SCNT), 1166 Somatic cell reprogramming efficiency, 745 Somatic cells, 898–899 Somatic cell therapy (SCT), 631, 644 Somatic stem cells (SSCs), 1231 Sonic hedgehog (Shh), 832 Sonomicrometric analysis, 301 Spheroid cultures, 568 Sphingosine-1 phosphate, 1190 Spike protein (S-protein), 167 Spinal cord-derived astrocytes, 396 Spinal cord injury, 399–402, 988–990 Spinal-derived oligodendrocytes, 397 Spinner flasks, 370 Sprague-Dawley rats, 555 SSEA-4, 137 Statins, 445–446 AMI, 452–460 combined statin and cell-based therapy, for AMI patients, 459–460 combined statin and MSCs-based cell therapy, 449–452 functional mechanism, 446 and in vitro experimental studies, 448–449 and in vivo experimental animal models, 449–459 on mesenchymal stem cells, 446–448 Steady-state hematopoiesis, 1186

Index Stem Cell Application Researches and Trials In Neurology-2 trial, 951 Stem cell-based therapy adult tissue-derived stem cells, 733–734 embryonic stem cells, 731–732 induced pluripotent stem cells, 732–733 myocardial repair, 441–442 Stem cell-based treatment, 610–612 Stem cells, 105, 1041–1043, 1392–1394 adult, 476 allogeneic sheep HSC, 926 asymmetric division, 1392 ciliary body, 593 ciliary pigment-epithelial, 588 clinical trials, 249–250 conjunctiva, 592 continuum, 1257–1258 cornea (see Cornea) embryonic, 111–112, 927 epithelial, 114–115 ESCs, 228, 1009 (see also Embryonic stem cells (ESCs)) exosomes (see Exosomes) functions, 247 heart diseases (see Heart diseases) hESCs, 168 hiPSCs, 176 induced pluripotent, 112–113, 231 iris, 592–593 IUT as tool (see In utero transplantation) large animal experimentation, 928 lens, 594–595 mesenchymal, 113–114 MSCs, 362 (see also Mesenchymal stem cells (MSCs) multipotentiality, 1392 NSCs (see Neural stem cells (NSCs)) in over-scarring, 117–118 paracrine activity, 1010 paracrine secretions, 229 plasticity, 934–939 and progenitor cell, 225 properties, 925 PSCs, 476 regenerative medicine, 608 regenerative potential of, 475–476 researchers, 227 in restoring heart function, 246–248 retina (see Retina) risks, 184–185 roles of, 105 somatic stem cell differentiation, 927 survival, 257

1523 symmetric divisions, 1392 therapy, 325 therapy in pathological scarring, 118 trabecular meshwork, 593–594 trafficking, 1190 transplantation, 232 trialled, 232 Stem/progenitor cells of apical papilla (SCAP), 1057–1058 Stem cells post-engraftment, 442–443 Stem cell therapeutics, heart failure bone marrow-derived cells, 518–519 cardiac stem cells, 517–518 direct regeneration, 528 embryonic stem cells, 520–521 false claims, 536–537 induced pluripotent stem cells, 515–516 mechanism of action, 527–528 mesenchymal stem cells, 519–520 paracrine signaling, 527–528 pluripotent stem cells, 520 skeletal myoblasts, 520 Stemness, 1282, 1283 Sterility, 879 Streptozotocin (STZ), 334 Stressed urinary incontinence (SUI), 676 Stress-induced regulated cell death, 1193 STRO-1, 135–136 Stroke extracellular vesicles (see Extracellular vesicles (EVs)) stem cell-based therapy, 949 Stromal cell-derived factor-1 (SDF-1), 496, 497, 1395 Stromal vascular fraction, 695–696 in plastic and cosmetic surgery, 704–714 regulatory considerations for the production and clinical application of, 700–701 soft tissue reconstruction, 704–709 Stromal vascular fraction (SVF), 30 Sub-retinal space (SRS), 339 Substitution therapy, 335 Suction of adipose tissue (SVF), 131 Sudden cardiac death (SCD), 878 Superoxide anion, 1452 Surface markers, 1306 Surgical trauma, 611 Sympathetic nervous system (SNS), 1181, 1188 Synovium fluid-derived mesenchymal stem cells (SMSCs/SFMSCs), 366 Synthetic microRNA, 865

1524 Synthetic scaffolds, 780 hydrogel based therapy, 781–783 patch-based cell therapy, 783–784 Systemic (intravenous) administration of MSCs, 210–213 T TAC-HFT-II trial, 485 TAC-HTF trial, 306 Targeted therapy, 1392 Target of rapamycin complex I, 1477 Tartrate-resistant acid phosphatase (TRAP), 1055 T-box protein expressed in T cells (T-bet), 417 T-cell metabolism, 1130 T-cell therapy, 181–182 TdT-mediated dUTP nick-end labelling (TUNEL) analysis, 1343 Telomere damage, 1457–1458 Tendon injuries, 1139–1140 Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, 301 Tetramethylrhodamine methyl ester perchlorate (TMRM), 741 Tetraspanins, 1109 Therapeutics, 1262, 1264, 1265, 1268, 1269, 1271 Therapy-related AML (t-AML), 1201–1203 Therapy-related myeloid neoplasms (t-MNs), 1202 Thermoregulation, 222 Thioredoxin-1, 84 3D anionic fibrillar cellulose, 572 3D-bioprinting, 785–787 bioinks for heart tissue, 789–790 cardiac tissue, complexity of, 785 μCOP, 788 heart tissue, techniques of, 787–789 pre-scaffold fabrication bioprinting, 787 process of, 787 simultaneous hybrid 3D bioprinting, 787 3D cardiac organoids, 751 analytical methods, 754 functional maturation analysis, 749–750 innovative methodologies for theranostic applications, 753 multicellular aggregates, 753 scaffold selection, 754 3D cell culture, cell fate decisions of MSCs, 569, 570, 576, 577 adipogenic differentiation, 574–575 cellular senescence, 572–573

Index chondrogenic differentiation, 575 MSC-secretome, immunomodulation and anti-inflammatory potential, 575–576 osteogenic differentiation, 573–574 viability and proliferation, 570–571 3D hydrogels, 568 3D hydroxypropylmethyl cellulose hydrogels, 575 3D nanofibrillar cellulose, 571 Thrombopenia, 7, 8 Thrombopoietin (TPO)-independent MK production, 1195 Thrombotic coronary occlusion, 271 TIR-domain-containing adapter-inducing interferon-β (TRIF), 1284, 1285 Tissue-engineered articular cartilage, 358 Tissue engineering, 497, 733, 734, 754, 1038 applications, 360 biomaterials, 367 cartilage, 353, 368 categories, 358 cell-scaffold construct, 358 cell source, 364 stem cell-based cartilage, 372 Tissue engineering/regenerative medicine (TERM), 775, 782 Tissue inhibitor of matrix metalloproteinase-3 (TIMP3), 988 Tissue inhibitor of metalloprotease 1 (TIMP-1), 1131 Tissue integration, 533 Tissue matrix metalloproteinase inhibitor-2 (TIMP-2), 1018 Tissue plasminogen activator (tPA), 861 Tissue regeneration, 35–36, 1041 Tissue repair, 70 T-lymphocytes, 1392 TNF-related apoptosis-inducing ligand (TRAIL), 75 Tolerogenic DCs, 420 Toll-interleukin-receptor domain (TIR), 1285 Toll-like receptor 3 (TLR3) and activation pathways, 1283–1286 miRNAs, 1292–1293 TLR3 mediated apoptosis and survival of MSCs, 1292 TLR3 priming and MSC differentiation, 1290–1292 TLR3 priming and MSC immunomodulation, 1288–1290 TLR3 priming and MSCs’ migration and proliferation, 1291–1292

Index Toll-like receptors (TLRs), 1056, 1110, 1282, 1284 Toll-like receptor (TLR)–NFκB, 1376 Toll-like receptor (TLR) signaling, 1199 TOPCARE-AMI trial, 254 Topological Associated Domains (TAD), 1460 Totipotent, 588 Toxic lipophilic substances, 1213 Trabecular meshwork, 593–594, 613 Traditional 2D cell culture, 572 Transcription activator-like effector nucleases (TALENS), 615, 865 Transcription coupled repair (TCR), 1449 Transcription factors, 386–387 Transdifferentiation, 938 Transendocardial injection, 306 Transendocardial route, 491 Transforming growth factor-β1 (TGF-β1), 980, 1131 Transforming growth factor-β (TGF-β) signaling, 731 Transgenic RGC loss models, 339–340 Transient immunosuppression, 445 Transient ischemia models, 338–339 Translational experimental studies, 1019–1023 Transmembrane serine protease 2 (TMPRSS2), 177 Transmission electron microscopy (TEM), 884 Transplantation, 113, 138, 141, 143, 230, 908, 1168 Transplant-related mortality (TRM), 1424 Traumatic brain injury (TBI), 402–403, 987–988 Trial and error screening of libraries, 388–390 Trialled stem cells, 232 Trinitrobenzene sulfonic acid, 199 Tronolab lentiviral production system, 1353 TruFit scaffold, 359 Tumor apoptosis, 1375 development, 1372 exosomes, 1376 microenvironment, 1372 suppression, 1373 Tumor-derived EVs (TEVs), breast cancer diagnostic and predictive markers, 1268 role, 1266–1267 treatment, 1267–1268 Tumor-derived exosomes, 1376–1378 Tumor necrosis factor-α (TNF-α), 247, 547, 671, 672, 1046 Tumorigenicity, 899–901

1525 Two-dimensional (2D) cell culture, 568, 570, 576 Type II diabetes, 666–667 drug discovery, MTT use in, 668 genetic diseases, multiple usage of MTT in, 668 human study, MTT in, 667–668 U UK Stem Cells Tool Kit (USCTK), 1170 Ulcerative colitis, 195, 199 allogeneic BM-MSCs, 211 hUC-MSCs in, 210 MSCs transplantation, 211 Ulex europaeus agglutinin 1 (UEA1), 1394 Ultrasonic bioreactor (USB), 372 Ultrasound-mediated localised direct reprogramming, 1354–1355 Ultrasound target bubble destruction (UTBD), 445 Ultrasound-targeted microbubble destruction (UTMD), 1338 advantages of lentiviral vectors for direct reprogramming, 1352 cardiac function improvement after, 1348 histological assessment of post-infarct ventricles after, 1343–1348 lentivirus–CMBs in gene delivery, 1351–1352 lentivirus delivery for cardiac repair, 1354–1355 microbubble/lentivirus hybrid delivery, 1358 reprogramming factors in direct cardiac reprogramming, 1353–1354 with CMB-lentiviral hybrid, 1348–1355 Umbilical cord, 132 Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), 67, 353, 362, 365–366 Umbilical cord blood (UCB), 26, 28, 31, 32, 1283 Umbilical cord-derived mesenchymal stem cells (UC-MSCs), 31–34, 256, 325, 328 Umbilical cord derived MSC-Exos (UC-MSC-Exos), 424, 425 Umbilical cord Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs), 353, 359, 364–365 Unfolded protein response (UPR), 1192 Universal stem cell, 1257 Unrestricted somatic stem cells (USSCs), 1465 Untranslated regions (UTRs), 1104

1526 Upper body treatment (UBT), 648 Urinary albumin creatinine ratio (UACR), 1077 Urine epithelial cells, 395–396 Urokinase-type plasminogen activator gene, 226 V Vaccination, 167–168 Valvular disease, 244 Vascular endothelial growth factor (VEGF), 181, 482, 496, 527, 575, 979, 1042, 1103, 1131 Vascular wall EPCs (VW-EPCs), 1397 Vascular wall-resident vascular stem cells (VW-VSCs), 1397 Vasculogenesis, 1044, 1395, 1397, 1403, 1406 Vasodilators, 246 Ventricular aneurysm, 658 Ventricular hypertrophy, 243 Ventricular myocardium, 657 Vertebrate muscular system, 631 Vesiclepedia, 1011 Vesicular activity, 1104 Virally-mediated lymphomas, 1272 Viruses (virulent), 168 Virus-mediated expression cloning method, 1338 Viscous fluid properties, 782 Visual acuity, 338 W Wernig lab, 389 Western blot analysis, 1357 Wharton’s jelly, 133–134 Wharton’s jelly-derived MSCs (WJ-derived MSCs), 118 advantages, 32 clinical applications, 34 isolation methods, 33–34 limitations, 33

Index Whole body treatment (WBT), DMD 25 billion myoblast WBT protocol, 649–650 50 billion myoblast WBT protocol, 650 donors, 648–649 dose escalation, 647 myoblast/placebo randomization, 648 myoblast preparation, 649 objectives, 647 rationale, 646 significance, 650 study design, 647–648 UBT/LBT randomization, 648 Wnt/beta-catenin signaling, 115 Wnt-pathway, 1286 Wnt signaling pathway, 75, 736–737, 832, 1310–1311 World Health Organization (WHO), 324, 513 Wound healing applications in defective, 115–117 excessive, 109–111 fetal vs. adult, 110 impaired, 108–109 normal, 105–108 stages of post-trauma, 106 stem cells in skin regeneration (see Stem cells) X Xenobiotic metabolism, 1197, 1198 Xenobiotic response elements (XREs), 1199 Xenobiotics, 1192, 1198 Xenogeneic mouse transplantation systems, 928

Z Zinc-finger nucleases (ZFNs), 865