Mesenchymal Stem Cells in Human Health and Diseases 9780128197134, 0128197137

Table of contents :
Cover
Mesenchymal Stem Cells in Human Health and Diseases
Copyright
Dedication
How To Use
Contributors
About the editor
Foreword by Alexzander A.A. Asea
Foreword by Junfeng Ji
Preface
Acknowledgments
1 - Mesenchymal stem cells in regenerative medicine and diseases: hope for better human health
References
Further reading
2 - Application of mesenchymal stem cells in human diseases
Introduction: background and driving forces
History of mesenchymal stem cells
Function of mesenchymal stem cells
Mesenchymal stem cells as seed cells participate in tissue regeneration
Mesenchymal stem cells for myocardial injury
Mesenchymal stem cells for osteoarthritis, lower back pain, and cartilage repair
Mesenchymal stem cells for pulmonary disease
Mesenchymal stem cells for liver disease
Mesenchymal stem cells for gastrointestinal disease
Mesenchymal stem cells as modulators participate in tissue homeostasis and regeneration
Controversy over and expectations for mesenchymal stem cells
Conclusion
References
3 - Immunoregulatory properties of mesenchymal stem cells and their application in immunotherapy
Immunomodulatory effects of mesenchymal stem cells
Mechanisms of antiinflammatory effects of mesenchymal stem cells
Prospective and concluding remarks
References
4 - Stem cell therapies in ocular repair, regeneration, and diseases
Introduction
Stem cell therapy for retinal diseases
Stem cell therapy for corneal diseases
Corneal stem cell types
Corneal and limbal stem cell markers
Regulatory factors of limbal stem cell proliferation
Stem cell therapeutic approaches for corneal diseases
Limbal stem cell deficiency
The management of patients with limbal stem cell deficiency
Conjunctival limbal autografts
Living related conjunctival limbal allograft
Cadaveric keratolimbal allografts
Simple limbal epithelial transplantation
Ex vivo expansion limbal stem cell transplantation
Cultivated oral mucosal epithelial transplantation
Conclusions, challenges and future directions
References
Further reading
5 - Applications of the stem cell secretome in regenerative medicine
Introduction
The potential of stem cells in regenerative medicine
The concept of the secretome
Modulation of the stem cell secretome profile
Three-dimensional cultures and biomaterials
Preconditioning of stem cells
Bioreactors
Application of the stem cell secretome in CNS disorders
Stroke
Traumatic brain injury
Parkinson disease
Spinal cord injury
Concluding remarks
References
6 - Innovation in induced mesenchymal stem cell uses in therapy
Introduction
Major sources of induced mesenchymal stromal/stem cells
Induced mesenchymal stromal/stem cell applications in cell therapy
Conclusion
References
7 - Role of mesenchymal stem cells in bone fracture repair and regeneration
Introduction
Sources of mesenchymal stem cells for bone regeneration
Mesenchymal stem cell homing in bone regeneration
Molecular mechanisms affecting mesenchymal stem cell homing
External factors affecting mesenchymal stem cell homing
The application of mesenchymal stem cells in bone tissue regeneration
Immunogenicity of mesenchymal stem cells
The isolation and treatment approach of mesenchymal stem cells
Genetically modified mesenchymal stem cells for bone regeneration
Application of biological materials and growth factors in mesenchymal stem cell bone repair
Clinical consequences
Conclusion
References
8 - Tendon stem cells and their interaction with microenvironments
Introduction
Isolation and culture of tendon stem/progenitor cells
Characterization of tendon stem/progenitor cells
Culture conditions
Cell passage and senescence
Growth factors
Mechanical stimulation
Extracellular matrix and scaffold
Clinical relevance: the role of tendon stem/progenitor cells in tendon disease
Conclusion and forecast
Acknowledgments
References
9 - Mesenchymal stem cell roles in osteoarthritis (joint) disease
Conclusion
References
Further reading
10 - Mesenchymal stem cells and cancer therapy
Introduction
The application of mesenchymal stem cells in cancer treatments
Limiting factors of the mesenchymal stem cell–mediated cancer therapies
Conclusions and future directions
References
11 - Mesenchymal stem cells in human health and diseases: general discussion, remarks, and future directions
References
Index
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Citation preview

Mesenchymal Stem Cells in Human Health and Diseases

Edited by Ahmed H.K. El-Hashash The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-819713-4 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Stacy Masucci Acquisition Editor: Elizabeth Brown Editorial Project Manager: Pat Gonzalez Production Project Manager: Punithavathy Govindaradjane Cover Designer: Matthew Limbert

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This book is dedicated to: Dr. Magdy Elhefnawy: You always have fingerprints on our lives. Thanks for lighting up our lives; My great parents, my brilliant wife Heba and her parents, and my children Hoor, Noor, and Lien; The memory of Dr. Gamal Madkour: Your inspiration, support, and words to me shan’t be forgotten. Ahmed H.K. El-Hashash, PhD

There are no incurable diseasesdonly the lack of will. There are no worthless herbsdonly the lack of knowledge. Medicine is the science by which we learn the various states of the human body in health and when not in health, and the means by which health is likely to be lost and, when lost, is likely to be restored back to health. In other words, it is the art whereby health is conserved and the art whereby it is restored after being lost. While some divide medicine into a theoretical and a practical [applied] science, others may assume that it is only theoretical because they see it as a pure science. But, in truth, every science has both a theoretical and a practical side. Medicine is, therefore, the preservation of health and the cause of disease which arises from causes that exist within the body. dIbn Sina (Avicenna); The Canon of Medicine

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Contributors Yangwu Chen, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Yishan Chen, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Xiao Chen, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Xiaotian Du, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Ahmed H.K. El-Hashash, The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC Mohamed Elalfy, Research Institute of Ophthalmology, Giza, Egypt; Queen Victoria Hospital and Eye Bank, East Grinstead & Maidstone and Tunbridge Wells NHS Trust, East Grinstead, United Kingdom Kareem Elsawah, Research Institute of Ophthalmology, Giza, Egypt Deming Jiang, Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Junxin Lin, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Ana Marote, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Cla´udia R. Marques, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Ba´rbara Mendes-Pinheiro, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Jorge Cibra˜o Ribeiro, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal

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xiv Contributors Deepak Rohila, Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Anto´nio J. Salgado, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Dmytro Shytikov, Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Eman E. Taher, Research Institute of Ophthalmology, Giza, Egypt; UPMC Eye and Ear Institute, Ophthalmology & Visual Sciences Research Center, University of Pittsburgh, Pittsburgh, PA, United States Fa´bio G. Teixeira, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Pengfei Wang, Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Yun Xu, Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Zi Yin, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Yeke Yu, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC Wenyan Zhou, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC

About the editor Professor Ahmed H.K. El-Hashash completed his PhD at Manchester University (UK). He is a Fellow of the California Institute for Regenerative Medicine and New York University School of Medicine (USA). Professor Ahmed H.K. El-Hashash has worked as a senior biomedical research scientist at the Mount Sinai School of Medicine of New York University and Children’s Hospital Los Angeles. He was an assistant professor and the Principal Investigator of Stem Cell and Regenerative Medicine at the Keck School of Medicine and the Herman Ostrow School of Dentistry of the University of Southern California (USA). Professor Ahmed H.K. El-Hashash has joined the University of Edinburgh’s Edinburgh-Zhejiang international campus as a tenure-track Associate Professor and Senior Principal Investigator of Biomedicine, Stem Cell and Regenerative Medicine. He is also an adjunct professor at the School of Basic Medical Science and School of Medicine, Zhejiang University. Professor El-Hashash has several breakthrough discoveries in genes and enzymes that control stem cell behavior and regenerative medicine. He has published more than 34 papers and abstracts in reputed international journals and is serving as an editorial board member of repute. Professor El-Hashash acts as a discussion leader at the prestigious Gordon Research Conferences in the United States and is a peer reviewer/international extramural reviewer for Medical Research Council grant applications (London, UK). Professor El-Hashash has been invited to cochair, coorganize, and/or present his research at several international conferences in the United States, Spain, Greece, Egypt, Germany, and China. He is the editor or author of several books on stem cell and regenerative medicine published by the prestigious publishers SpringereNature, Elsevier, and Imperial College London Press.

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Foreword by Alexzander A.A. Asea It is with great pleasure that I pen this foreword to Mesenchymal Stem Cells in Human Health and Diseases. The field of stem cell biology and its application in human diseases is moving extremely rapidly, as the concept and potential practical applications have moved from theoretical concepts into human clinical trials with often outstanding results, and has thus entered the mainstream. Despite this worldwide intensity and diversity of endeavor, there remain a small number of book volumes that are focused almost entirely on mesenchymal stem cells in human health and diseases, and this volume is an example of this kind of book that also brings the novel scientific findings of most of the leaders of this research field together. The concept of stem and progenitor cells has been around for a long time. However, it was the progress in embryonic stem cells that truly led the field of stem cell research and related fields such as regenerative medicine. Research on mouse embryonic stem cells originally came from many studies that aimed at the identification of mechanisms in the control and progression of embryonic differentiation. Despite being magnificent, cell differentiation in culture was overshadowed experimentally by their use as a vector to the germ line and hence as a vehicle for experimental mammalian genetics. These studies led to research on targeted mutations in up to one-third of gene loci and an ongoing international program to provide mutations in every locus of the mouse genome. A positive outcome of these studies has been to greatly illuminate our understanding of human genetics. In addition, promising research that focuses on discovering the equivalent human embryonic stem cells will certainly provide a universal source of a diversity of tissue-specific precursors, as a resource for tissue repair and regenerative medicine. Progress toward understanding various aspects of mesenchymal stem cell biology and development, including self-renewal, cellular differentiation, and pluripotentiality, that are fundamental to developmental biology at the cell and molecular level now stands as a gateway to major clinical applications, both now and in the future. This book provides a timely, up-to-date, state-of-the-art reference on mesenchymal stem cell roles in human health and disease. Understanding the functions of endogenous tissue-specific stem and progenitor cells in various organs will greatly enhance the use of stem cells in

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xviii Foreword by Alexzander A.A. Asea

tissue repair and regeneration, and as a therapy for a wide range of human diseases. The rapid advances in different stem cell types, including induced pluripotent stem cell research, and their potential clinical applications point to the great possibilities for patient-specific treatment. As Sir Martin Evans, who is credited with discovering embryonic stem cells and who received the Nobel Prize for Physiology or Medicine in 2007, said, “this patient-specific ad hominem treatment will help to open the door to personalized medicine, in which patients are stratified into different groups and therapeutics are tailored based on the individual patient’s response.” However, thus far the high costs associated with this technology may not allow it to be commercially viable, as Sir Evans said. Quite properly, much of this book concentrates on the roles of mesenchymal stem cells in human health and disease, from which the solid clinical applications will arise. Professor Alexzander A.A. Asea University of Texas M.D. Anderson Cancer Center Houston, Texas, United States

Professor Alexzander A.A. Asea is currently a professor and the director of the Precision Therapeutics Proteogenomics Diagnostics Center at the University of Toledo College of Medicine (USA). Professor Asea was a professor and consultant immunologist at the University of Texas M.D. Anderson Cancer Center (Houston, USA). Professor Asea obtained his PhD from the University of Gothenburg (Sweden), where his studies formed the basis for clinical trials of combined histamine and interleukin-2, a drug now known as Ceplene, currently prescribed for patients with metastatic melanoma and high-risk acute myelogenous leukemia. Professor Asea is a highly innovative and accomplished world-renowned research scientist and visionary executive academic leader, with exceptional executive leadership experience spearheading strategic planning, research, training, education, and commercialization initiatives. Professor Asea has received numerous honors and awards, and has received grant funding from the federal government, industry, private foundations, and local community groups. Professor Asea currently has five pending patents and over 255 scientific publications, books, reviews, news headlines, and editorials in a wide range of medical disciplines, including stem cell biotherapeutics, cancer, diabetes, obesity, neuroscience, cardiovascular disease, exercise immunophysiology, aging, nanotechnology, thermal therapy, medicinal plants, and biomarker discovery.

Foreword by Junfeng Ji It is with great pleasure that I compose this foreword to Mesenchymal Stem Cells in Human Health and Diseases. Stem cells are found in almost all organisms from the early stages of development to the end of life. There are several types of stem cell that have been reported. These different stem cell types may prove useful for medical research; however, each of the different types has both promise and limitations. Stem cell is a fast-growing field of research. Sir Martin Evans, who is credited with discovering embryonic stem cells, received the Nobel Prize for Physiology or Medicine in 2007; and Shinya Yamanaka, who discovered how to reprogram differentiated cells into induced pluripotent stem cells (iPSCs), won the Nobel Prize in 2012 for the achievement. Much more research has been done, and a lot of discoveries have been published. Much has been learned in a relatively short time on the biology of stem and progenitor cells and their potential applications in tissue repair and regeneration as well as in the clinic. This book covers the latest advances in mesenchymal stem cells in human health and diseases. Regenerative medicine is a branch of translational research in tissue engineering and molecular biology, which deals with the “process of replacing, engineering, or regenerating human cells, tissues, or organs to restore or establish normal function.” This field holds the promise of engineering damaged tissues and organs by stimulating the body’s own repair mechanisms to functionally heal previously irreparable tissues or organs. Applying recent discoveries in stem cell biology and regenerative medicine, in the lung for example, to the betterment of human diseases has brought forth much hope but continues to present many challenges. The hope for cures has motivated different states and countries worldwide to invest in stem cell and regenerative medicine research. Since 2000, major advances have occurred in both the stem cell and the regenerative medicine fields, with several new classes of stem cells being described. Thus, if one were to do a PubMed search with the term “stem cells,” it would yield at least 200,000 entries related to scientific research articles published in peer-reviewed journals covering the past 5 decades. Notably, a similar search for the prior 5 decades yields only a total of less than 100 references. It is obvious that the stem cell research field is fast growing, and that this rapid growth has major advantages and implications for the future of stem cell knowledge and clinical translation. The characterization of xix

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mesenchymal stem cells and iPSCs, for example, brought many more avenues of research and discovery. Many clinical trials were initiated and conducted with multiple different stem cell types, including those derived from embryonic, cord, bone marrow, amnion, and fat sources, and manufacturing processes are now in place for a wide range of stem cell types. Thus, in 2012, the first paper showing results of two patients treated with human embryonic stem cells, which were first isolated just 14 years previously, in 1998, was published. Many studies have been carried out on endogenous stem cells, including mesenchymal stem cells, in different organs to achieve a greater understanding of tissue turnover and responses to injury. A wide range of these studies is focused on how we can harness the power of endogenous stem cells as a source for regenerative medicine. Successful clinical application has been achieved for some organs, including the hematopoietic system. However, difficulties in identification, isolation, and expansion of many of these cell types ex vivo in other organs have limited their widespread application. Finally, mesenchymal stem cells, like other stem cell types, have attracted much attention largely because of their potential therapeutic use in regenerative medicine and tissue repair and for developing therapies for a wide range of diseases, such as in cancer to eliminate cancer stem cells. Understanding the basic molecular, cellular, and genetic mechanisms that regulate stem biology, such as cell proliferation, self-renewal, and differentiation, is a very hot topic in stem cell biology and medicine and developmental biology, and will lead to harnessing the ability of these cells in tissue repair and regeneration after injury as well as in human disease. Professor Junfeng Ji Chairman, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

Preface The research on stem cells can be traced back almost 40 years to when two British pioneer scientists, Sir Martin Evans and Matthew Kaufman, together with the American scientist Gail Marin, were the first to culture and derive embryonic stem cells (ESCs) from mouse embryos in 1981 in a laboratory. This was followed by the important discovery of human ESCs in 1998. Stem cell research is a fast-growing field that has remarkably expanded as new research and experience broaden our knowledge about different aspects of stem cell biology and applications, making stem cell research one of the most exciting aspects of biomedical research. Mesenchymal stem/stromal cells (MSCs) are widely distributed in the body and can therefore be isolated from many tissues and organs, such as the bone marrow, skin, bodily fluids, heart, and perinatal tissues. MSCs are one of the most commonly investigated stem cell populations, and the most commonly used cell-based therapy for several diseases, such as neurodegenerative, cardiovascular, and other diseases, in clinical trials because of their regenerative effects. At this writing, 695 US clinical trials are testing the utility of MSCs as therapeutic agents for an array of medical conditions. Scientists worldwide continue to apply new stem cellebased discoveries, including mesenchymal stem cells, to the betterment of human diseases, which has clearly brought forth much hope for better human life. Since 2000, there is an increased number of publications and discoveries on mesenchymal stem cells from a wide range of research universities and institutes in the United States, Europe, Japan, Australia, Canada, China, and many other countries, which have established centers for stem cell research and regenerative medicine. This book on mesenchymal stem cells brings together several important topics that are related to mesenchymal stem cell biology, properties, and applications in tissue repair and regeneration to treat diseases. It aims to provide an important updated resource for undergraduate students, graduate students, researchers, and clinicians in key and recent applications of mesenchymal stem cells for some selected and important human diseases. The book has 11 chapters, covering key applications of mesenchymal stem cells in tissue repair and regeneration to treat human diseases. Chapters 1 and 2 introduce the importance and general application of mesenchymal stem cells in tissue repair and regeneration to treat human diseases, while Chapter 3 describes the immunoregulatory properties of mesenchymal stem cells and their application in immunotherapy.

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xxii Preface

Chapter 4 focuses on the application of stem cells in ocular repair, regeneration, and diseases, while Chapter 5 discusses the applications of the stem cell secretome in regenerative medicine and Chapter 6 covers recent innovations in induced mesenchymal stem cell uses in therapy. Chapters 7e10 introduce the applications of mesenchymal stem cells in the repair and regeneration of damaged or diseased tissues or organs, including bone fracture (Chapter 7), tendon diseases (Chapter 8), osteoarthritis (joint disease) (Chapter 9), and cancer (Chapter 10). Chapter 11 describes conclusions drawn from research investigations on the role of mesenchymal stem cells in tissue repair and regeneration to treat human diseases that are described in Chapters 1e10, and both proposes future work and discusses future directions for the application of mesenchymal stem cells in tissue repair and regeneration. Although we could not hope to be comprehensive in the coverage of mesenchymal stem cells of different types of tissues and organs, our main goal in compiling this book was to bring together a selection of works on the current progress in understanding the properties and applications of mesenchymal stem cells in tissue repair and regeneration to treat human diseases. In preparing this mesenchymal stem cellefocused book, we aimed at making it accessible not only to those working in the stem cell and regenerative medicine field, but also to nonexperts with a broad interest in mesenchymal stem cell biology and regeneration biology/medicine in human health. Our hope is that this book will be of value to all concerned with mesenchymal stem cell application in medicine. We are indebted to our authors, who graciously accepted their assignments and who have infused the text with their energetic contributions. We are incredibly thankful to Elizabeth Brown, Patricia Gonzalez, and other staff at Elsevier, who published this book. Professor Ahmed H.K. El-Hashash, PhD The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC

Acknowledgments The editors would like to acknowledge: Esam I. Agamy College of Medicine, University of Sharjah, Sharjah, Sharjah, Untied Arab Emirates Wadah AlHassan California State Polytechnic University, Pomona, CA, United States Alexzander A.A. Asea Center for Radiation Oncology Research, Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Mohamed Berika Anatomy Department, Mansoura University Faculty of Medicine, Egypt and Applied Medical Science College, King Saud University, Rehyad, Kingdom of Saudi Arabia Karen Ek California State University San Bernardino, San Bernardino, CA, United States Magdy Elhefnawy Gharbia Medical Syndicate, Tanta University Medical School, Tanta, Egypt Thoria Roshdy Haggag Department of Internal Medicine, Mansoura University Hospital, Mansoura, Egypt Ali Kotab Hasan Faculty of Education, Tanta University, Tanta, Gharbia, Egypt Haifen Huang California State Polytechnic University, Pomona, CA, United States Junfeng Ji Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China John Ku California State Polytechnic University, Pomona, CA, United States Karol Lu University of Southern California, University Park, Los Angeles, CA, United States Gamal Madkour Department of Zoology, Tanta University School of Science, Tanta University, Tanta, Gharbia, Egypt Moustafa Mahmoud Department of Surgery, Tanta University School of Medicine, Tanta University, Tanta, Gharbia, Egypt Mohamed Labib Salem Center of Excellence in Cancer Research, Tanta University, Gharbia, Egypt, and Medical University of South Carolina, Charleston, SC, United States

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xxiv Acknowledgments

Osama Saad Salama Department of Clinical Pathology, Mansoura University Faculty of Medicine, Mansoura, Dakahlia, Egypt Mohamed Sharaf-Eldin Department of Tropical Medicine and Infectious Diseases, Faculty of Medicine, Tanta University, Tanta, Gharbia, Egypt

Chapter 1

Mesenchymal stem cells in regenerative medicine and diseases: hope for better human health Ahmed H.K. El-Hashash1, 2 1 The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; 2Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC

The stem cell research field has grown fast since the beginning of the 21st century, with many new and astonishing discoveries, representing the most exciting aspects of biological and biomedical research. Remarkably, the stem cell research field is growing over twice as fast (7%) as the reported world average growth in research, which is 2.9%. Since 2008, the annual growth rate of studies on one rapidly grown type of stem cell, induced pluripotent stem cells, the discovery of which was awarded the Nobel Prize in Physiology or Medicine in 2012, is an astonishing 77%. A significant increase in the volume of research output and publications has been reported in all areas of stem cell research. Major advances have been achieved in the stem cell research field by the generation of the first functioning whole organ, the thymus, in the laboratory, and the first documented human baby girl from in vitro fertilization, who now has children of her own. Stem cell and biomedical research is under way in various national and international laboratories with ambitious goals of generating several other functioning whole organs, including kidney and intestine. Advances in stem cell biology have seen the rise of an exciting new field of regenerative medicine and research. Regenerative medicine is a multidisciplinary branch of translational research in tissue engineering and molecular biology, which deals with the “process of replacing, engineering, or regenerating human cells, tissues, or organs to restore or establish normal function” Regenerative medicine, therefore, aims at repairing injured tissues to restore normal cellular function. This field holds the promise of engineering damaged Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00001-3 Copyright © 2020 Elsevier Inc. All rights reserved.

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tissues/organs via stimulation of the body’s own repair mechanisms to functionally heal previously irreparable tissues/organs. Several classes of stem cells that originate from different mesenchymal compartments of the body, including the marrow, amnion, adipose tissue, and amniotic fluid, exert promising therapeutic effects in some important human fibrotic and inflammatory diseases, identifying the next generation of cellbased therapy of human diseases based on mesenchymal stem cells. As of this writing, the cell population that is most commonly studied in clinical trials comprises mesenchymal stem/stromal cells (MSCs), which are self-renewing multipotent cells. MSCs are, therefore, the most commonly used cell-based therapy in clinical trials because of their regenerative effects. Experimental and clinical studies have provided promising results using MSCs to treat diabetes. The therapeutic potential of MSCs is based on their remarkable ability to differentiate into multiple cell types, ease of isolation, low immunogenicity, and capacity to secrete multiple biologic factors that can restore, repair, and alleviate injured or impaired tissues. Preclinical and clinical evidence has substantiated the therapeutic benefit of MSCs in various medical conditions. MSCs are widely distributed in the body and can therefore be isolated from multiple sources, including the bone marrow, heart, bodily fluids, skin, and perinatal tissues. They can react to microenvironmental changes (stress, pH, oxygen) by releasing immune modulatory and trophic factors known to regenerate injured cells and tissues. Experimental findings in neurodegenerative and cardiovascular disease have supported the rapid growth of cell-based research. At this writing, 695 US clinical trials are testing the utility of MSCs as therapeutic agents for an array of medical conditions. Understanding the biology of MSCs and their mechanisms of development and function could identify innovative solutions for the treatment of many stem-based diseases/disorders, restoring normal morphogenesis and/or regeneration of various organs. There are also likely to be advances in the potential applications of these stem cells in the repair and regeneration of various organs after injury, as well as in the treatments of various human diseases. Applying recent discoveries in MSC biology and regenerative medicine for the betterment of human diseases has brought forth much hope, but continues to present many challenges. The hope for cures has motivated different states and countries worldwide to invest in stem cell and regenerative medicine research. For instance, the people of California have been prompted to strongly mandate an amendment to the state’s constitution establishing the California Institute for Regenerative Medicine (California Institute for Regenerative Medicine Annual Report, 2018) with a $3 billion bond issue. This state-based funding has significantly boosted research on stem cells and regenerative medicine in California and other states (Alberta et al., 2015). Similar interests and investments in the stem cell, regenerative medicine, and cellular

Mesenchymal stem cells in regenerative medicine and diseases Chapter | 1

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therapeutics field are now in process in Japan, Germany, Australia, the United Kingdom, and elsewhere. Therefore, stem cell research growth is twice as fast (7%) as the world average growth in research, which is almost 3%, and stem cell-based published articles are cited 50% more often than the world average for all related subject areas, according to the report Stem Cell Research: Trends and perspectives on the evolving international landscape (2013). This book contains a global collection of monograph essays from collaborating research scientists at various research institutes and countries worldwide. They describe exciting progress in MSC biology and regenerative medicine, including MSC applications in the treatment of human diseases. The book includes insights ranging from MSC biology and development through the derivation and characterization of MSCs, as well as their applications in tissue repair/regeneration and important human diseases. This book, therefore, discusses the fact-based promise of MSC therapeutics and regenerative medicine in the real world.

References Alberta, H.B., Cheng, A., Jackson, E.L., Pjecha, M., Levine, A.D., 2015. Assessing state stem cell programs in the United States: how has state funding affected publication trends? Cell Stem Cell 16 (2), 115e118. California Institute for Regenerative Medicine, 2018. California Stem Cell Report 2018. https:// www.cirm.ca.gov/sites/default/files/files/about_cirm/CIRM%202018%20Annual%20Report.pdf. Stem Cell Research: Trends and perspectives on the evolving international landscape, 2013. A Joint Report by Elsevier, EuroStemCell and Kyoto University’s Institute for Integrated Cell-Material Sciences (iCeMS) Including Scientists’ and Other Stakeholders’ Views on Current Progress and Future Expectations for the Field. https://www.elsevier.com/__data/assets/ pdf_file/0005/53177/Stem-Cell-Report-Trends-and-Perspectives-on-the-Evolving-InternationalLandscape_Dec2013.pdf.

Further reading Ralston, M., 2008. Stem Cell Research Around the World. https://www.pewforum.org/2008/07/17/ stem-cell-research-around-the-world/.

Chapter 2

Application of mesenchymal stem cells in human diseases Wenyan Zhou1, Yun Xu2 1 Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC; 2Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC

Introduction: background and driving forces Adult tissue stem cells lose pluripotency through aging and disease, which hinders normal tissue function. Mesenchymal stem cells (MSCs), found in a variety of adult tissues, have the ability to differentiate into various mesenchymal lineages in vitro and also show a mighty immunomodulatory ability; these properties make MSCs potential therapy candidates for tissue engineering and regeneration medicine.

History of mesenchymal stem cells Friedenstein transplanted bone fragments and found that nonhematopoietic mesenchymal tissue could form in the heterotopic area; he named the cells osteoblasts (Friedenstein et al., 1968). Subsequently, a number of studies were carried out to confirm the colony formation and plastic adherent ability (Friedenstein et al., 1970, 1976). Similar cells were found in human bone marrow (Castro-Malaspina et al., 1980); these cells with the capability of selfrenewal and in vitro multilineage differentiation potential were named “mesenchymal stem cells” by Caplan (Caplan, 1991). The next decade, a series of studies focused on verifying the stem cell properties of MSCs, but not until 1997 was the in vivo bone formation capacity of human bone marrow MSCs (BMSCs) verified (Kuznetsov et al., 1997). Ever since the nomenclature MSCs was adopted, the correctness of the name has been a matter of concern. According to the antigen expression of the adherent cells of bone marrow, “mesenchymal” was accredited, but there is no uniform answer to which antigen (a single antigen or a small panel of antigens) could be used to define “stem” (Horwitz and Keating, 2000). To solve this puzzle, the International Society for Cellular Therapy (ISCT) gave a new Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00002-5 Copyright © 2020 Elsevier Inc. All rights reserved.

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terminology, mesenchymal stromal cells, to all of the plastic-adherent mesenchymal cells, no matter the tissue origin, and only when the stemness is demonstrated by clearly stated criteria can these cells be called mesenchymal stem cells (Horwitz et al., 2005), but the acronym MSC is still kept in use. The next year, the ISCT issued minimal criteria to define human MSCs (here referring to mesenchymal stromal cells), including adherence to plastic, specific surface antigen expression, and multipotent differentiation potential (Dominici et al., 2006). Although the ISCT had proposed the difference between mesenchymal stromal cells and MSCs, these two terminologies were still confused. Researchers use the minimal criteria to define MSCs without evidence of stem cell activity (Le et al., 2007; Soleimani and Nadri, 2009). During the next decade, investigators cared more about the function of MSCs; transplantation of MSCs can enhance the engraftment of hematopoietic stem cells (HSCs) (Chao et al., 2012; Le et al., 2007). Due to their multipotential capacity, MSCs can be used to repair tissue injury (Han et al., 2015; Martino et al., 2016). Since the highly active cytokine-secreting property was found, the role of MSCs in immunomodulation and homeostasis maintenance has been studied (Davies et al., 2017; Zhao et al., 2015). This makes MSCs a potential therapeutic cell, which may become a commercialized product, so it is vital to produce MSCs in batches as a standard therapeutic during largescale proliferation. In 2016, the ISCT made a statement on this issue, highlighting that culture conditions can influence MSC functions, so the clinical application should be the guidance for large-scale proliferation, to optimize the culture system (Martin et al., 2016).

Function of mesenchymal stem cells Mesenchymal stem cells as seed cells participate in tissue regeneration Mesenchymal stem cells for myocardial injury MSCs were treated with hypoxia, preconditioned for 24 h, and tested in a monkey’s infarcted heart. Ninety days after the first injection the infarct size and left ventricular (LV) function, as well as cardiomyocyte proliferation, vascular density, and myocardial glucose uptake, were significantly improved compared with the control group (Hu et al., 2016a,b). Human MSCs treated with 5% O2 were administered intravenously into mice with LV dysfunction. LV ejection fraction and infarct size were significantly improved in the MSC group after 21 days (Luger et al., 2017). A double-blind, placebo-controlled, dose-ranging trial was conducted in 53 patients. The result showed that the forced expiratory volume in 1 s and LV ejection fraction were improved in patients treated with human MSCs (Hare et al., 2009).

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Mesenchymal stem cells for osteoarthritis, lower back pain, and cartilage repair Chondrogenic progenitor cell-based stem cell therapy has been studied for cartilage regeneration in osteoarthritis (OA) (Koelling and Miosge, 2009). In an animal model tumor necrosis factor a (TNFa) inducing leucine-rich a2glycoprotein 1 (LRG1) secretion contributed to angiogenesis coupling with aberrant bone formation; the LRG1 inhibitor lenalidomide could be a potential therapeutic approach for OA treatment (Wang et al., 2017). Recombinant Atsttrin, which expressed by MSCs is a novel TNFa blocker and play a chondroprotective role in ameliorating OA development (Xia et al., 2015). Human MSCs injected intraarticularly were activated to express high levels of Hedgehog and trigger type II collagen to enhance rat meniscal regeneration (Horie et al., 2012). Delayed fracture union can be prevented by MSCs mixed with platelet-rich plasma (Liebergall et al., 2013). Orozco et al. presented a phase I/II study of 12 patients with clinical and objective follow-up coverage for 1 year after intraarticular MSC injection. MSC administration appears to be safe, and the result showed that 65%e78% of patients exhibited rapid and progressive improvement and most of them emerged with improvement of cartilage quality (Orozco et al., 2013). Two-year follow-up results showed that the quality of cartilage had further improved (Orozco et al., 2014). 71% of patients with degenerative disc disease transplanted with MSCs exhibited rapid improvement of pain and disability, but without disc height recovery (Orozco et al., 2011). How to prolong the cells’ intraarticular longevity became a focus of attention. MSC pretreatment with inflammatory factors or hypoxia does not influence migration or adhesion to osteoarthritic cartilage and synovium (Leijs et al., 2017). Alternative approaches should be developed to improve the therapeutic effect. Mesenchymal stem cells for pulmonary disease A phase I study has shown that in severe chronic obstructive pulmonary disease patients, combined one-way endobronchial valve insertion and MSC treatment can decrease levels of circulating C-reactive protein at 30 and 90 days, as well as the BODE (body mass index, airway obstruction, dyspnea, exercise) index and modified Medical Research Council score (de Oliveira et al., 2017). A phase II study showed that bronchopulmonary dysplasia patients receiving intratracheal transplantation with allogeneic human umbilical cord blood-derived MSCs have lower severity than the control group (Chang et al., 2014). MSCbased therapies were used to treat acute respiratory distress syndrome (ARDS) patients; serious adverse events (SAEs) were subsequently observed in three of nine patients, even they thought those SAEs were not related to the MSCs. Phase II is already under way to treat ARDS (Wilson et al., 2015).

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Mesenchymal stem cells for liver disease Adipose-derived MSCs (ADSCs) and BMSCs seeded in regenerated silk fibroin (RSF) scaffolds were evaluated in a CCl4-induced liver injury mouse model. Results showed that neovascularization, a bile canaliculus-like structure, and some hepatocyte-like cells were observed after transplantation of RSF MSCs (Xu et al., 2017). A phase II trial evaluated the effects of BMSCs transplanted in cirrhotic patients. Three and six months after surgery patients exhibited partial improvement of liver function (El-Ansary et al., 2012). Thirty to fifty million MSCs were injected into eight patients with end-stage liver disease. According to the results, serum albumin, bilirubin, and liver function score were all improved (Kharaziha et al., 2009). In 54.5% of alcoholic cirrhosis patients histological improvements were observed after BMSC injection, and the levels of transforming growth factor b1 (TGFb), type I collagen, and a-smooth muscle actin were decreased (Jang et al., 2014). MSC transplantation can be used as a potential treatment for liver injury. Mesenchymal stem cells for gastrointestinal disease Silanized hydroxypropylmethylcellulose hydrogeleloaded ADSCs improve colonic epithelial structure as well as hyperpermeability in a rat model of radiation-induced severe colonic damage (Moussa et al., 2017). Crohn’s disease patients were given intravenous infusions of allogeneic MSCs weekly for 4 weeks. Forty-two days after the first MSC administration, 8/15 had clinical remission, and 7/15 had endoscopic improvement (Forbes et al., 2014). In a phase III randomized, double-blind controlled trial using ADSCs for complex perianal fistulas in Crohn’s disease, 212 patients were randomly divided into two groups with MSCs or placebo. After 24 weeks, a significant remission was found in the MSC group compared with the placebo group (53 of 107 vs. 36 of 105) (Pane´s et al., 2016). Mesenchymal stem cells as modulators participate in tissue homeostasis and regeneration MSCs usually work through systemic infusion to cure disease. Although this method can show the effectiveness of treatment, there is little direct evidence that MSCs differentiate into injured tissue cells, so there must be another mechanism by which MSCs cure diseases. In a review, Horwitz and Dominici summarized the role of MSCs cytokine secretion in tissue regeneration (Horwitz and Dominici, 2008), and since then, more and more studies have begun to focus on the secretome of MSCs. BMSCs are the earliest kind of MSCs that appear to have the tissue regulation function, mainly in the maintenance of HSC microenvironment. Human MSCs have been demonstrated to reconstitute a functional human hematopoietic microenvironment in NOD-SCID mice (Muguruma et al., 2006). CD146þ

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MSCs are thought to be the specific population that contributes to hematopoietic maintenance, as they have the capability of forming a hematopoietic niche in heterotopic sites after transplantation and highly express angiopoietin-1, which is a pivotal molecule of the HSC niche (Sacchetti et al., 2007). However, in 2016 a study reported that CD146þ MSCs can only form bone after transplantation, suggesting that mouse CD146þ cells are osteo-progenitors committed to osteoblastic fate, and they identified a Sca1þ population that could generate CD146þ cells as the progenitor of the HSC niche (Hu et al., 2016a,b). This divergence may be due to differences in cell species; human and mouse MSCs may possess different characteristics. Since MSCs can secrete a variety of chemotactic factors and express low level of major histocompatibility complex II, the immunomodulatory ability of MSCs has drawn much attention in this field. Considerable literature shows that MSCs are involved in both innate immune and adaptive immune responses, through interaction with monocytes, polarization of macrophages, and promoting the formation of regulatory T cells, not just suppressing the immune response, but also displaying proinflammatory effects depending on the specific inflammatory environment. The innate immune system is the first line of nonspecific defense to resist invasion by pathogenic germs, during which the Toll-like receptors (TLRs) play an important role. TLRs are a group of receptor molecules expressed on the surface of innate effector cells, which can recognize danger signals such as lipopolysaccharide (LPS), double-stranded RNA, and endotoxin when tissue is injured (Kawai and Akira, 2011; Medzhitov, 2001). MSCs express abundant TLRs, especially TLR3 and TLR4 (Hwa et al., 2006; Pevsner-Fischer et al., 2007). An interesting study demonstrated that TLR3 and TLR4 play different roles in the immunomodulatory properties of MSCs. In their research, TLR3-primed MSCs show an antiinflammatory profile (MSC2), while TLR4-primed MSCs are proinflammatory (MSC1) (Waterman et al., 2010). However, that is not always the case. By using poly(I:C) and LPS to stimulate TLR3 and TLR4, respectively, of MSCs, both methods can inhibit T cell modulatory activity by impairing Notch signaling (Liotta et al., 2008). On the other hand, both TLR3 and TLR4 activation of MSCs can increase T regulatory cell induction; that is to say, both TLR3-primed and TLR4-primed MSCs show antiinflammatory characteristics (Rashedi et al., 2017). Different experiments may use different concentrations of TLR ligands to stimulate MSCs, which may influence the proinflammatory and antiinflammatory properties of MSCs (Ren et al., 2008), and as the molecular mechanisms of TLR-primed immunomodulatory actions remain unclear, this may be the cause of divergence. Macrophages are an important population in the immune response. They interact with MSCs to participate in tissue repair (Wang et al., 2015). At the early stage of infection or injury, macrophages respond rapidly and release inflammatory factors to kill pathogens. At this stage, the levels of

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inflammatory factors in the tissue are relatively low; MSCs perceive the low inflammatory level, and are polarized into the proinflammatory type MSC (MSC1), releasing growth factors such as interleukin-6 (IL-6) and IL-8 to enhance inflammation. When there are sufficient levels of proinflammatory cytokines, MSCs adopt the antiinflammatory type (MSC2) and release TGFb, IL-10, and indoleamine 2,3-dioxygenase, which polarize the proinflammatory macrophages (M1) to antiinflammatory macrophages (M2). In a model of colitis and sepsis, ADSCs induced a distinct regulatory activation state of macrophages, which possess potent immunomodulatory ability and therapeutic potential in inflammatory bowel diseases and sepsis (Anderson et al., 2013). In addition, through an extracellular vesicle-mediated mitochondrial transfer mechanism, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype (Morrison et al., 2017).

Controversy over and expectations for mesenchymal stem cells There is still a lot of controversies about MSCs, although they show numerous benefits. A most basic question is, what is the MSCs? Mesenchymal stromal cells and mesenchymal stem cells are always confused. Since there is no clear definition to distinguish between the two terminologies, they are used to refer to the same cell population, which defined by the ISCT in 2006 (Dominici et al., 2006). According to this manual, researchers use CD73, CD90, and CD105 to judge whether the cells they cultured are MSCs. However, there is the problem that using the conventional panel of CD markers to characterize MSCs has its limitations, especially when large-scale amplification is needed. During MSC expansion, replicative senescence will accumulate (Yang et al., 2017), but the CD markers (CD73, CD90, CD105) are rarely affected (Kundrotas et al., 2016), it is inaccurate to predict the function of MSCs according to these markers. What’s more, different researchers use different markers to identify subpopulations of MSCs, such as CD146, Nestin, and PDGFR-a, but without a definite hierarchical relationship like the hematopoietic system. A vast number of documents indicate that MSCs exist in various tissues of the body, but the difference between MSCs derived from different tissues is still not clear Scientists never stop exploring the in situ form of MSCs. Thus provoking another controversy. In 2008, Crisan and colleagues found that MSCs possess a perivascular localization for the first time (Crisan et al., 2008). In this research they isolated pericytes and found that cultured pericytes show multipotency as well as MSC markers. Caplan also made his point that all MSCs were pericytes (Caplan, 2008, 2017). Another work using lineage tracing demonstrated that pericytes never contribute to tissue regeneration and wound healing during aging and injury (Guimara˜es-Camboa et al., 2017). It is still difficult to make a conclusion about what is the relationship between MSCs

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and pericytes. It could be said that some pericytes but not all can develop into MSC-like cells during in vitro culture, and cell property will change with long term in vitro culture. All the efforts of MSCs researchers have been made to better utilize MSCs for disease treatment, and the results of several clinical trials with MSCs are inspiring (Butler et al., 2017; Staff et al., 2016). There are several factors need to be considered in MSCs clinical application. For example, it is necessary to choose a suitable tissue source and culture condition for MSCs when treating a specific disease. Moreover, both the safety and the effectiveness are important for the clinical applications of MSCs. In addition, tracking the results of clinical trials is important no matter the results are, positive or not.

Conclusion In summary, MSCs possess huge clinical application prospects, for their strong activities in tissue regeneration, immunoregulation, and other paracrine effects. However, until now scientists have not given a universal definition of MSCs. Moreover, MSCs are proved to be a heterogeneous cell population with the advancing research. Thus new technologies, such as single-cell RNA sequencing, proteomics, and epigenomics, should be adopted to investigate the complexity of MSCs. So far, the clinical application of MSCs is largely behind the level of laboratory research. Therefore, the combination of basic research and clinical trials is a significant factor to improve the development of MSCs. In future studies, new technologies should be used to encode the heterogeneity of MSCs, and appropriate clinical trials are needed to clarify the curative effect of MSCs. The prospects of MSCs are worth expecting.

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14 Mesenchymal Stem Cells in Human Health and Diseases Liebergall, M., Schroeder, J., Mosheiff, R., Gazit, Z., Yoram, Z., Rasooly, L., Daskal, A., Khoury, A., Weil, Y., Beyth, S., 2013. Stem cell-based therapy for prevention of delayed fracture union: a randomized and prospective preliminary study. Mol. Ther. 21, 1631e1638. Liotta, F., Angeli, R., Cosmi, L., Filı`, L., Manuelli, C., Frosali, F., Mazzinghi, B., Maggi, L., Pasini, A., Lisi, V., Santarlasci, V., Consoloni, L., Angelotti, M.L., Romagnani, P., Parronchi, P., Krampera, M., Maggi, E., Romagnani, S., Annunziato, F., 2008. Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling. Stem Cells 26, 279e289. Luger, D., Lipinski, M.J., Westman, P.C., Glover, D.K., Dimastromatteo, J., Frias, J.C., Albelda, M.T., Sikora, S., Kharazi, A., Vertelov, G., Waksman, R., Epstein, S.E., 2017. Intravenously delivered mesenchymal stem cells: systemic anti-inflammatory effects improve left ventricular dysfunction in acute myocardial infarction and ischemic cardiomyopathy. Circ. Res. 120, 1598e1613. Martin, I., De Boer, J., Sensebe, L., 2016. A relativity concept in mesenchymal stromal cell manufacturing. Cytotherapy 18, 613e620. Martino, M.M., Maruyama, K., Kuhn, G.A., Satoh, T., Takeuchi, O., Mu¨ller, R., Akira, S., 2016. Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem cell-driven tissue regeneration. Nat. Commun. 7, 11051. Medzhitov, R., 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135e145. Morrison, T.J., Jackson, M.V., Cunningham, E.K., Kissenpfennig, A., McAuley, D.F., O’Kane, C.M., Krasnodembskaya, A.D., 2017. Mesenchymal stromal cells modulate macrophages in clinically relevant lung injury models by extracellular vesicle mitochondrial transfer. Am. J. Respir. Crit. Care Med. 196 (10), 1275e1286. Moussa, L., Pattappa, G., Doix, B., Benselama, S.L., Demarquay, C., Benderitter, M., Se´mont, A., Tamarat, R., Guicheux, J., Weiss, P., Re´thore´, G., Mathieu, N., 2017. A biomaterial-assisted mesenchymal stromal cell therapy alleviates colonic radiation-induced damage. Biomaterials 115, 40e52. Muguruma, Y., Yahata, T., Miyatake, H., Sato, T., Uno, T., Itoh, J., Kato, S., Ito, M., Hotta, T., Ando, K., 2006. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 107, 1878e1887. Orozco, L., Soler, R., Morera, C., Alberca, M., Sa´nchez, A., Garcı´a-Sancho, J., 2011. Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation 92, 822e828. Orozco, L., Munar, A., Soler, R., Alberca, M., Soler, F., Huguet, M., Sentı´s, J., Sa´nchez, A., Garcı´a-Sancho, J., 2013. Treatment of knee osteoarthritis with autologous mesenchymal stem cells: a pilot study. Transplantation 95, 1535e1541. Orozco, L., Munar, A., Soler, R., Alberca, M., Soler, F., Huguet, M., Sentı´s, J., Sa´nchez, A., Garcı´a-Sancho, J., 2014. Treatment of knee osteoarthritis with autologous mesenchymal stem cells: two-year follow-up results. Transplantation 97, e66e68. Pane´s, J., Garcı´a-Olmo, D., Van Assche, G., Colombel, J.F., Reinisch, W., Baumgart, D.C., Dignass, A., Nachury, M., Ferrante, M., Kazemi-Shirazi, L., Grimaud, J.C., de la Portilla, F., Goldin, E., Richard, M.P., Leselbaum, A., Danese, S., 2016. Expanded allogeneic adiposederived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn’s disease: a phase 3 randomised, double-blind controlled trial. Lancet 388, 1281e1290.

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Pevsner-Fischer, M., Morad, V., Cohen-Sfady, M., Rousso-Noori, L., Zanin-Zhorov, A., Cohen, S., Cohen, I.R., Zipori, D., 2007. Toll-like receptors and their ligands control mesenchymal stem cell functions. Blood 109, 1422e1432. Rashedi, I., Go´mez-Aristiza´bal, A., Wang, X.H., Viswanathan, S., Keating, A., 2017. TLR3 or TLR4 activation enhances mesenchymal stromal cell-mediated Treg induction via Notch signaling. Stem Cells 35, 265e275. Ren, G., Zhang, L., Zhao, X., Xu, G., Zhang, Y., Roberts, A.I., Zhao, R.C., Shi, Y., 2008. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2, 141e150. Sacchetti, B., Funari, A., Michienzi, S., Di, C.S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, P.G., Riminucci, M., Bianco, P., 2007. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324e336. Soleimani, M., Nadri, S., 2009. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat. Protoc. 4, 102e106. Staff, N.P., Madigan, N.N., Morris, J., Jentoft, M., Sorenson, E.J., Butler, G., Gastineau, D., Dietz, A., Windebank, A.J., 2016. Safety of intrathecal autologous adipose-derived mesenchymal stromal cells in patients with ALS. Neurology 87, 2230e2234. Wang, M., Zhang, G., Wang, Y., Liu, T., Zhang, Y., An, Y., Li, Y., 2015. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int. J. Biochem. Cell Biol. 58, 53e61. Wang, Y., Xu, J., Zhang, X., Wang, C., Huang, Y., Dai, K., Zhang, X., 2017. TNF-a-induced LRG1 promotes angiogenesis and mesenchymal stem cell migration in the subchondral bone during osteoarthritis. Cell Death Dis. 8, e2715. Waterman, R.S., Tomchuck, S.L., Henkle, S.L., Betancourt, A.M., 2010. A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an immunosuppressive MSC2 phenotype. PLoS One 5, e10088. Wilson, J.G., Liu, K.D., Zhuo, H., Caballero, L., McMillan, M., Fang, X., Cosgrove, K., Vojnik, R., Calfee, C.S., Lee, J.W., Rogers, A.J., Levitt, J., Wiener-Kronish, J., Bajwa, E.K., Leavitt, A., McKenna, D., Thompson, B.T., Matthay, M.A., 2015. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet Respir. Med. 3, 24e32. Xia, Q., Zhu, S., Wu, Y., Wang, J., Cai, Y., Chen, P., Li, J., Heng, B.C., Ouyang, H.W., Lu, P., 2015. Intra-articular transplantation of atsttrin-transduced mesenchymal stem cells ameliorate osteoarthritis development. Stem Cells Transl. Med. 4, 523e531. Xu, L., Wang, S., Sui, X., Wang, Y., Su, Y., Huang, L., Zhang, Y., Chen, Z., Chen, Q., Du, H., Zhang, Y., Yan, L., 2017. Mesenchymal stem cell-seeded regenerated silk fibroin complex matrices for liver regeneration in an animal model of acute liver failure. ACS Appl. Mater. Interfaces 9, 14716e14723. Yang, F., Yang, L., Li, Y., Yan, G., Feng, C., Liu, T., Gong, R., Yuan, Y., Wang, N., Idiiatullina, E., Bikkuzin, T., Pavlov, V., Li, Y., Dong, C., Wang, D., Cao, Y., Han, Z., Zhang, L., Huang, Q., Ding, F., Bi, Z., Cai, B., 2017. Melatonin protects bone marrow mesenchymal stem cells against iron overload-induced aberrant differentiation and senescence. J. Pineal Res. Zhao, K., Lou, R., Huang, F., Peng, Y., Jiang, Z., Huang, K., Wu, X., Zhang, Y., Fan, Z., Zhou, H., Liu, C., Xiao, Y., Sun, J., Li, Y., Xiang, P., Liu, Q., 2015. Immunomodulation effects of mesenchymal stromal cells on acute graft-versus-host disease after hematopoietic stem cell transplantation. Biol. Blood Marrow Transplant. 21, 97e104.

Chapter 3

Immunoregulatory properties of mesenchymal stem cells and their application in immunotherapy Dmytro Shytikov, Pengfei Wang, Deepak Rohila Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC

Chronic and unresolved inflammation is one of the major components of metabolic disorders, obesity, type 2 diabetes, cardiovascular diseases, and autoimmune diseases. Moreover, the prevalence of these and other chronic inflammatory disorders has a tendency to increase with time in developed countries. The cessation of inflammation includes multiple mechanisms, among which the most widely studied are T-regulatory cells (Tregs), myeloid regulatory cells, and alternatively activated macrophages (MVs) with regulatory and reparatory properties. However, data indicate that mesenchymal stem cells (MSCs) themselves play an important role in the mitigation of inflammation. MSCs are multipotent progenitor cells, a major source of which is the adipose tissue and the bone marrow, but they have been isolated from multiple organs (heart, skeletal muscles, umbilical cord, spleen, amniotic fluid, etc.). Since their first discovery in the 1960s by Ernest A. McCulloch and James E. Till as bone marrow stromal cells, the differentiation potential of MSCs has been shown to also include osteogenic cells, adipocytes, neuronlike cells, myocytes, etc. Despite the extensive studies on these cells, further investigations into their nature, phenotype, and differentiation potential need to be made. It is accepted that MSCs should lack expression of lineage differentiation markers (CD45, CD34, CD14, CD11b, and major histocompatibility complex [MHC] class II proteins) but should express stromal markers, like CD73, CD90, and CD105, as well as possessing an ability to differentiate into a broad spectrum of cell lineages (Marion and Mao, 2006; Rodrı´guez-Fuentes et al., 2015; Secunda et al., 2014). MSCs attract attention as a promising tool in regenerative medicine. Some of the Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00003-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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main applications include tissue engineering and replacement of damaged cells in the sites of injury. Bone marrow MSCs can give rise to chondrocytes and osteoblasts (Shafiee et al., 2011; Jin et al., 2013); adipose tissueederived MSCs and umbilical cord blood MSCs contribute successfully to the formation of capillary- or vessel-like networks in vitro (Freiman et al., 2016) as well as in vivo (Rohban et al. 2013, 2016). Amniotic membraneederived MSCs participate successfully in neurogenesis (Kim et al., 2014; Uchida et al., 2000). Bone morphogenetic proteins, WNTs, fibroblast growth factors, and some other heparan sulfateesensitive morphogens and growth factors can stimulate osteogenesis (Bhakta et al., 2012; Bramono et al., 2012); dexamethasone, insulin, indomethacin, and isobutylmethylxanthine stimulate adipogenesis (Scott et al., 2011). Ascorbate, insulin, transferrin, selenic acid, and transforming growth factor-b (TGFb) are inducers of chondrogenesis (Johnstone et al., 1998; Mackay et al., 1998; Barry et al., 2001). MSCs can differentiate into neural, myocyte, and epithelial cells, thereby demonstrating their endodermic and neuroectodermic potential (Petersen et al., 1999; Pittenger et al., 1999). The potential to differentiate toward distinct cell lineages is not equal for MSCs from different locations (Cook and Genever, 2013: Pevsner-Fischer et al., 2011), but this issue currently is under scrutiny. MSCs were able to improve outcomes in multiple experimental injuries and clinical conditions: thermal burns (Maranda et al., 2017); traumatic injuries (Thurairajah et al., 2017; Libro et al., 2017); cartilage damage (Kurth et al., 2011; Plaas et al., 2011); autoimmune diseases, including type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, and others (Munir and McGettrick, 2015; Bai et al., 2009; Miller et al., 2010; Miller and Bai, 2007; Fathollahi et al., 2018; Scuteri and Monfrini, 2018); cardiovascular diseases (Karantalis and Hare, 2015 Price et al., 2006; Miyahara et al., 2006); organ failure (Mohamadnejad et al., 2007; Kharaziha et al., 2009; Peired et al., 2016; Li et al., 2017; Akram et al., 2013); graft-versus-host disease (GVHD; Amorin et al., 2014) and many other settings. Tissue engineering, as well as plastic surgery, considers MSCs as a promising tool (Yorukoglu et al., 2017; Eun, 2014). Dexter et al. (1977) were one of the first teams to show the ability of MSCs to support growth and survival of hematopoietic stem cells. Later other investigators demonstrated the abilities of MSCs to promote neurotrophic influence and support functional recovery of patients with stroke; MSCs promoted repair of injured cardiac tissue, restoration of knee cartilage, and recovery from other injuries (Aggarwal and Pittenger, 2005; Di Nicola et al., 2002; Shabbir et al., 2010; Centeno et al., 2008; Andrews et al., 2008). MSCs facilitate normal tissue healing by cell-to-cell contact and/or secretion of bioactive factors. But despite showing actual efficiency in clinical and experimental settings, many studies showed little to no engraftment or differentiation of transplanted cells within host tissue (Le Blanc et al., 2008; Ortiz et al., 2007; Shabbir et al., 2009).

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Horwitz et al. (1999) showed improvements in the condition of babies suffering from osteogenesis imperfecta when allogeneic bone marrow cells were transplanted. Bone mineral density was increased, and the frequency of bone fractures was reduced, even though less than 2% of the donor MSCs were found to be engrafted. Only a few implanted MSCs survived 6 weeks after transplantation in a rat ectopic model (Luong-Van et al., 2007). Anjos-Afonso and colleagues injected green fluorescent protein (GFP)-expressing MSCs into mice and checked engraftment into the host’s tissues. Primarily, GFP-labeled MSCs were detected in the lungs, and later in some other tissues (mostly lungs) at low frequencies without giving rise to tissue-specific cell types. Further, no evidence of cell expansion was observed within the examined tissues (Anjos-Afonso et al., 2004; Lee et al., 2009b). Intravenous (i.v.) injection of MSCs in a model of acute myocardial infarction showed the beneficial effect and faster recovery of experimental animals, but at the same time, they were almost undetectable in the heart, neither within 24 h postinjection nor later. Toma et al. (2002) also observed relatively few MSCs engrafted into injured myocardium after i.v. injection, while the positive effect of this procedure was evident. Thus, it is assumed that MSCs can exert their positive and reparative influence not only by replacing dead cells but also by secreting some paracrine factors, modulating the local tissue microenvironment and shifting it from an inflammatory microenvironment into a reparative one. Here we will discuss the immunomodulatory effects of MSCs, mechanisms underlying these effects, and perspective on the possible therapeutic use of MSCs as immunomodulatory agents.

Immunomodulatory effects of mesenchymal stem cells In various kinds of injuries, injection of MSCs within the early stages of induction of experimental pathology showed a significantly better outcome in MSC-treated animals compared with untreated animals. Just a few studies concerning the capability of MSCs to influence the innate and adaptive immune system will be mentioned. As few as 0.5
106 bone marrowederived MSCs injected in the early stage of bleomycin-induced lung injury prevented lung fibrosis. The authors stated that the activation of interleukin-1 (IL-1) receptor antagonist (IL-1ra) secretion was one of the most important mechanisms that helped to prevent lung injury in the tested conditions. More vigorous experiments showed that MSCs were able to prevent injury only if they were injected at the same time with bleomycin, thus decreasing the acute inflammation. In vitro experiments showed that activated MSCs were able to inhibit the production of IL-1 and tumor necrosis factor a (TNFa) by an activated MV cell line, RAW 264.7 (Ortiz et al 2003, 2007).

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In a model of lipopolysaccharide (LPS)-induced systemic inflammation, human MSCs injected intraperitoneally into LPS-treated mice were able to dampen inflammation by secretion of soluble TNFa receptor-1 (sTNFR1; Yagi et al., 2010). There is a number of studies that demonstrate the beneficial effect of MSC injection in a model of acute myocardial infarction due to antiinflammatory activity. Lee and colleagues injected human MSCs intravenously into mice during the early stage of artery ligation and found a beneficial effect, but the authors did not observe stable engraftment of the MSCs, which disappeared from the recipient body completely within 48 h. Most of the injected cells appeared to be retained in the lungs. Nevertheless, the injected MSCs displayed an activated phenotype and the gene expression analysis revealed differential expression of more than 50 genes, among which TNFa-induced secreted protein-6 (TSG6) was found. TSG6-deficient MSCs were unable to exert their beneficial effect (Lee et al., 2009a). Luger and colleagues demonstrated reduced dysfunction rate of the left ventricle during myocardial ischemia and the effect was caused, at least partly, by systemic antiinflammatory activities of the injected MSCs. Human MSCs (2
106) injected 24 h after artery ligation in mice demonstrated just a little retention of MSCs in the injured tissue, but the authors observed a significant reduction of myocardium inflammation and also improved cardiac function compared with control mice (Luger et al., 2017). MSCs were able to induce antiinflammatory effects in a model of cornea damage in rats. The cornea of the animals was exposed to alcohol and mechanical damage. If the animals were subsequently injected with MSCs, either intravenously or intraperitoneally, or were injected with recombinant TSG6, corneal inflammation and opacity showed significant reduction compared with control animals. No engraftment of MSCs in the site of injury was found (Roddy et al., 2011; Oh et al., 2010). Several models of peritonitis and sepsis showed a positive effect of MSCs and their cross talk with MVs. Choi and colleagues showed that zymosan-induced systemic inflammation was significantly ameliorated if MSCs were injected. Again, according to in vivo and in vitro experiments, TSG6 generated by MSCs was proposed as one of the main factors in dampening MV activation (Choi et al., 2011). Ne´meth and colleagues showed that in the case of sepsis induction (ligation and puncture of cecum), mice that received MSCs intravenously showed better survival, lower levels of TNFa and IL-6, and a higher level of IL10. No engraftment was observed, as injected MSCs were detectable only in the lungs 24 h after injection (Ne´meth et al., 2009). Mei et al. (2010), in a similar model, showed improved survival as well as increased bacterial clearance in mice injected with MSCs. Intraarticular injection of MSCs reduced inflammation and cartilage damage in murine antigen-induced arthritis as was shown in the study of Kehoe and colleagues. Bone marrowederived MSCs (0.5
106) were injected

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during the early stage of collagen-induced autoimmune arthritis, locally at the site of injury. The authors observed reduced swelling of the injured limb, declined inflammatory cytokine level, and only moderate MSC incorporation in the injured tissue, although the long-term survival of incorporated cells was not analyzed (Kehoe et al., 2014). Bartholomew and colleagues showed that systemically injected MSCs were able to suppress allogeneic skin graft rejection in baboons (although they did not prevent it completely). An in vitro study revealed the significant inhibitory effect of MSCs on T cell proliferation in the mixed lymphocyte reaction (MLR) (Bartholomew et al., 2002). Haploidentical MSCs showed a significant positive effect on acute steroid-resistant GVHD (Le Blanc et al., 2008). Duijvestein and colleagues showed improvement of Crohn disease activity index in some of the patients injected with autologous bone marrowederived MSCs, with no serious adverse events occurring during the administration. In this study, 1e2
106 cells per kilogram of patient body weight were injected intravenously twice (Duijvestein et al., 2010). Application of MSCs for treatment of autoimmune diseases, clinical or experimentally induced, also showed the antiinflammatory properties of MSCs, and a similar kind of effect was observed in some other studies (Yamout et al., 2010; Zappia et al., 2005; Munir and McGettrick, 2015). In conclusion, it is evident that MSCs exerted their antiinflammatory effects in multiple experimental models and clinical investigations. The mechanism of tissue repair by MSCs is complex and relies on multiple factors such as replacement of injured cells in organs and secretion of factors that govern tissue repair and vascular growth (growth factors and chemokines), and the immunosuppressive and antiinflammatory activity is another important component of the reparative influence. MSCs are able to interact with a broad spectrum of immune system cells, and this is the subject of the next part of the chapter.

Mechanisms of antiinflammatory effects of mesenchymal stem cells Inhibition of inflammation by MSCs can be achieved through the inhibition of the innate immune system (prevention of maturation of antigen-presenting cells [APCs]; conversion of MVs to the alternatively activated, reparative phenotype; inhibition of natural killer [NK] cells) as well as the adaptive immune system (suppression of T and B lymphocytes, induction of Tregs). The main way to achieve these effects lies in the secretion of immunomodulatory molecules such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), cytokines (TGFb, IL-10), or other soluble factors (TSG6, IL-1ra, sTNFR1). Intercellular contacts have lower importance, since most of the studies reported that soluble factors were able to inhibit the immune system cells under in vitro conditions. This idea is also supported at least in

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part by the observation that systemically infused MSCs are usually trapped in the lung capillary system and are rarely preserved in the injured tissues, while the effects of these injections are usually preserved for a considerable time. Initial steps of the immune response utilize the innate branch of the immune system, which can be activated through the engagement of pathogenassociated molecules with the respective Toll-like receptors. As a result, the massive release of proinflammatory cytokines such as TNFa and IL-1b takes place (Dayer, 2002). However, a highly efficient immune response can be achieved by activation of the adaptive immune system cells (T lymphocytes in particular), and the presentation of a cognate antigen to T lymphocytes is the critical step. T cells require antigens to be captured and digested into smaller molecules, which should be presented by the MHC molecules. Highly specialized professional APCs include dendritic cells (DCs), MVs, and B lymphocytes. However, APCs require prior activation and maturation to be able to mount the immune response (Brutkiewicz, 2016). MSCs were reported to be able to inhibit the maturation of APCs. Human monocytes cultured under conditions supporting their maturation (culture medium supplemented with TNFa, GM-CSF, and IL-4) together with MSCs showed reduced expression of MHC class II and costimulatory molecules, lower endocytosis, and IL-12 secretion (Jiang et al., 2005; Nauta et al., 2006; Li et al., 2008; Saeidi et al., 2013; English et al., 2008). The ability to stimulate allogeneic T cells in the MLR or T cell antigenedependent proliferation was also decreased. DCs cultured in the presence of MSCs or MSC conditioned medium showed reduced maturation. Similar results were observed on murine DCs isolated from spleen (Sadeghi et al., 2014). The inhibitory effect of MSCs on DCs appeared to be reversible and dependent on soluble factors because MSC conditioned medium was able to suppress DCs almost as efficiently as the addition of MSCs into the in vitro system (Sadeghi et al., 2014). PGE2 was proposed to be one of the key factors that mediate this inhibitory effect (Spaggiari et al., 2009). However, a number of other soluble factors were proposed to mediate the inhibition (Djouad et al., 2007; Jiang et al., 2005) as well as intercellular contacts through the Notch pathway (the addition of neutralizing antibodies against TGFb or Notch inhibitors to the in vitro system was able to reverse the inhibitory effects of MSCs). Another possible mechanism of MSC action on DCs may lie in the induction of Tregs, which may suppress activation of other T cells and DCs (Li et al., 2008). Another type of professional APC is the MV. Under normal conditions, their function is to clear tissue from dead cells or debris (Gordon and Plu¨ddemann, 2017), but upon stimulation by the danger signals, inflammatory cytokines or interferon-g (IFN-g), they upregulate antigen presentation together with costimulatory molecules (Zhang and Wang, 2014). There are

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several types of MVs, which differ in their tissue distribution, origin, and properties (Murray et al., 2014). Under normal conditions MVs patrol tissues and capture and digest debris, dead or apoptotic cells, or pathogens. This cell type has several phenotypes, which are activated under different conditions depending on the environmental signals. Inflammatory, type I MVs arise after encountering bacterial or viral products or inflammatory cytokines and are able to secrete cytokines that favor the T helper cell 1 (Th1) immune response (IL-12, TNFa, IFN type I, IL-6, etc.); on the other hand, MVs activated in the presence of IL-4/IL-13 and/or immune complexes are able to secrete Th2 cytokines (such as IL-10), renew extracellular matrix, stimulate blood vessel growth and migration of stem cells at the site of tissue damage, and stimulate tissue repair (Mantovani et al., 2013; Sica and Mantovani, 2012). M2 MVs can be further divided into M2a-, M2b-, and M2c-type MVs. M1 MVs appear at early phases of wound healing (1e3 days) and are later replaced by M2 MVs (4e7 days). Despite there being no clear demarcation of the M2 types of NVs, each of which expresses the M2 markers CD206 and CD163, they are expected to have distinct functions in the tissue repair process by secreting a variety of antiinflammatory cytokines and chemokines. In a variety of tissue injury models, the participation of M2 MVs seems to be important for promoting tissue repair (Lucas et al., 2010; Mirza et al., 2009; Summan et al., 2006; van Amerongen et al., 2007). It should be noted that the differentiation toward all of these phenotypes is reversible and can be achieved by changes in the external conditions. One of the first studies that identified the interaction between MSCs and MVs was done by Kim and colleagues. They showed that MVs isolated from human peripheral blood and cocultured with human bone marrowederived MSCs (Kim and Hematti, 2009) increased expression of CD206, and high phagocytic activity was observed in MVs cocultured with MSCs. They were also characterized by reduced TNFa expression, but high expression of IL-10 and CCL18 and high phagocytic activity. As per previous studies, interaction of MSCs and MVs plays a vital role in the immunoregulatory properties of MSCs. Inflammatory cytokines secreted by MVs stimulate MSCs to secrete antiinflammatory compounds like TSG6, PGE2, and nitric oxide (NO). In vitro studies revealed that TNFa-activated MSCs can interfere with zymosan-induced inflammation driven by MVs, and secreted TSG6 plays a key role in it (Choi et al., 2011). Intravenously infused human MSCs improved a mouse model of myocardial infarction in part because the MSCs, trapped in the lung as microemboli, became activated and secreted TSG6 protein and other proteins, which suppressed inflammatory reactions triggered by ischemia (Ortiz et al., 2007). Ne´meth and colleagues investigated the effect of mouse bone marrowederived MSCs in a murine model of septicemia and showed that LPS-stimulated MVs produced more IL-10 when cocultured with bone marrowederived MSCs. The beneficial effect of MSCs was eliminated by MV depletion or pretreatment with

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antibodies specific for IL-10 or IL-10 receptor (Ne´meth et al., 2009). Li and colleagues showed successful treatment of corneal wounds with autologous MSCs, which induced vascular regeneration and tissue repair. At the same time clodronate-containing liposomes, which deplete MVs, completely blocked the effect of MSCs, and transfused peritoneal MVs successfully recovered the defect of corneal wound healing. Even in the presence of transplanted MSCs, attenuated vascularization and angiogenesis were observed with reduced numbers of myofibroblasts and pericytes in the corneal stroma (Li et al., 2013). Soluble factors play a major role in the inhibition of NVs by MSCs. LPSactivated MVs express high levels of CD11c, inducible NO synthase (iNOS), and TNFa. However, direct coculture with bone marrowederived MSCs reversed this effect and, moreover, increased expression of Arg-1, IL-10, and TGFb, markers of type 2 differentiation (Zheng et al., 2018). Another study demonstrated increased percentage of CD206þ and Arg-1-expressing MVs during activation in MSC conditioned medium compared with the cells cultured under standard conditions (Wolfe et al., 2016). Moreover, Lu et al. (2013) demonstrated that MVs cultured in the presence of dead MSCs (cell death was induced by culturing under hypoxic conditions) gained the expression of tissue-repairing factors and were able to significantly improve viability and survival of hypoxic cardiomyocytes. Ortiz and colleagues showed that MSC conditioned medium can inhibit the secretion of TNFa by activated RAW-264.7 cells. IL-1ra was one of the potential mediators of this effect (Ortiz et al., 2007; Gao et al., 2014). Acetylsalicylic acid was able to reduce the inhibitory influence of MSCs, suggesting that PGE2 may play an important role (Maggini et al., 2010). Vasandan et al. (2016) observed that MSCs promoted MV differentiation from M1 to M2 type during in vitro coculture by the action of IDO and PGE2 (Franc¸ois et al. 2012). These observations suggest that the cooperative actions of MSCs and MVs are required for the completion of tissue regeneration and revascularization during the wound healing process. The overall effect of MSCs on MVs relies not only on the reduction of synthesis of inflammatory cytokines but also on the shift towards the antiinflammatory, tissue-repairing phenotype. However, MSCs are also able to interact with other immune system cells and modulate their activity. Having some similarities with T cells, such as clonal expansion and longlasting survival of successful clones, NK cells are the other members of the innate immune system (Sun and Lanier, 2011; Peng and Tian, 2017). NK cells possess cytotoxicity without prior activation or sensitization against cells that lack the expression of MHC class I proteins, which often happens with virusinfected, transformed, or stressed cells. An important feature of NK cells is their fine-tuning according to the expression of MHC proteins by the host (Bozzano et al., 2017; Geiger and Sun, 2016). Each organism has an individual combination of genes encoding

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MHC class I proteins, and NK cells during maturation adjust the expression of inhibitory MHC receptors on their surface to be able to recognize the combination of MHC I products of their particular organism (He and Tian, 2017). If in the case of MVs and DCs the influence of MSCs was demonstrated in multiple studies, much less is known about interactions of MSCs and NK cells. The available information suggests that MSCs are able to inhibit NK cell activation, but the data are incomplete. MSCs can prevent the activation of NK cells, their IFN-g and TNFa production, and their cytotoxic effect (Aggarwal and Pittenger, 2005; Spaggiari et al. 2006, 2008; Poggi et al., 2005; Selmani et al., 2008). Spaggiari et al. (2006) suggested that MSCs are first stimulated by nonactivated NK cells with a moderate IFN-g release, and these MSCs, in turn, block NK cell lytic ability. Coculturing NK cells with IL-2 or target cells (tumor or virus-infected cells) together with MSCs downregulates receptors CD132, CD56, NKR2B4, NKG2D, NK p30-related protein (NKp30), and NKp44 on NK cells (Aggarwal and Pittenger, 2005; Poggi et al., 2005; Selmani et al., 2008), thereby preventing them from exerting their cytotoxicity on target cells. IDO, TGFb1, PGE2, and human leukocyte antigen G5 assume the mediation of the inhibitory function of MSCs (Sotiropoulou et al., 2006; Spaggiari et al., 2008; Selmani et al., 2008). However, prior activation of NK cells is able to prevent the suppressive effect of MSCs. It should be noted that the influence of MSCs on NK cells is less well characterized and probably less significant than on other cell types. It should also be noted that low expression of MHC class I proteins on MSCs makes them possible targets of NK cells. When the immunosuppressive effect of MSCs on recipient anti-donor reactivity was examined before and after human kidney transplantation, MSCs inhibited the proliferation not only of CD4þ and CD8þ T cell lymphocytes in pre- and posttransplanted donor-directed MLR, but also of NK cells (Crop et al., 2009). The inhibition of NK cell proliferation was shown to be dependent only on soluble factors. The antiproliferative effect of MSCs is induced under inflammatory conditions, such as IFN-g-rich microenvironments. We also need to consider that NK cells are able to kill autologous as well as heterologous MSCs and that NK cell activation is thought to be mediated by NKp30 and a receptoreligand pair of lymphocyte function-associated antigen 1 with the intercellular adhesion molecule 1 (Poggi et al., 2005). IL-2 or IL15-cultured NK cells can efficiently kill MSCs (Crop et al., 2011). However, exposure of MSCs to IFN-g is able to upregulate their MHC class I proteins and improve their survival in the presence of NK cells (Crop et al., 2011). Interactions of MSCs and NK cells may have a significant implication in organ transplantation. The original findings suggested that acute transplant rejection should not be related to NK cells; however, NK cells can play a role in this process, by recognizing the incorrect transplant MHC proteins and

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secreting large amounts of immunostimulatory cytokines (Benichou et al., 2011). MSCs may be considered as suppressors of NK cells, but they themselves are still targets for NK cells. The adaptive immune system consists of B and T cells, which are able to recognize antigens through their antigen-specific receptors and subsequently mount a highly specific immune response. The role of these cells during the immune response and the mechanisms of their functions are described elsewhere. B cells are reported to be inhibited by MSCs in in vitro conditions in a dosedependent way. It is reported that B cells activated by IL-2, IL-4, IL-10, CD40, LPS, and CpG oligonucleotides in in vitro conditions have reduced immunoglobulin production and proliferation in the presence of MSCs (Corcione et al., 2006). Addition of the conditioned medium from MSCs cultured together with activated B cells elicited an inhibitory effect on B cells as well, but not the conditioned medium from cultured, but nonactivated, MSCs (Augello et al., 2005). MSCs appeared to be able to inhibit B cells even if they were physically separated by transwell filters, which suggests the role of soluble factors as main mediators of these interactions (Asari et al., 2009; Corcione et al., 2006). The fact that MSCs require prior contact with activated B cells shows that there is a cross talk between the two cell populations, and factors synthesized by B cells are required for licensing the immunosuppressive properties of MSCs. Cho et al. (2017) demonstrated inhibitory effects of MSC infusion on the estradiol-2induced B cell response in vivo, thus indicating that MSCs may restrain pathological responses of B cells and prevent autoimmunity. T cells were one of the first types of cells reported to be influenced by MSCs (Bartholomew et al., 2002). Bartholomew and colleagues showed that MSCs injected systemically are able to prolong skin allograft survival and inhibit the T cell allospecific response. Then it appeared that MSCs were potent T cell suppressors and could inhibit T cell proliferation in response to mitogens (phytohemagglutinin, antibodies against CD3ε) or allogeneic cells in MLR in a dose-dependent manner (Bartholomew et al., 2002; Di Nicola et al., 2002; Krampera et al., 2003; Sheng et al., 2008; Le Blanc et al., 2004; Meisel et al., 2004; Angoulvant et al., 2004), while not having sufficient immunogenic potential (Rasmusson et al., 2007). And again, previous exposure of MSCs to proinflammatory cytokines from activated T cells (namely, IFN-g) was sufficient to induce their antiinflammatory properties (Sheng et al., 2008; Krampera et al., 2006). And the blockage of IFN-g receptors was sufficient to abrogate immunosuppressive properties of MSCs (Polchert et al., 2008; Krampera et al., 2006; Schurgers et al., 2010), but it should be noted that there are results that argue the dependency of functions of MSCs on IFN-g (Gieseke et al., 2007; Ryan et al., 2007; English et al., 2007). Injection of MSCs in several experimental conditions (experimental autoimmune encephalomyelitis, allergy induction, and skin graft transplantation) showed induction of Tregs in in vivo models (Bai et al., 2009; Luz-Crawford et al., 2013; Kavanagh and Mahon, 2011; Barry et al., 2005).

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This may be caused by direct suppressions of T cells, promoting Treg accumulation, and by inhibiting maturation of APCs, which altogether can abrogate the successful immune response (Cutler et al., 2010; Wang et al., 2008; English et al., 2009; Aksu et al., 2008). As seen in previous parts of this chapter, MSCs use soluble factors to inhibit multiple immune system cell types and so they use a number of soluble factors to inhibit T cells as well. Here we will briefly describe the major mediators of the immunosuppressive effects of MSCs aimed at T cells. Reported roles for both cellecell contact and release of soluble factors in MSC-mediated T cell suppression are evident throughout the literature, and numerous candidate mediators have been reported: PGE2, IDO, NO, IL-10, IL-27, TGFb, monocyte chemotactic protein 1, human leukocyte antigen G, and intracellular adhesion molecule 1, among others (English et al., 2009; Tatara et al., 2011; Zhao et al., 2008; Ren et al., 2010; Lim et al., 2010; Ge et al., 2010). Groh et al. (2005) have suggested that CD14þ cells activate MSCs through IL-1b when cocultured, and then MSCs inhibit the T cell response through TGFb, which is shown to be able to inhibit T cell responses if added to the reaction (Yoshimura and Muto, 2010). However, the roles of different soluble factors in experimental settings are sometimes controversial; for example, Min et al. (2007) were able to prevent GVHD by injecting IL-10-transgenic MSCs, while injection of IL-10 alone was not sufficient to achieve this goal. Djouad et al. (2005), in a model of autoimmune arthritis, also showed that IL-10-transgenic MSCs were not able to prevent collagen-induced arthritis, while they were still potent T cell suppressors in MLR in vitro. Addition of COX inhibitors is able to reverse inhibitory influence of MSCs, which also suggests the implication of PGE2 in MSC-mediated inhibition of T cells in vitro (Yan˜ez et al., 2010; Schurgers et al., 2010). Another mechanism of immunosuppressive action of MSCs on T cells is the secretion of IL-6. Xu et al. (2009) showed that IFN-g and TNFa act synergistically to induce high levels of expression of IL-6 and several other immune-related molecules in MSCs under in vitro conditions. Another way to suppress T cells is secretion of IDO. MSCs do not constitutively express IDO, but they do when exposed to IFNg (Ling et al., 2014; He et al., 2015; Kim et al., 2018). When an IDO inhibitor or IDO/ MSCs were used, partial reduction of the MSC suppression effect both in vitro and in vivo was observed (Hong et al., 2016; Zhang et al., 2015). Secretion of IDO by MSCs is also related to an increased number of Tregs as shown in the paper by Ge et al. (2010). MSCs from wild-type or IDO knockout C57BL/6 mice were injected intravenously into BALB/c recipients 24 h after they received a life-supporting orthotopic C57BL/6 renal graft. Wild-typeMSC-treated recipients achieved allograft tolerance with normal histology, undetectable anti-donor antibody levels, impaired CD4þ T cell responses, and increased frequencies of CD4þCD25þFoxp3þ regulatory T cells found in

28 Mesenchymal Stem Cells in Human Health and Diseases

recipient spleens and donor grafts. NO is another important mediator of the immunosuppressive properties of MSCs. Mouse MSCs have been proved to upregulate the expression of iNOS after stimulation with IFN-g and either TNFa or IL-1 (Ren et al., 2008; Xu et al., 2009). When L-NMMA, an iNOS inhibitor, or iNOS/ MSCs are used, reduced suppression of T cells is detected (Sato et al., 2007). Furthermore, in vivo GVHD experiments demonstrated amelioration of the disease by wild-type MSCs, compared with the failure of IFN-bR1/ or iNOS/ MSCs to protect recipients (Ren et al., 2008). Galectins have emerged as soluble factors secreted by human and mouse MSCs and are upregulated when stimulated in the MLR. They are capable of suppressing T cell proliferation. Several studies demonstrated that small interfering RNA against galectins 1 and 3 reversed the suppressive effects of human MSCs on T cells (Lepelletier et al., 2010; Sioud et al. 2010, 2011). However, whether the suppression is mediated by a direct influence on T cells or through the inhibition of APCs or through the induction of Tregs is still under investigation as of this writing. MSCs have been reported to increase the proliferation of Tregs both in vitro and in vivo. For example, the percentage of Tregs increased when MSCs were added to an MLR (Selmani et al., 2008; Maccario et al., 2005; English et al., 2009). This effect was contact dependent when purified CD4þ T cells were used, while direct contacts were not required in the case of complete peripheral blood mononuclear lymphocytes. This suggests a role for APC mediation (English et al., 2009). In vitro, PGE2, HLA-G5, TGFb1, and IDO have been proposed as the effector molecules responsible for the induction of Tregs by MSCs (Selmani et al., 2008; English et al., 2009; Ge et al., 2010). In vivo, MSCs injected intravenously prior to allogeneic organ transplantation increased the number of Tregs, which in turn suppressed rejection and improved graft survival rate (Casiraghi et al., 2008; Ge et al., 2010). In contrast, some authors have not found any induction of Tregs by MSCs in vitro (Carrio´n et al., 2011; Krampera et al., 2006; Sheng et al., 2008). Summarized data about the interactions of MSCs and the immune system cells are presented in Table 3.1. The presented data suggest a significant impact of MSCs on the innate and adaptive branches of the immune system. The ability to influence APCs (MVs and DCs) and the effector cells (NK cells, T cells, B cells) and to perform tissue regeneration makes them a prospective agent for regenerative medicine and as an immunomodulatory agent. MSCs have low immunogenicity as well as the potential to be expanded in vitro, thus making them a prospective instrument for the immunotherapy of autoimmune diseases and in organ transplantation.

Prospective and concluding remarks One of the most targeted diseases for which the immunomodulatory function of MSCs is utilized is acute GVHD. According to Le Blanc and colleagues,

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TABLE 3.1 Key partners of mesenchymal stem cells among the immune system cells. The effects of interactions

Key effector molecules

Dendritic cells

Inhibition of maturation Decreased expression of costimulatory molecules

TGFb IL-6 Expression of Jagged (ligand for Notch receptors) PGE2 Induction of Tregs

Spaggiari et al., (2009); Djouad et al., (2007); Jiang et al., (2005); Li et al. (2008).

Macrophages

Decreased expression of proinflammatory cytokines Increased phagocytic activity Induction of type 2 phenotype

TSG6 IL-10 IL-1ra sTNFR1 PGE2 NO

Choi et al., (2011); Ortiz et al., (2007); Ne´meth et al., (2009); Zheng et al., (2018); Maggini et al., (2010); Vasandan et al., (2016); Franc¸ois et al. 2012.

Natural killer cells

Decreased expression of CD132, CD56, NKR2B4, NKG2D, NKp30, and NKp44 Reduced cytotoxicity However, the effects are less well studied

TGFb PGE2 IDO Probably cell-to-cell interactions

Aggarwal and Pittenger (2005); Poggi et al., (2005); Selmani et al., (2008); Sotiropoulou et al., (2006); Spaggiari et al., (2008); Selmani et al. (2008).

B cells

Lower proliferation Impaired immunoglobulin synthesis Decreased expression of chemokine receptors

Soluble factors to be determined

Augello et al., (2005); Asari et al., (2009); Corcione et al. (2006).

Cell type

References

Continued

30 Mesenchymal Stem Cells in Human Health and Diseases

TABLE 3.1 Key partners of mesenchymal stem cells among the immune system cells.dcont’d Cell type T cells

The effects of interactions

Key effector molecules

Inhibition of proliferation Suppression of cytotoxicity and cytokine release Induction of anergy Induction of Tregs

HGF TGFb IL-10 IL-27; Expression of PDL1 and PDL2 Galectin-1 and -3 PGE2 IDO Inhibition of APCs

References English et al., (2009); Tatara et al., (2011); Zhao et al., (2008); Ren et al., (2010); Lim et al., (2010); Ge et al., (2010); Groh et al., (2005); Yan˜ez et al., (2010); Schurgers et al., (2010); Aggarwal and Pittenger (2005).

APC, antigen-presenting cell; IDO, indoleamine 2,3-dioxygenase; HGF, hepathocyte growth factor; IL, interleukin; NO, nitric oxide; PDL, programmed death ligand; PGE2, prostaglandin E2; sTNFR1, soluble TNFa receptor 1; TSG6, TNFa-induced secreted protein-6; TGFb, transforming growth factor-b; Treg, T-regulatory cell.

several clinical trials showed a positive effect of infusions of MSCs in patients with steroid-resistant GVHD. After a few infusions of haploidentical MSCs patients showed a response to treatment or had an improvement in their condition (Le Blanc et al. 2003, 2004, 2008). Kebriaei et al. (2009) also showed the prospective effect of MSC infusions in the case of acute GVHD. More information about this subject can be found in the literature review and metaanalysis by Chen et al. (2015). MSC infusions showed positive or promising response without severe adverse effects in patients suffering from Crohn disease. Although the response was not complete, the disease index in patients was decreased (Duijvestein et al., 2010; Forbes et al., 2014; Forbes, 2017). MSCs demonstrated the potential use as an immunosuppressive mediator in multiple sclerosis. A huge body of data obtained from animal models suggested a positive effect of MSCs; at the same time clinical trial data are much less clear (Dulamea, 2015): Yamout et al. (2010) and Harris et al. (2018) demonstrated some improvements in patients receiving MSCs, while Bonab et al. (2012) and Llufriu et al., (2014) did not show a strong response in patients to the treatments with MSCs. Jiang et al. (2011) showed a significant reduction in the insulin daily dose and C-reactive peptide level of type 2 diabetes patients receiving MSCs.

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As of this writing, there is a lack of clinical studies regarding the therapeutic potential of MSCs in the treatment of systemic lupus erythematosus, but the available studies showed promising effects of MSC infusion and no acute adverse effects (Sun et al. 2009, 2010; Liang et al., 2010; Wang et al., 2018). Preclinical and clinical trial data have shown efficacy in the treatment of inflammatory disorders with MSCs, although the results are sometimes controversial and clinical benefits vary between trials. Differences in experimental design, cell dosages, evaluation of clinical benefits, and cell isolation and cell propagation protocols might influence the results, but overall, it is evident that MSCs may serve as a potent immunomodulatory treatment. Good manufacturing practice protocols and the consensus concerning the definition of MSCs are important steps in using MSCs as a therapeutic agent. To test the true efficacy of MSCs, the treatment protocol and ways of cell isolation and expansion of MSCs, if needed, require high standardization to minimize the off-target effects and safety risks. Another question to be addressed is the long-term safety. In vitro data strongly suggest that MSCs can act as endogenous regulators of tissue inflammation, but whether this translates to clinical efficacy remains to be seen.

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Selmani, Z., Naji, A., Zidi, I., Favier, B., Gaiffe, E., Obert, L., et al., 2008. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4þCD25highFOXP3þ regulatory T cells. Stem Cells 26 (1), 212e222. https://doi.org/10.1634/stemcells.2007-0554. Shabbir, A., Zisa, D., Lin, H., Mastri, M., Roloff, G., et al., 2010. Activation of host tissue trophic factors through JAK-STAT3 signaling: a mechanism of mesenchymal stem cell-mediated cardiac repair. Am. J. Physiol. Heart Circ. Physiol. 299 (5), H1428eH1438. http://doi.org/ 10.1152/ajpheart.00488.2010. Shabbir, A., Zisa, D., Suzuki, G., Lee, T., 2009. Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen. Am. J. Physiol. Heart Circ. Physiol. 296 (6), H1888eH1897. http://doi.org/10.1152/ajpheart.00186.2009. Shafiee, A., Seyedjafari, E., Soleimani, M., Ahmadbeigi, N., Dinarvand, P., Ghaemi, N., 2011. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnol. Lett. 33 (6), 1257e1264. http://doi.org/10.1007/s10529-011-0541-8. Sheng, H., Wang, Y., Jin, Y., Zhang, Q., Zhang, Y., Wang, L., et al., 2008. A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through upregulation of B7-H1. Cell Res. 18 (8), 846e857. http://doi.org/10.1038/cr.2008.80. Sica, A., Mantovani, A., 2012. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Investig. 122 (3), 787e795. https://doi.org/10.1172/JCI59643. Sioud, M., Mobergslien, A., Boudabous, A., Fløisand, Y., 2010. Evidence for the involvement of galectin-3 in mesenchymal stem cell suppression of allogeneic T-cell proliferation. Scand. J. Immunol. 71 (4), 267e274. http://doi.org/10.1111/j.1365-3083.2010.02378.x. Sioud, M., Mobergslien, A., Boudabous, A., Fløisand, Y., 2011. Mesenchymal stem cell-mediated T cell suppression occurs through secreted galectins. Int. J. Oncol. 38 (2), 385e390. http://doi. org/10.3892/ijo.2010.869. Sotiropoulou, P.A., Perez, S.A., Gritzapis, A.D., Baxevanis, C.N., Papamichail, M., 2006. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 24 (1), 74e85. https://doi.org/10.1634/stemcells.2004-0359. Spaggiari, G.M., Abdelrazik, H., Becchetti, F., Moretta, L., 2009. MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Blood 113 (26), 6576e6583. http://doi.org/10. 1182/blood-2009-02-203943. Spaggiari, G.M., Capobianco, A., Abdelrazik, H., Becchetti, F., Mingari, M.C., 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), 1327e1333. https://doi.org/10.1182/blood-2007-02-074997. Spaggiari, G.M., Capobianco, A., Becchetti, S., Mingari, M.C., Moretta, L., 2006. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107 (4), 1484e1490. http://doi.org/10.1182/blood-2005-07-2775. Summan, M., Warren, G.L., Mercer, R.R., Chapman, R., Hulderman, T., et al., 2006. Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290 (6), R1488eR1495. http://doi.org/10.1152/ajpregu.00465.2005. Sun, J.C., Lanier, L.L., 2011. NK cell development, homeostasis and function: parallels with CD8þ T cells. Nat. Rev. Immunol. 11 (10), 645e657. http://doi.org/10.1038/nri3044.

42 Mesenchymal Stem Cells in Human Health and Diseases Sun, L., Akiyama, K., Zhang, H., Yamaza, T., Hou, Y., Zhao, S., et al., 2009. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells 27 (6), 1421e1432. http://doi.org/10.1002/stem.68. Sun, L., Wang, D., Liang, J., Zhang, H., Feng, X., Wang, H., et al., 2010. Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus. Arthritis Rheum. 62 (8), 2467e2475. http://doi.org/10.1002/art.27548. Tatara, R., Ozaki, K., Kikuchi, Y., Hatanaka, K., Oh, I., Meguro, A., et al., 2011. Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation. Cytotherapy 13 (6), 686e694. http://doi.org/10.3109/14653249.2010.542456. Thurairajah, K., Broadhead, M.L., Balogh, Z.J., 2017. Trauma and stem cells: biology and potential therapeutic implications. Int. J. Mol. Sci. http://doi.org/10.3390/ijms18030577. Toma, C., Pittenger, M.F., Cahill, K.S., Byrne, B.J., Kessler, P.D., 2002. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105 (1), 93e98. Uchida, S., Inanaga, Y., Kobayashi, M., Hurukawa, S., Araie, M., Sakuragawa, N., 2000. Neurotrophic function of conditioned medium from human amniotic epithelial cells. J. Neurosci. Res. 62 (4), 585e590. https://doi.org/10.1002/1097-4547(20001115)62:43.0.CO;2-U. van Amerongen, M.J., Harmsen, M.C., van Rooijen, N., Petersen, A.H., van Luyn, M.J., 2007. Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice. Am. J. Pathol. 170 (3), 818e829. https://doi.org/10.2353/ ajpath.2007.060547. Vasandan, A.B., Jahnavi, S., Shashank, C., Prasad, P., Kumar, A., Prasanna, S.J., 2016. Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE2-dependent mechanism. Sci. Rep. http://doi.org/10.1038/srep38308. Wang, D., Zhang, H., Liang, J., Wang, H., Hua, B., Feng, X., et al., 2018. A long-term follow-up study of allogeneic mesenchymal stem/stromal cell transplantation in patients with drugresistant systemic lupus erythematosus. Stem Cell Reports 10 (3), 933e941. https://doi.org/ 10.1016/j.stemcr.2018.01.029. Wang, Q., Sun, B., Wang, D., Ji, Y., Kong, Q., Wang, G., et al., 2008. Murine bone marrow mesenchymal stem cells cause mature dendritic cells to promote T-cell tolerance. Scand. J. Immunol. 68 (6), 607e615. http://doi.org/10.1111/j.1365-3083.2008.02180.x. Wolfe, A.R., Trenton, N.J., Debeb, B.G., Larson, R., Ruffell, B., Chu, K., et al., 2016. Mesenchymal stem cells and macrophages interact through IL-6 to promote inflammatory breast cancer in pre-clinical models. Oncotarget 7 (50), 82482e82492. http://doi.org/10.18632/ oncotarget.12694. Xu, G., Zhang, Y., Zhang, L., Roberts, A.I., Shi, Y., 2009. C/EBPbeta mediates synergistic upregulation of gene expression by interferon-gamma and tumor necrosis factor-alpha in bone marrowderived mesenchymal stem cells. Stem Cells 27 (4), 942e948. http://doi.org/10.1002/stem.22. Yagi, H., Soto-Gutierrez, A., Navarro-Alvarez, N., Nahmias, Y., Goldwasser, Y., Kitagawa, Y., 2010. Reactive bone marrow stromal cells attenuate systemic inflammation via sTNFR1. Mol. Ther. 18 (10), 1857e1864. http://doi.org/10.1038/mt.2010.155. Yamout, B., Hourani, R., Salti, H., Barada, W., El-Hajj, T., Al-Kutoubi, A., et al., 2010. Bone marrow mesenchymal stem cell transplantation in patients with multiple sclerosis: a pilot study. J. Neuroimmunol. 227 (1e2), 185e189. http://doi.org/10.1016/j.jneuroim.2010.07.013.

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Yan˜ez, R., Oviedo, A., Aldea, M., Bueren, J.A., Lamana, M.L., 2010. Prostaglandin E2 plays a key role in the immunosuppressive properties of adipose and bone marrow tissue-derived mesenchymal stromal cells. Exp. Cell Res. 316 (19), 3109e3123. http://doi.org/10.1016/j.yexcr.2010.08.008. Yorukoglu, A.C., Kiter, A.E., Akkaya, S., Satiroglu-Tufan, N.L., Tufan, A.C., 2017. A concise review on the use of mesenchymal stem cells in cell sheet-based tissue engineering with special emphasis on bone tissue regeneration. Stem Cell. Int. http://doi.org/10.1155/2017/2374161. Yoshimura, A., Muto, G., 2010. TGF-b function in immune suppression. In: Ahmed, R., Honjo, T. (Eds.), Negative Co-receptors and Ligands. Current Topics in Microbiology and Immunology, vol. 350. Springer, Berlin, Heidelberg, pp. 127e147. Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., et al., 2005. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing Tcell anergy. Blood 106 (5), 1755e1761. http://doi.org/10.1182/blood-2005-04-1496. Zhang, L., Wang, C.C., 2014. Inflammatory response of macrophages in infection. Hepatobiliary Pancreat. Dis. Int. 13 (2), 138e152. https://doi.org/10.1016/S1499-3872(14)60024-2. Zhang, Z., Han, Y., Song, J., Luo, R., Jin, X., Mu, D., et al., 2015. Interferon-g regulates the function of mesenchymal stem cells from oral lichen planus via indoleamine 2,3-dioxygenase activity. J. Oral Pathol. Med. 44 (1), 15e27. http://doi.org/10.1111/jop.12224. Zhao, W., Wang, Y., Wang, D., Sun, B., Wang, G., Wang, J., et al., 2008. TGF-beta expression by allogeneic bone marrow stromal cells ameliorates diabetes in NOD mice through modulating the distribution of CD4þ T cell subsets. Cell. Immunol. 253 (1e2), 23e30. http://doi.org/10. 1016/j.cellimm.2008.06.009. Zheng, Y.H., Deng, Y.Y., Lai, W., Zheng, S.Y., Bian, H.N., Liu, Z.A., et al., 2018. Effect of bone marrow mesenchymal stem cells on the polarization of macrophages. Mol. Med. Rep. 17 (3), 4449e4459. http://doi.org/10.3892/mmr.2018.8457.

Chapter 4

Stem cell therapies in ocular repair, regeneration, and diseases Eman E. Taher1, 2, Mohamed Elalfy1, 3, Kareem Elsawah1 1 Research Institute of Ophthalmology, Giza, Egypt; 2UPMC Eye and Ear Institute, Ophthalmology & Visual Sciences Research Center, University of Pittsburgh, Pittsburgh, PA, United States; 3 Queen Victoria Hospital and Eye Bank, East Grinstead & Maidstone and Tunbridge Wells NHS Trust, East Grinstead, United Kingdom

Introduction Stem cells are naive cells with a composite structure and are functionally undifferentiated. They can self-renew and differentiate into other cell types in the body. In addition, stem cells can repair tissue and restore function after injury. When relocated into a suitable environment, they either propagate and proliferate into their own native type or differentiate into varied types of cells and generate cell populations of those types. Replacement therapy by stem cells has become a promising mode of treatment for various pathologies and diseases that may lead to profound visual loss and ultimately blindness (Chagastelles and Nardi, 2011; Kalra and Tomar, 2014). Stem cells have unique properties; they have the ability to proliferate by splitting and proliferating over extended periods of time. They are selfrenewable, with the ability to continue as a stem cell similar to the mother cell. Stem cells can differentiate, bringing about specialized cells of different types (Bennicelli and Bennett, 2013). Stem cell differentiation is affected by internal stimuli, controlled by the cell’s genetic material, and external factors that are managed by other cells in the surroundings, by secreted chemical elements and/or the physical influence of adjacent cells, and by other molecules in the surrounding environment (Bennicelli and Bennett, 2013). In addition, stem cells can repair tissue and restore function after injury. When relocated into a suitable environment, they either propagate and proliferate

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into their own native type or differentiate into varied types of cells and generate cell populations of those types. Because of this unique potential, stem cells may be capable of either replacing or restoring destroyed cells as a curative option for many diseases. However, many regulatory, scientific, and ethical challenges are still present for stem cell therapy (Zarbin, 2016). Stem cell types include the pluripotent embryonic stem cells (ESCs), which can differentiate into any type of body cell that is ectodermal, mesodermal, or endodermal in origin. ESCs can be produced in culture from the intrinsic cell mass of an embryo (blastocyst) removed in the first 3e5 days of early embryogenesis, or can be removed without destroying the embryo (Zarbin, 2016). Adult mesenchymal stromal/stem cells (MSCs) are found in adult tissues such as blood vessels, skin, teeth, bone marrow, skeletal muscles, fat, and cartilage, and are isolated from these tissues in vitro. MSCs are multipotent and, therefore, can differentiate into many species of specialized cells in the body. The most commonly used MSCs are those derived from the bone marrow of fat tissues (Gnecchi et al., 2016). Induced pluripotent stem cells (iPSCs) are another type derived by conferring ESC characteristics to cells that are derived from adult tissues through in vitro genetic formulation. iPSCs are pluripotent, like ESCs, and have the advantage of harboring the properties of ESCs with being autologous, requiring less immunosuppression, and expressing cell markers (Fields et al., 2016). iPSCs can produce different cell types (Singh et al., 2015). Other types and sources of stem cells include cord blood stem cells, which are derived in vitro from cells collected from umbilical cord blood following delivery, and amniotic fluid stem cells, which are isolated in vitro from cells collected from the amniotic fluid (Lund et al., 2006; Kalra and Tomar, 2014). The mechanisms of stem cell action are variable, as healthy cells have the capability to replace unhealthy or lost stem cells. Nutritional support to adjacent cells is provided by secreting specific growth factors. In addition, reports have described the stem cell monitoring ability of the decay of vessels and cells by prohibiting apoptosis, and they can establish new synaptic connections (Zarbin, 2016). In contrast to other organs, the eye is considered a good applicant for stem cell clinical research, mainly because of it being a relatively immuneprivileged site and the clear ocular medium, which allows direct visualization of the transplanted cells. Furthermore, smaller quantities of therapeutic tissue are required due to the relatively smaller size of the eye compared with other organs of the body (Whiting et al., 2015). The surgical approach is easy for trained ophthalmic surgeons, with easy monitoring by routine imaging in clinical practice; the healthy eye can be used as the control without the need for long-term immunosuppressive therapy because of the characteristic ocular immune privilege (Bennicelli and Bennett, 2013).

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Stem cell therapy for retinal diseases Degenerative retinal diseases are the leading cause of irreversible vision loss; they include age-related macular degeneration, retinitis pigmentosa, and Stargardt macular dystrophy. These diseases can cause intensifying visual decline due to continuous loss of photoreceptors in addition to the outer nuclear layers. Stem cells have been considered as a promising therapy for such conditions. Stem cells can both restore visual function and perform functions as neurotrophins, inhibiting apoptosis of neurons, and in immunoregulation secretion. Experimental and clinical studies on stem cell therapeutic approaches ¨ ner, 2018). for retinal diseases have been reviewed (Garg et al., 2017; O Stem cells can differentiate into many other cell types of the body and can also repair and restore destroyed retinal cells and functions after injury. In the eye, stem cells have two main functions. One is a regeneration function that can replace ganglion cells of the retina. The other function is the production of some growth factors and cytokines, such as nerve-derived growth factor, which can induce an effect on the surrounding cell types in macular degeneration (Schwartz et al., 2016). Stem cell transplantation in the retina, especially the subretinal space, is superior since the eye is approximately immune entitled. The bloodeocular barrier preserves the subretinal space through antigenspecific prohibition of the reactions of the cellular and humoral immune systems, presuming that it is not physically compromised during transplantation or due to a developed disease pathology (Kaplan et al., 1999; Garg et al., 2017). However, stem cell therapies of the eye still face many regulatory, ¨ ner, 2018). scientific, and ethical challenges (O Many studies have evaluated the applications of different stem cell types in retinal diseases. MSCs (from the bone marrow or adipose tissues), iPSCs, and ESCs are used for the treatment of retinal diseases (Whiting et al., 2015). Experimental research has recorded that application of healthy stem cells in the place of degenerated retinal cells promotes the formation of new intercellular connections, cell regeneration, and progression of different visual functions (Whiting et al., 2015). Moreover, other experimental studies have shown that stem cells are very compatible with the retina and can adapt to amacrine, bipolar, horizontal, Mu¨ller, and glial cells as well as photoreceptors (Tucker et al., 2014). Many studies have demonstrated the great advantages of the high proliferative capacity of MSCs (Garg et al., 2017). MSCs can differentiate into many cell types of mesodermal, ectodermal, and endodermal origin, and can be isolated from various tissues, including the cord blood, peripheral blood, liver, teeth, nervous system, and, specifically, both adipose and bone marrow tissues (He et al., 2014). MSCs that are derived from the adipose or bone marrow tissue are the most commonly used in tissue repair and regeneration. Moreover, MSCs can differentiate into neuron-like cells, have a neuroprotective effect, and secrete growth factors, as well as repairing synaptic

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connections and damaged cells through their paracrine action (Emre et al., 2015). They can also provoke the construction of practical connections (Emre et al., 2015), and promote functional connection development (Konno et al., 2013). MSCs have a vigorous immune-repressive effect and prohibit the discharge of proinflammatory cytokines. They do not induce the formation of tumors and their use is not associated with any ethical conflict. Due to their unique characteristics, MSCs were at first tested experimentally, after which clinical testing was implemented for various disease groups in humans (Guan ¨ ner, 2018). et al., 2013; O Experimental studies have revealed that MSCs can differentiate to various cell types of the retina. In an experimental study carried out by Castanheira et al. (2008), MSCs were injected into the vitreous chamber in a laser-induced retinal damage model. This study found that most of the MSCs had shifted to the ganglion cell layer and both inner and outer nuclear layers (Castanheira et al., 2008). It also showed that the injected MSCs expressed the markers of photoreceptors, bipolar cells, amacrine cells, and Mu¨ller glial cells (Castanheira et al., 2008). This study did not record tumor creation, and the ethical argument regarding the use of MSCs does not exist, as recorded in other studies (Chen et al., 2011). In addition, Huang et al. (2012) reported MSC differentiation into retinal pigmented epithelium (RPE)-like cells with identical morphological features that could replace the damaged cells. Moreover, it was reported that MSCs survived for 90 days in rat vitreous and for 6 months in other retinal tissues and, therefore, MSCs are thought of as an upcoming therapeutic choice for degenerative retinal diseases (HaddadMashadrizeh et al., 2013). Furthermore, Konno et al. (2013) revealed that rat MSCs can activate Mu¨ller cell differentiation and exert a paracrine effect by secreting growth factors in culture. Interestingly, Park et al. (2014) have also recorded that the injection of MSCs does not result in intraocular inflammation, proliferation, or deterioration on electroretinogram (ERG). Other experimental studies reported that the subretinal application of MSCs can reconstruct disintegrated retinas in retinal degeneration models in rats, and the secreted elements from human MSCs can prevent light-induced retinal damage (Jian et al., 2015). Human ESCs (hESCs) are a very important tool of regenerative medicine. Similarly, iPSCs have advanced the field of regenerative medicine and enable the progress of retinal stem cell therapy (Garg et al., 2017). iPSCs are derived by conferring ESC characters to cells that are derived from adults through in vitro genetic formulation, and are pluripotent like ESCs (Garg et al., 2017). Current retinal differentiation protocols for iPSCs can be classified into default differentiation, where cells are cultured without extrinsic growth factors, or directed differentiation, that is, dependent on the addition of extrinsic transcription elements, small molecules, and proteins. The selection of the appropriate protocol relies on the purification goals and research aims, as one differentiation protocol administers complete proficiency in producing retinal

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cells (Borooah et al., 2013). The role of iPSCs in retinal conditions therapy lies in their success in creating the makeup of the retina, but with the disadvantages of induction of the T cell-mediated immune reaction and suppression of tumor-suppressive genes, which gives these cells an oncogenic potential (Zhao et al., 2011). Many groups of retinal cells, such as photoreceptors as well as those of the RPE and ganglion cells, were successfully differentiated from iPSCs (Lamba et al., 2010). The RPE cells are monolayers with pigments, which make them easier to purify and separate than other groups of cells (Corneo and Temple, 2009). The RPE cells are the first retinal cell groups to be differentiated from iPSCs. RPE transplantation using iPSCs is effective since the transplanted iPSCs have the advantage of harboring the properties of ESCs with being autologous, having less need for immunosuppression, expressing RPE-specific markers, and showing the ability to process retinoids like native RPE (Fields et al., 2016). Tucker et al. (2011) have reported the enhancement of retinal functions in rats using iPSCs as measured with ERG. In addition, the experimental study of Li et al. (2012) has demonstrated the ability of human iPSCs to differentiate into RPE cell types and increase retinal functions in rats. Remarkably, the iPSCderived RPE cells express the markers of RPE cells and there is an increase in the rat ERG responses, compared with the control group, indicating that these cells are functionally and morphologically RPE-like cells without tumor development in rats (Li et al., 2012). These promising results of experimental studies encourage testing this approach in humans in clinical trials. In humans, studies had taken different approaches for the application of RPE transplantation using stem cells in ophthalmology (Schwartz et al., 2016). The first human studies of stem cell-based RPE transplants were published in 2012 for atrophic macular disease (AMD) and Stargardt disease (Schwartz et al., 2012). In addition, Schwartz et al. (2016) have performed clinical trials in patients with degenerative retinal diseases. In this study, subretinal transplantations of RPE cells derived from hESCs were performed, in combination with immunosuppression at the location of the transplant (Schwartz et al., 2016). Following the transplantation, the patients showed an increase in subretinal pigmentation, implying the existence of the injected cells (Schwartz et al., 2016). Moreover, there were no signs of rejection, aberrant tissue formation, tumorigenicity, or hyperproliferation for several months after transplantation (Schwartz et al., 2016). In addition, there was no detectable serious adverse outcome in visual field, visual acuity, static perimetry, electroretinography, or reading speed (Schwartz et al., 2016). Even after 4 years, none of the eyes experienced proliferative vitreoretinopathy or a retinal detachment nor developed abnormal growth evocative of a teratoma (a tumor composed of two or more germ layers, which could originate from stem cells) (Schwartz et al., 2016). However, there was an increase in various functional endpoints, such as best-corrected visual acuity and degrees of life aspects, while the requirement

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for more structuralefunctional correlation with clinical investigations, including microperimetry, autofluorescence imaging, and optical coherence tomography scanning, was admitted (Schwartz et al., 2016, Siqueira et al., 2015). Song et al. (2015) published preliminary results of subretinal hESC-derived RPE transplantation on a coated, synthetic basement membrane in several patients: two with advanced macular dystrophy and two with Stargardt disease. This study revealed the success and feasibility of grafting RPE cells on the synthetic membrane for the therapy of different eye diseases. Despite the rapid progress in cell-based therapy of retinal diseases, some problems have been recorded, including retinal separation due to proliferative vitreoretinopathy, dislocation of a fluocinolone implant used for immunosuppression, and worsening of diabetes from oral steroids treatment (Uy et al., 2013). In addition, other studies have utilized iPSCs in two trials for different patients, using an autologous graft in a single patient with Stargardt disease that led to vision stabilization. However, there were disadvantages of this autologous graft, mostly because of the expense and length of time (3 months for preparation, and gene mutation occurred in the second panel of cells). In the second trial, they used an allogeneic type of cell with the complications of both rejection and immunosuppression (Garg et al., 2017). MSCs have a great advantage and, therefore, have been used in many cellbased therapy studies, since they can be used for both allogeneic and autologous transplantations (Chen et al., 2011). In potential phase I studies, a single dose of intravitreal analogous bone marrowederived MSCs was tested in three patients with retinitis pigmentosa and two patients with coneerod dystrophy (Siqueira et al., 2011). No constitutional or operative toxicity was detected in the retinas in 10 months of follow-up of the patients in these studies (Siqueira et al., 2011). In addition, Siqueira et al. (2015) applied MSCs in the vitreous of degenerative retinal disease patients and observed no indicative basic or functional toxicity in the retinas. A statistically significant improvement in the vision of the patients was correlated with quality of life at 3 months. However, the scores returned to basic levels at 12 months (Park et al., 2014). Other studies have reported MSC injection intravitreally as a well-accepted treatment for eyes with permanent vision loss. This injection resulted in no intraocular irritation or proliferation, and there was no decrease in ERG and best-corrected visual acuity (BCVA) results in no recorded systemic side issues after a follow-up of 6 months. One patient only developed a complication, which was treated with a dose of antivascular endothelial growth factor agent (Oner et al., 2016). Notably, the preferential use of MSCs in eye therapy is associated with an increased record of ocular complications due to MSC therapy. For example, Kuriyan et al. (2017) have described several complications after the intravitreal utilization of adipose tissueederived MSCs, including an elevated intraocular pressure, hemorrhagic retinopathy, vitreous hemorrhage, and tractional detachment of the retina during the follow-up periods, with the loss of vision in patients. In addition, other complications

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such as the total tractional detachment of the retina and vision loss within 3 months were also reported (Satarian et al., 2017). Interestingly, the introduction of therapy into the suprachoroidal space might prohibit complications reported after intravitreal and subretinal applications (Limoli et al., 2016). In addition, no complications were recorded in 36 eyes of 25 patients with dry AMD, who also showed an improvement in visual function, at 6 months after the administration of adipose-derived MSCs under a deep scleral flap in the suprachoroidal area (Satarian et al., 2017). Several complications of iPSC transplants were related to photoreceptor graft orientation, as they should be adjusted to where the inner synapse and the outer photoreceptive portions are located, facing the host inner retina and RPE, respectively (Nakano et al., 2012). This obstacle could be overcome by the advancement of a three-dimensional multilayered autonomous optic cup consisting of both rods and cones that curves spontaneously in an apically convex configuration. In addition, other studies have reported the creation of retinal cups containing all major cell types, which are configured properly and capable of demonstrating the beginning of outer-segment disc formation and photosensitivity (Zhong et al., 2014). Furthermore, Assawachananont et al. (2014) have described a successful therapy of retinal sheet transplantation by implanting an exterior nuclear layer in a model with advanced retinal degeneration. Other complications of the iPSC-based therapy of eye diseases include epigenetic profile, DNA sequence, and copy number heterogeneity between cell lines produced at different laboratories (Garg et al., 2017). In addition, tumorigenesis is of significant concern in iPSC-based therapy, since it is mostly caused by the perseverance of indiscriminate iPSCs during cell reprogramming and differentiation protocols. This might be alleviated by applying a combination of reprogramming transcription elements or adjusting DNA methylation status that allows for the quality and heterogeneity of iPSC lines (Watanabe et al., 2013). Moreover, the pluripotency is a great strength for iPSCs. However, pluripotency can be viewed also as a principal disadvantage for iPSCs, since the recognition and separation of desired cell types is challenging. If it is not achieved properly, unwanted cell types will contaminate the same dish (Gamm et al., 2013).

Stem cell therapy for corneal diseases Stem cells’ ability to differentiate into numerous lineages of cells has a major potentiating effect on regenerative medicine. At this writing, the most common stem cell sources for clinical applications are embryonic, adult, and iPSCs. The cornea is the clear front window of the eye. Its clarity is essential for normal vision. The cornea plays various roles in light refraction and transportation, along with safeguarding internal eye structures from environmental injuries. It is characterized by avascularity and transparency, which both are vital for normal vision (Gonzalez et al., 2018).

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Histologically, the cornea contains various tissues, including the epithelium, stroma, and endothelium, separated by two membranes, the Bowman membrane, which is located between the epithelial and the stromal layers, and the Descemet membrane, which is between the endothelial and the stromal layers. A recently discovered pre-Descemet layer, also known as the Dua layer after Professor Harminder Dua of Nottingham University, who discovered this layer, lies posterior to the corneal stroma and anterior to the Descemet membrane (Dua et al., 2013). The corneal epithelium is the outermost layer, forming 10% of the thickness of the cornea, and is continuous with the conjunctival epithelial cells at the corneoscleral limbus. An indicative aspect of the corneal epithelial tissue is its immense developmental potentiality and capability for the rapid regeneration of the ocular surface through propagation and central movement of progenitor cell populations residing at the corneae sclera border in a location called the limbus. The corneal epithelial tissue is nonkeratinized, stratified, and squamous, and of ectodermal origin (Gonzalez et al., 2018). The major functional role of the corneal epithelial tissue is protective by acting as a defensive boundary that prevents pathogen infiltration. In addition, it is responsible for the smoothness and cohesion of the corneal anterior surface in addition to nutrient and oxygen consumption (Sasamoto et al., 2018). The corneal epithelial tissue has a remarkably stable process of cell rejuvenation and restoration characterized by dynamic equilibrium. The corneal epithelial basal cells are characterized by their proliferation capacity, while their daughter cells show a differentiation capacity to give rise to wing cells, and subsequently form the superficial cells that localize at the surface of the cornea (Wagoner, 1997). Both the death and the desquamation of these superficial cells are induced by multiple factors, including exposure to ultraviolet radiation, mechanical friction, and hypoxia (Estil et al., 2000; Esco et al., 2001). The shed corneal epithelium is regenerated by mitosis from basal cells accompanied by emergence from stem cells localized at the limbus. Therefore, a severe limbal stem cell (LSC) deficiency (LSCD) may cause conjunctival invasion onto the corneal surface due to loss of the cellular barrier, with subsequent corneal vascularization, which in turn leads to corneal opacification (Nishida et al., 2004; Sasamoto et al., 2018). Pathophysiologically, corneal transparency is crucial for normal vision, and thus the exterior defensive stratified corneal epithelial cell layer is under continuous, accelerated rejuvenation with intense restoration mechanisms. These processes are vital since the cornea is continually exfoliating every 3e10 days, and any epithelial cell damage or loss should be replenished instantly (Ebrahimi et al., 2009). This repair process is critical to both avoid infection and conserve vision. The corneal stem cells are located in the corneal periphery at the limbus, in the basal layer of cells located in pigmented crypts, named the palisades of Vogt (Ebrahimi et al., 2009). This pigmentation can probably support stem cell protection from potential damage from exposure to ultraviolet light. The restoration of normal, nondiseased cornea starts from the

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basal cells and is associated with stem cell migration centripetally (Hertsenberg and Funderburgh, 2015). These stem cells, together with their progenitors, take their vascular nourishment from blood vessels in the stromal vasculature of the conjunctiva and limbus outside the cornea, hence their location at the corneal periphery (Ramos et al., 2015). Reciprocally, the cornea is an avascular tissue. Corneal avascularity is crucial to avoid interference of vascular structures with light propagation and thus vision. The limbus acts as a barrier prohibiting vascularization of the cornea from the conjunctiva. Damage of the limbal integrity results in conjunctival cell migration to the cornea, which could in turn progress to corneal neovascularization (Ramos et al., 2015). The Bowman layer is an acellular, membrane-like zone located immediately posterior to the basement membrane of the epithelium. The corneal stroma is avascular and represents most of the corneal thickness (90%). It contains glycosaminoglycans and proteoglycans, water, and collagen fibrils, with spindle-shaped keratocytes abundant in between the collagen fibrils. Corneal transparency relies on the organization of the collagen fibers (Meek and Knupp, 2015). The stroma of the cornea plays essential roles in corneal functions, mainly the transfer and refraction of light. The mean diameter of collagen fibrils (22.5e35.0 nm) and the mean distance between them (w41.4 nm) are relatively homogeneous and less than half of the visible light wavelength (400e700 nm). This arrangement is responsible for the fact that scattering of incident light by each collagen fibril is canceled by interference from other scattered rays, and that is the major determinant of corneal transparency (Maurice,1957; Hassell and Birk, 2010). The Descemet membrane is the corneal endothelial basement membrane. The endothelial cells are well arranged in a mosaic pattern of polygonal cells (mainly hexagonal); they pump excess water outside the stroma, maintaining the corneal transparency. These cells are the innermost part of the cornea that is responsible for fluid and solute transportation between the aqueous humor and the corneal stroma. Notably, the endothelial cells cannot regenerate. If the endothelial cell density decreases to a pathological level, this results in increased imbibition of water by the corneal stroma and loss of its transparency, which may lead to corneal edema and opacity and thus impair vision (Hassell and Birk 2010; Ramos et al., 2015).

Corneal stem cell types The limbus is placed at a 2-mm-wide area between the bulbar conjunctiva and the cornea and was suggested to be the niche of corneal epithelial stem cells (Hsu et al., 2015). LSCs (corneal epithelial stem cells) are located at the basal layer of the corneal limbus in a definite and well-constructed niche of cells called the palisades of Vogt, which characteristically exists between the sclera and the cornea (Hsu et al., 2015). LSCs perform a vital role in corneal homeostasis and represent a quiescent cell populace with high proliferative

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potential, which facilitates competent corneal restoration and repair (Nowell and Radtke, 2017). In addition, limbal epithelial stem cells (LESCs) have several characteristic features, including the ability to form colonies, slow cell cycling, cell propagation capacity, expression of stem cell-specific markers, LESC reproduction ability, and the capability to reconstruct the entire corneal epithelium (Saghizadeh et al., 2017). The garland-like pattern of the limbal corneal epithelial stem cells surrounding the limbus represents a barrier that protects the clear cornea from the invasion of conjunctival epithelial cells. This invasion can cause a neovascularization of the cornea, an obvious clouding, and a detectable blurred vision or even total vision loss (Gonzalez et al., 2018). The basal epithelium near the limbus is not composed of a homogeneous cell population. The basal epithelial cells are mixed with a diverse population of stem cells, named transient amplifying cells (TACs), and other cells that are terminally differentiated (Hsu et al., 2015). The TACs are first generated by stem cells. They then migrate to the central cornea and proliferate rapidly afterward (Hsu et al., 2015; Nowell and Radtke, 2017). Little is known about the mechanisms that help to maintain the stemness of the stem cells in the limbus. However, several external and/or internal mechanisms and factors may be involved in maintaining the stemness of these cells, and these extrinsic factors or mechanisms are produced by the surrounding stem cell environment (Dua et al., 2000; Saghizadeh et al., 2017). The limbal zone is different from the central cornea since it has blood vessels derived from the palisades of Vogt, which provide nutrition to the limbus, and shows a greater interaction with blood cytokines (Saghizadeh et al., 2017). It also has anchoring fibrils, which extend from the basement membrane and intersect with other anchoring fibrils extending through the stromal pegs. This intersection forms a niche promoting the adherence of the LSCs, protecting them from physical injury. The limbal basement membrane is composed of collagen type IV, which is absent from the central cornea and enhances the attachment of epithelial cells in the limbal area (Saghizadeh et al., 2017). The basement membrane was also suggested to influence stem cell differentiation in areas (the stem cell niche) and contains high levels of type IV collagen and low levels of AE27 binding antigen (Dua et al., 2000). The intrinsic and characteristic properties of LSCs include the high rate of proliferation in culture, and both growth factors and calcium ions affect the cell type differently. They also characteristically contain high levels of several proteins such as metabolic enzymes (a-enolase), cytochrome oxidase, Na/K-ATPase, and carbonic anhydrase; interestingly, the intermediate filaments such as vimentin and keratin 19 may be responsible for anchoring the stem cells into a certain environment. Notably, the limbal epithelium is more resistant to tumor promoters, and transplantation of limbal epithelial cells can result in the growth of a limbus-like epithelium (Di Girolamo et al., 2015).

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Corneal and limbal stem cell markers Unique stem cell markers are those that enable the recognition of distinct stem regions within the epithelium and facilitate the isolation, enrichment, and characterization of viable stem cells at the molecular level. Many candidate markers have been identified in LSCs, where they regulate cell proliferation and other biological processes at different stages of development (Yoon et al., 2014; Saghizadeh et al., 2017). One of the common and conserved stem cell markers in various tissues is ABCG2, which exists in the cell plasma membrane and belongs to the ATP binding cassette (ABC) group of transporters (Saghizadeh et al., 2017). In addition, the transcription factor P63 is a suggested LSC nuclear marker and plays a major role in maintaining cell proliferation. P63 also functions as a tumor suppressor and plays a key role in tissue morphogenesis in different body organs. A 2018 transplantation study by Rama and coworkers using cultivated limbal epithelial cells demonstrated that success in the generation of normal epithelium on the patients’ stroma was associated with the percentage of P63-bright stem cells in culture (Sacchetti et al., 2018). Thus, in most (78%) cases, the transplantation was more successful if these cells were 4% or more of the clonogenic cell total number. The transplantation was successful in only 10% of cases when these cells were 3% or less of the clonogenic cell total number (Sacchetti et al., 2005; Rama et al., 2010). Most abundant cytosolic proteins in the corneal epithelial cells include both aldehyde dehydrogenase and transketolase. Indeed, staining methods of corneal sections such as lectin staining can help in characterizing different changes that occur in basal cells during their transition across the limbale corneal margin in experimental animals (Saghizadeh et al., 2017). Other proteins that are predominantly nuclear such as PAX6 protein play important functional roles in both ocular and other tissues (Li et al., 2015). For example, a heterozygous deletion of PAX6 in humans can lead to some ocular developmental defects/disorders, including the aniridia (Li et al., 2015). The cadherin family mediates cell-to-cell adhesion and other biological functions in different tissue types (Yoon et al., 2014). N-cadherin, a member of the cadherin family, was detected in putative stem and progenitor cells in the niche of LESCs in humans, in which it inhibits cell proliferation. As a cell adhesion molecule, N-cadherin is critical for cellecell adhesion between corneal epithelial progenitors and their surrounding niche cells (Yoon et al., 2014). In addition, another adhesion molecule, E-cadherin, plays an important role in mediating cellecell contact that can lead to cell activation and an enhancement of essential signaling molecules regulating both cellular proliferation and survival (Ramos et al., 2015). E-cadherin is easily detected in all cell layers of the corneal epithelium in rat (Bardag-Gorce et al., 2016), and in the suprabasal layers (but not in the basal layers) at the limbus in humans. The cellecell adhesion molecule P-cadherin is expressed in the basal cells of the

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ocular surface epithelia in humans, with a small P-cadherin-negative cell population existing in the limbal basal epithelium (Sacchetti et al., 2018). Many immunohistochemical studies have identified several subunits of integrins, such as integrins a2, a3, a4, a5, a6, and av, in addition to integrins ß1, ß4, and ß5, as present in corneal epithelial cells of humans, whereas integrins a1 and ß3 were not detected in these cells (Hsu et al., 2015). The expression of the transferring receptor CD71 was detected in both corneal and limbal epithelial cell types. Notably, the basal cells of the corneal epithelium show a positive staining for the CD71-specific antibody, while the limbal basal cells are negative for this antibody (Hsu et al., 2015). The octamer-binding transcription factor 4 (OCT4) is crucial for the selfrenewal and maintenance of ESCs. Zhou and coworkers found that the OCT4 gene is mainly expressed in the corneal epithelial basal layer, especially in the limbal area in humans. The OCT4-positive corneal epithelial basal cells show robust proliferation. In addition, OCT4 could probably control the phenotype of different layers of corneal epithelium (Zhou et al., 2010; Hsu et al., 2015). Vitronectin (VN) is a key extracellular matrix protein that is mainly detected in the basement membrane of the human limbus, but not the corneal basement membrane. Ordonez and collaborators have postulated that the VN receptors, integrins avb3/5, can be successfully used to identify and isolate the stem cells of the limbal epithelium (LESCs) (Ordonez et al., 2013).

Regulatory factors of limbal stem cell proliferation Several factors and molecules are important regulators of LSC proliferation. For instance, nerve growth factor (NGF) belongs to the neurotrophin family, which has many biological effects on stem cells outside the nervous system. NGF functions through a specific tyrosine kinase receptor (TrkA) to induce the proliferation of corneal epithelial cells. TrkA receptor is also considered a potential marker of corneal LSCs since it is preferentially localized to the limbal basal epithelium (Hsu et al., 2015). The growth factor receptor (GFR) family, including the epithelial (EGFR), hepatocyte, and keratinocyte GFRs, is highly expressed at the limbal basal cell membrane. In addition, undifferentiated limbal basal epithelial cells express more GFRs than suprabasal cells in the cornea of experimental animals. The expression level of the EGFR decreases in differentiated cells (Hsu et al., 2015; Gonzalez et al., 2018). b-Catenin is a central component of the cadherin cell adhesion complex, which functions as a key regulator of epithelial cell proliferation and differentiation in different tissues. In addition, b-catenin functions as a maintenance factor for keratinocyte stem cells. Moreover, in the cornea of experimental animals (e.g., rabbit and rat), b-catenin is strongly expressed in the limbal epithelial basal layer and is relatively weakly expressed in the corneal basal layer. Other regulatory factors of stem cell proliferation in the eye include the OCT4 transcription factor and cadherins as described earlier (Tian et al., 2011; Pipparelli et al., 2013; Hsu et al., 2015).

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Stem cell therapeutic approaches for corneal diseases Corneal surface reconstruction after an epithelial injury compromises division, migration, and maturation of specific stem cell groups that exist in the limbus. Several factors, both internal and external, can trigger the disruption of the microenvironment of the stem cell niche, resulting in LSCD. In LSCD, reepithelialization fails, with the conjunctiva extending across the cornea, resulting in vascularization, persistent epithelial defects, and chronic inflammation (Hertsenberg and Funderburgh, 2015; Gonzalez et al., 2018). ESCs and iPSCs represent an important source for the stem cell therapy in diseased or severely denuded cornea. These stem cell types are renewable, are easily expandable, and can be directed to limbal differentiation with some amount of stimulation, but this process is not yet fully optimized (Saghizadeh et al., 2017). Corneal LSCs are normally situated at the limbal stroma, mixed with complex limbal niche cells and extracellular matrix, mainly type IV collagen. The functions and stemness of LSCs are highly associated with the microenvironment, including the extracellular matrix. Adjusting the microenvironment of iPSCs or ESCs provides a way to guide their epithelial differentiation to be like the corneal LSC niche (Saghizadeh et al., 2017). hESCs have allowed significant advancements in the field of regenerative medicine. Although ESCs can give rise to multiple lineage cells such as corneal progenitors and their subtypes of cells, there are always ethical and immunological issues surrounding ESC therapies. Direct transplantation of ESCs to damaged or dysfunctional sites is a possible method for the treatment of degenerative diseases. However, both immune rejection and formation of teratomas remain great concerns (Hsu et al., 2015). This was revealed in a study by Ahmad et al. (2007), where positive detection of a differentiation marker was noted in some hESC-derived corneal epithelial-like cells, implying a possible future differentiation or oncogenesis during ESC therapy. Meanwhile, a lifelong immunosuppression is required in patients receiving cell therapy using hESCs due to their immunogenic properties (Ahmad et al., 2007; Whiting et al., 2015). Some adult stem cell types also show ESC-like properties and could potentially be used as alternative cells for regenerative medicine. For example, Yang et al. (2007) have isolated adult epidermal stem cells expressing ESC immunological markers from the skin of goat’s ear. Mature somatic cells (adult stem cells) or their progenitor cells that are differentiated from iPSCs might be safer for clinical use, compared with ESCs. This process can be done by inducing a stable change in the nucleus of a mature cell, which can then be maintained and replicated as the cell divides through mitosis (Whiting et al., 2015). These changes are most frequently associated with the reacquisition of a pluripotent state, thereby endowing the cell with developmental potential. Based on the iPSC production concept, there should be different sources of somatic cells for reprogramming, including skin fibroblasts, the most widely used cells. Different lineages of

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somatic cells for iPSCs showed various propensities for further differentiation of corneal epithelial cells in Nishida and colleagues’ study demonstrating that cell sources from corneal limbal epithelial cells are better than those from skin fibroblasts to produce epithelial-like cells (Nishida et al., 2004). This finding was due to the epigenetic modification on the genes dealing with iPSC differentiation (Whiting et al., 2015). Other studies directly transplanted ESCs or spontaneously differentiated ESCs and let these cells differentiate naturally in an in vivo microenvironment. Hanson et al. (2012) had simply cultured hESCs in a differentiation medium to facilitate their spontaneous differentiation (Hanson et al., 2012). Then, they transplanted these differentiated hESCs onto wounded human corneal buttons in culture, and successfully generated one to four layers of epithelial cells expressing Pax6 on day 3 and Pax6 with CK3 on day 6 (Hanson et al., 2012). Zhang and colleagues have developed a quick method to form scaffold-free ECS sheets (SESCSs) for transplantation (Zhang et al., 2014). They suspended ESCs in a glycerin medium and constructed cell layers on an amniotic membrane (AM). When SESCSs were transplanted in a rabbit with LSCD, corneal damage was healed, and the ESC sheets further differentiated into three groups of cells: corneal LSCs, corneal TACs, and terminally differentiated cells. These results demonstrated that the stem cell microenvironment is conducive to ESC differentiation into epithelial cells in vivo (Zhang et al., 2014; Gonzalez et al., 2018). The in vitro coculture of limbal epithelial progenitors with limbal niche cells led to the maintenance of clonal growth of corneal progenitors and a reduced epithelial cell differentiation. The most popular method for in vitro epithelial differentiation is to use collagen IV-coated plates and conditioned medium of limbal fibroblasts (Gonzalez et al., 2018). The conditioned medium of fibroblasts contains the keratinocyte growth factor, which is a stimulator for LSCs (Gonzalez et al., 2018). With the assistance of collagen IV-coated plates and limbal fibroblast-conditioned medium or DMEM/F12-conditioned medium, corneal epithelial progenitors expressing E-cadherin, CD44, P63a, and ABCG2 can be generated (Hayashi et al., 2007). These corneal epithelial progenitor cells can form a monolayer or multilayers of epithelial-like cells that adhere on corneal stroma in animal models (Gonzalez et al., 2018). The epithelial progenitors can be directly transplanted (Hayashi et al., 2007) or seeded on an acellular porcine corneal matrix as stratified epithelial cell sheets first and utilized later for transplantation (Saghizadeh et al., 2017). Ueno et al. tried transfecting the Pax6 gene to differentiate mouse ESCs into corneal epithelium-like cells (Ueno et al., 2007). The transcription factor Pax6 is essential for embryonic development and formation of both the cornea and the anterior segment or anterior cavity of the eye (Gonzalez et al., 2018). When these Pax6-transfected cells were transplanted onto damaged corneas, they could adapt to the injured cornea and remained alive on it (Gonzalez et al., 2018).

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Limbal stem cell deficiency LSCs are important for maintaining corneal epithelium integrity. Several insults, either intrinsic or extrinsic, can lead to the destruction of the delicate microenvironment of the stem cell niche. This absence of structural support leads to the death of the stem cell population in a condition known as LSCD, and the cornea cannot therefore regenerate itself, leading to scarring and loss of transparency. LSCD can be complete or incomplete, unilateral or bilateral (Ahmad, 2012; Barut Selver et al., 2017). The etiology of LSCD can be genetic, acquired, or idiopathic. As for genetic causes, LSCD has been associated with PAX6 gene mutations, which are also implicated in both aniridia (Skeens et al., 2011) and Peters anomaly (Hatch and Dana, 2009). Other genetic disorders that have been reported with LSCD include ectrodactylyeectodermal dysplasiaeclefting syndrome (Di Iorio et al., 2012), keratitiseichthyosisedeafness syndrome (Messmer et al., 2005), xeroderma pigmentosum (Fernandes et al., 2004), dominantly inherited keratitis (Lim et al., 2009), Turner syndrome (Strungaru et al., 2014), and dyskeratosis congenita (Aslan et al., 2009). The acquired causes of LSCD may be either inflammatory, as those seen in SteveneJohnsons syndrome (Puangsricharern and Tseng, 1995), ocular cicatricial pemphigoid (Tsai et al., 2000), and graft versus host disease (Meller et al., 2009). Chronic ocular allergy such as vernal keratoconjunctivitis is another reported cause (Sangwan et al., 2011a,b). Neurotrophic keratopathy, whether neuronal or ischemic, can also lead to this disease as well (Dua et al., 2000), as can bullous keratopathy (Uchino et al., 2006). Acquired causes may also be due to any infection of the corneal surface, such as herpes keratitis (Dua et al., 2000) and trachoma (Dua and Azuara-Blanco, 1999). In addition, the acquired causes also include trauma from chemical or thermal burns, and patients who have undergone prior ocular surgeries or cryotherapies at the limbus are, therefore, more susceptible to the disease (Dua et al., 2000; Sridhar et al., 2001). Moreover, both radiation and chemotherapy, as well as systemic and topical chemotherapeutic medications, may cause LSCD (Ding et al., 2009; Lichtinger et al., 2010). LSCD has also been seen with benzalkonium chloride toxicity with glaucoma medications (Kim et al., 2014). The inappropriate use of contact lenses, with consequent hypoxia and ocular irritation with destruction of the limbus, may also contribute to both focal and total LSCD (Jeng et al., 2011; Chan and Holand, 2013). It is worth saying that ocular surface tumors are a known cause of LSCD (Dua et al., 2000). The pterygium may also cause a focal acquired absence of LSCs (Tseng, 1985). LSCD can eventually lead to the encroachment of the conjunctiva over the cornea (conjunctivalization), which is indicated by goblet cell detection in the cornea, and recurrent/persistent epithelium defects that lead to chronic keratitis, neovascularization, and calcification, if untreated. These defects can also lead to subepithelial scarring, corneal opacity, and vision loss. In severe cases, LSCD can lead to corneal melting and perforation (Saghizadeh et al., 2017).

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The complaint of patients with LSCD is usually pain, photophobia, and blurry vision due to repeated epithelial defects. Other symptoms vary according to the etiology. They include contact lens intolerance, blepharospasm, and infectious keratitis. Patients mostly have tear film dysfunction, either mild or moderate (Gonzalez et al., 2018). On examination, there is loss of the palisades of Vogt, a whorled corneal epithelium of frank conjunctivalization, scarring, and neovascularization in advanced cases. Recurrent epithelial defects can also get secondarily infected (Sacchetti et al., 2005; Lim et al., 2009). Some modalities can help in the diagnosis of LSCD, including impression cytology, which reveals conjunctivalization of the cornea by absence of keratin Ck3 and the presence of goblet cells. However, inadequate goblet cells might be detected in nearly one-third of patients. The mixture of conjunctival and corneal epithelial cells (or mainly conjunctival epithelial cells) is greatly improving LSCD treatment. On histopathology, there is infiltration of the conjunctival epithelium to the affected part, neovascularization, interruption of the basement membrane, and inflammatory cell infiltrates (Saghizadeh et al., 2017; Gonzalez et al., 2018). The application of in vivo confocal microscopy has helped in the diagnosis of LSCD. Detected changes may include the absence of the palisades of Vogt in the affected sector, decreased basal epithelial cell density, basal nerve density, and vascular fibrotic tissues replacing normal limbal epithelial cells in late stages (Ramos et al., 2015).

The management of patients with limbal stem cell deficiency The management of patients with LSCD relies upon the extent of involvement of the limbus (sectorial vs. total), and whether the LSCD is unilateral or bilateral. LSCD is commonly dealt with clinically by transplanting an autologous or allogeneic limbal graft or cultured LESCs, since the transplantation of LSCs is the only available treatment for LSCD (Saghizadeh et al., 2017). Disease management is typically symptomatic early in the disease. Conservative medical measures might be satisfactory when LSC damage is transient (LSC disease or LSC distress; Atallah et al., 2016). However, the management of total LSCD must be surgical (Fernandez-Buenaga et al., 2018). In cases of partial or total LSCD, prior to implantation a better microenvironment of the ocular surface can help the remaining LSCs or the transplanted limbal graft to survive. Conservative first-line procedures are established on two general principles: controlling causative factors as mentioned previously and controlling comorbid conditions. Controlling such issues will promote differentiation of healthy epithelium (Gonzalez et al., 2018). For the causative factors, controlling includes immunosuppression for autoimmune diseases or chronic inflammation, antibiotics for infection, tumor resection, and stoppage of any iatrogenic stimulus. Comorbid condition management necessitates addressing the associated adnexal problems; eyelid

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problems like cicatricial changes, trichiasis, lagophthalmos, and ankyloblepharon; and aqueous tear deficiency (Liang et al., 2009). Punctual occlusion, topical vitamin A ointment, and salivary gland implants may help in improving the wetting of the ocular surface. Autologous serum eye drops might also be used. Scleral lens is another option to optimize the health of the ocular surface (Atallah et al., 2016; Sacchetti et al., 2018). In partial LSCD, the conjunctival epithelium is mechanically debrided from the corneal surface using a crescent blade, which will be enough to regain a stable ocular surface since limbal cells can migrate from healthy sectors and cover the defect. This scraping can be coupled with the transplantation of the AM for faster healing of the ocular surface (Atallah et al., 2016). The AM can promote corneal reepithelialization by preserving and maintaining the epithelial progenitors as well as reducing both angiogenesis and inflammation (Nguyen et al., 2018). The postoperative treatment includes topical antibiotics, artificial teardrops, and bandage contact lens. Follow-up examinations will reveal increase in visual acuity, decreased pain, and progressive epitheliopathy (Nguyen et al., 2018). The management of patients with total LSCD is quite challenging. At this writing, penetrating keratoplasty is a prohibited therapy for LSCD. Treatment involves autologous (in unilateral LSCD patients) or allogeneic keratolimbal grafts for total bilateral LSCD (Atallah et al., 2016). The Cornea Society has classified various stem cellebased transplantation techniques based on the anatomic source of the transplanted tissue (conjunctival, keratolimbal, or mucosal) and whether the stem cells were obtained from the patient’s eye (i.e., autograft), a cadaver (i.e., allograft), or a living related donor (i.e., allograft). Various methods and techniques have been used in preparing transplant sheets, e.g., a sheet of corneal epithelial cells alone or cells that were cultured on a polymer substrate such as collagen and fibrin or on a biomaterial such as the AM (Nguyen et al., 2018).

Conjunctival limbal autografts The conjunctival limbal autograft is considered the best alternative as of this writing for restoration of corneal epithelium in unilateral LSCD. From a healthy eye, part of the limbal tissue can be transplanted into the eye area with LSCD in the fellow eye of the same patient. This method was first introduced for unilateral burn victims in 1965 by Barraquer (1965). Over a decade later, Kenyon and Tseng (1989) introduced the first modern-day limbal autograft: two large grafts extending 5e7 mm in limbal arc length were taken from the healthy eye to the diseased one. The benefit of this technique is that systemic immunosuppression is unnecessary (Cristina et al., 2017). This technique showed a high success rate in the short and intermediate follow-up periods (success rate of 80%e100%, and 25%e100% improvement in visual acuity) (Nubile et al., 2013). Long-term follow-up showed a survival rate of 76% and 62% at 3 and 6 years after surgery (Cauchi et al., 2008). The tissues are

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collected from donors under topical, local, or general anesthesia and should not contain Tenon’s capsule. In the preparation of the recipient eye, a peribulbar, sub-Tenon, or general anesthesia is performed, followed by a 360degree peritomy, 1e2 mm peripheral to the visible limbus, with the removal of the fibrovascular pannus that covers the cornea. It is important to find the plane of least resistance and extend that across the whole cornea. The thin abnormal tissue is mechanically scraped off the cornea (Cristina et al., 2017). The AM can be utilized as a graft, if the bed is scarred and unhealthy, or as a patch. For the success of this technique, large grafts should be considered (Cristina et al., 2017). Vision was improved in 90% of 39 patients who had unilateral LSCD and the ocular surface was restored in 94% of them when large grafts (120 degrees) were used. Visual improvement dropped down to 60% in 22 cases when smaller grafts were attempted (Liang et al., 2009). This was confirmed in 2012 by Baradaran-Rafii et al., who concluded that most common complications are associated with suboptimal graft (size, thickness, and position) and chronic surface exposure (Baradaran-Rafii et al., 2012). The focus of this technique is to find an equilibrium between taking a sufficiently large graft and avoiding risk to the healthy eye.

Living related conjunctival limbal allograft The living related conjunctival limbal allograft technique requires immunosuppression for a long time and was first presented by Kenyon and Tseng (1989) for the management of LSCD by using, at first, a conjunctival allograft from a living related donor, which could be then modified to include limbal tissue (Baradaran-Rafii et al., 2012). Recent advances use eccentric trephination of the donor’s cornea (Yin and Jurkunas, 2018). Cadaveric keratolimbal allografts The cadaveric keratolimbal allograft (KLAL) procedure utilizes cadaveric limbal tissue to provide a large stem cell supply to the diseased cornea. Indications for this technique are bilateral cases with absence of or unwilling living related donors and unilateral cases in which the patient or doctor does not want to jeopardize the healthy eye (Holland, 1996; Croasdale et al., 1999; Meisler et al., 2005). The current technique of KLAL utilizes two donor corneoscleral rims and adds them all around the affected cornea in 360 degrees. Due to the lack of fresh conjunctival tissue in this procedure, a new combined procedure was introduced by Holland that takes fresh conjunctival tissue from a living related donor and a keratolimbal graft from a cadaveric one, transplanting them simultaneously (Biber et al., 2011; Holland, 2015; Haagdorens et al., 2016). The first series of patients who underwent allografts reported by Tsai and Tseng (1994) with follow-up over a period of 18
5 months, showed an improvement of visual acuity in 13 eyes (81.3%) and a rapid (within 1 week)

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surface healing in 10 eyes (62.5%), with no graft failure or rejection. Tan et al. (1996) reported success in seven cases of nine with a follow-up over 14.7 months. Tsubota et al. (1995) also reported the same results over a short follow-up period. Long-term results of allografts are still in question, as many studies with a long-term follow-up showed a decline in graft survival. Ilari and Daya (2002) followed up 33 KLAL procedures in 23 patients with different etiologies for 60 months (a range of 15e96 months). Eight eyes (24.2%) were never reepithelialized and were considered primary failures. The remaining 25 grafts initially restored a phenotypic corneal epithelium, but at last follow-up only 7 (21.2%) were stable. The graft survival rate was 54.4% at 1 year, 33.3% at 2 years, and 27.3% at 3 years. Visual acuity improved or was unchanged in 19 eyes (82.6%) and decreased in 4 eyes (17.4%; Ilari and Daya, 2002). A similar long-term follows-up showed comparable results in other studies (Solomon et al., 2002; Han et al., 2011; Moreira et al., 2015). The grafts in these cases were allogeneic, and graft rejection is the main issue in this type of procedure, which requires both systemic and long-term immunosuppression. This is often accompanied by many potential side effects such as anemia, liver and kidney function impairment, and worsening or initiation of diabetes mellitus (Holland, 2015; Haadorgens et al., 2016; Vazirani et al., 2016; Holland et al., 2012). Another association of immunosuppression is continuing or developing elevated intraocular pressure postoperatively. This requires further intervention, either medical or surgical in the form of cyclophotocoagulation or shunt surgery (Tsubota et al., 1995; Solomon et al., 2002; Han et al., 2011; De La Paz et al., 2008; Nassiri et al., 2011).

Simple limbal epithelial transplantation Sangwan et al. (2012) used the simple limbal epithelial transplantation (SLET) technique for the first time to treat patients with unilateral LSCD. In this technique, they took a small portion (2  2 mm) of the limbal tissue from a donor. Then, they cut this small portion into small pieces (up to 15) and distributed them on the affected cornea before gluing a graft of AM over them. This technique allowed the in vivo expansion of these small pieces to cover the defect (Sangwan et al., 2012). Amescua et al. (2014) modified the SLET technique by using two AMs that can sandwich the small pieces of tissue to protect them (Amescua et al., 2014). Many studies successfully adopted the SLET technique (Queiroz et al., 2016; Mittal et al., 2015; Arya et al., 2016; Vazirani et al., 2016). For example, in a series of research studies by Mittal et al. (2015) on a small number (5) of patients, the ocular surface epithelialization was complete in all cases within 14 days, and two lines of improvement in the visual acuity was observed in three of the five patients (Mittal et al., 2015). In another study, Vazirani et al. (2016) retrospectively investigated 68 eyes of 68 patients, who underwent autologous SLET, performed across eight centers in three countries. Clinical

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success was achieved in 57 cases (83.8%). With a median follow-up of 12 months, survival probability exceeded 80% and 44 eyes (64.7%) gained two or more Snellen lines. In addition, 125 cases of autologous SLET in unilateral LSCD were analyzed in a single center in the same year, in which a success rate of 76% was achieved (Basu et al., 2016). The risk factors for SLET failure are the presence of symblepharon, doing a combined keratoplasty procedure, a history of acid burn, and the postoperative loss of the small pieces (Basu et al., 2016). The operation does not significantly do harm to the donor site except for small subconjunctival hemorrhage noted, which resolves spontaneously (Basu et al., 2016). In addition, two cases of focal LSCD were discovered in donor eyes (Vazirani et al., 2016). The most frequent complication has been focal recurrence of LSCD in the recipient tissue; this can just be monitored closely if not progressing or encroaching over the visual axis (Vazirani et al., 2016). If this complication happens repeatedly, SLET can be performed (Vazirani et al., 2013, 2015, 2016; Basu et al., 2016). Other complications include microbial keratitis, progressive conjunctivalization and symblepharon, hemorrhage under the AM, and the eventual loss or detachment of the transplanted tissue, either the explants or the AM (Basu et al., 2016). SLET is a promising new LSCD technique in which limbal epithelial cells presume in vivo expansion, with a minimal amount of tissue taken from the donor eye. It is highly successful in moderate cases, but its role in severe cases and long-term success still need more investigations (Vazirani et al., 2016).

Ex vivo expansion limbal stem cell transplantation Ex vivo expansion limbal stem cell transplantation is also known as cultivated limbal epithelial transplantation (CLET). This technique was proposed in 1997 by Pallegrini et al., who reported cultivation of a small biopsy sample from the limbus of healthy eyes of patients who had alkali burn in the other eye (Pellegrini et al., 1997). As of this writing, the ex vivo culture of epithelial stem cells from the cornea is the most promising technique in limbal transplantation (Atallah et al., 2016). This technique is used to overcome some of the main complications brought about by limbal tissue transplants and to diminish the loss of donor limbal tissue with subsequent possibility of inducing LSCD. This process has turned into the treatment of choice for LSCD (Atallah et al., 2016; Saghizadeh et al., 2017). In CLET, a small (2  2 mm) piece of limbal tissue (1 mm from either side of the cornealescleral junction) is excised and processed for ex vivo culture. These limbal epithelial cells can be taken from the other healthy eye in unilateral LSCD, from living related donors, or from cadaveric eyes. Several culture techniques have been developed, but they are generally divided into two main categories: explant or suspension methods. Variations of the explant

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culture system have been used in many studies (Grueterich et al., 2003; Nakamura et al., 2003, 2006; Sangwan et al., 2011a,b). Grueterich et al. (2003) demonstrated that culturing limbal epithelial cells (LECs) on the amnion with an intact amniotic epithelium may result in a more stem cellelike phenotype than the deepithelialized amnion. The AM epithelial cells are eliminated by cryopreservation and are then removed by enzymatic digestion, chemical treatment, or physical scraping of the membrane prior to use (Grueterich et al., 2003; Nakamura et al., 2003, 2006). The limbal biopsy is placed on and allowed to adhere to the basement membrane surface of the AM. After attachment, the biopsy and AM are submerged in culture medium. Then, limbal epithelial cells can proliferate and migrate out of the biopsy to cover the surface of the AM. This takes between 2 weeks and a month. An air-lifting technique was used additionally in some studies by lowering the level of the surface epithelium to allow stratification of the epithelium (Ban et al., 2003; Cooper et al., 2004; Nakamura et al., 2006). A variant of this technique is called the 3T3 explant coculture system ((Nakamura et al., 2003; Nakamura et al., 2006). This method uses an additional feeder layer of growtharrested 3T3 fibroblasts, which have a high proliferative capacity, in the bottom of the cell culture well. The growth arrest promotes the production of growth factors that stimulate epithelial growth and can be done either by irradiation or by a treatment with mitomycin C prior to use. Both the AM and the growth-arrested 3T3 fibroblasts inhibit the differentiation of corneal epithelial cells in vitro, which allows the expansion of the population of LESCs (Pellegrini et al., 1999; Grueterich et al., 2003). The suspension culture system method employs the enzymes dispase, which digests the basement membrane collagen and separates epithelial cells from the stroma, and trypsin, leading to the separation of clumps of limbal epithelial cells into a suspension of single cells. This suspension is then seeded either onto the AM or onto a plastic tissue culture dish that contains a feeder layer of growth-arrested 3T3 fibroblasts. Culture medium is added, and the cells are incubated for 14e21 days. When the cultures reach confluence, the epithelial sheet is transferred to the ocular surface using either a contact lens, paraffin gauze collagen shield, or fibrin gel (Pellegrini et al., 1997; Schwab, 1999). Rama et al. (2001) demonstrated that the use of fibrin as a carrier supports the maintenance of stem cells. When the suspension of single limbal epithelial cells is seeded onto the AM, they are cocultured with a layer of growth-arrested 3T3 fibroblasts in the bottom of the dish and the amnion serves as a carrier (Nakamura et al, 2003, 2006). Xenobiotic-free cultures were established in 2017 using either serum-free or human serumesupplemented medium as a feeder-free system (Behaegel et al., 2017). A metaanalysis by Zhao and Ma (2015) focused on CLET with the AM as a substrate (total of 562 patients) and demonstrated a 67% success rate and 62% two-line visual improvement without significant difference between autografts and allografts (Zhao and Ma, 2015). Another study that represented one of the

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largest reviews, of 1164 patients by Haagdorens et al. in 2016, demonstrated a slightly lower success rate of 70% and an overall 55% two-line visual improvement (Haagdorens et al., 2016). The complications of CLET on the recipient eye include hemorrhage under the graft in one-third of cases, which can resolve spontaneously (Rama et al., 2010; Sangwan et al., 2011a; Basu et al., 2012; Sejpal et al., 2013). Infection accounts for around 10% of the postoperative complications (Fasolo et al., 2017). The most common infection was bacterial in more than 50% of all infectious cases (Sejpal et al., 2013). Herpetic infection was observed in only one study (Rama et al., 2010). Some of these cases responded to antibiotic therapy (Shortt et al., 2010), while the rest needed urgent keratoplasty (Sejpal et al., 2013). Postoperative ocular surface inflammation is considered a negative prognostic factor for graft survival (Rama et al., 2010). Corneal perforation secondary to persistent thinning or persistent epithelial defects (PED) is associated with eyelid and eyelash abnormalities (Fasolo et al., 2017). Cyclosporine-related systemic symptoms are noted in cases of allogeneic grafts (Shortt et al., 2010; Fasolo et al., 2017). The main advantage of CLET is that it is a successful procedure with immediate covering of the ocular surface. It is well known to be a reproducible procedure. The disadvantage of CLET is that its expense limits its utilization on a wide scale. Another disadvantage is that using animal and nonhuman feeder cells has a risk of transmitting prion disease (Schwab et al., 2006).

Cultivated oral mucosal epithelial transplantation In this technique we rely on the autologous epithelium of the oral mucosa rather than the ocular epithelium to reconstruct the corneal surface, to eliminate the need for long-term immunosuppression as in KLAL or allogenic CLET (Nakamura et al., 2011). A healthy oral mucosa is examined by a dentist or maxillofacial surgeon and a 2- to 3-mm-diameter biopsy is cut into small explants and cultured on AM for 14e21 days, producing a confluent epithelial sheet. At the time of the procedure, the pannus and conjunctivalized tissues are removed using mitomycin C (0.04%) for 5 min, followed by adding the AM with the explants, securing them with 10-0 nylon sutures at the limbus area. A bandage contact lens is applied afterward (Nakamura et al., 2011; Burillon et al., 2012; Gaddipati et al., 2014). Nakamura et al. (2011) studied the long-term effects of cultivated oral mucosal epithelial transplantation (COMET) in the scar phase of LSCD in 19 eyes of 17 patients. Autologous cultivated oral mucosal epithelial and patients were followed up for more than 36 months (Nakamura et al., 2011). The generation of the epithelial sheet was successful in all patients. In addition, during the long-term follow-up period, postoperative conjunctivalization and symblepharon were significantly inhibited (Nakamura et al., 2011). All eyes

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manifested various degrees of postoperative corneal neovascularization, but it gradually abated, and its activity was stable at 6 months after surgery (Nakamura et al., 2011). The best-corrected visual acuity was improved in 18 eyes (95%) during the follow-up periods, and visual acuity at the postoperative 36th month was improved in 10 eyes (53%). In addition, Sotozono et al. (2014) had reported similar results (mean follow-up 2 years) in about 50% of 15 patients who underwent COMET for bilateral LSCD. Moreover, Satake et al. (2014) performed COMET on 40 eyes and achieved a 57.5% overall success rate. The failure in this study was due to persistent epithelial defects in nine eyes and gradual fibrovascular tissue invasion of the corneal surface in eyes with mucous membrane pemphigoid (Satake et al., 2014). Nevertheless, COMET is a still rising technique and there are not enough studies comparing it with previous regular techniques like ex vivo expansion (CLET) or KLAL (Fernandez-Buenaga et al., 2018; Gonzalez et al., 2018).

Conclusions, challenges and future directions Stem cellebased therapy embraces an exceptional potential in improving the lives of people with sight-threatening visual disorders because its capability of differentiation into numerous cell lineages potentiates regenerative medicine. Research in the field of stem cell therapy will continue to flourish, with a promising future for new biological factors implemented in the treatment of the vision loss. The most common stem cell sources that can be used in clinics are mesenchymal, embryonic, adult, and iPSCs. These stem cells represent an important source for stem cellebased therapies in eye diseases. They are revivable, easily expandable, and bankable and can be directed to cellular discrimination. Its capability of differentiation into numerous cell lineages potentiates regenerative medicine. Nevertheless, certain critical issues should be fixed before introducing stem cellebased therapies into clinical practice, such as high cost and reproducibility of differentiation in many ESC or iPSC clones, in addition to the hazards of mutagenesis and tumorigenesis. The therapeutic applications of various MSCs may be beneficial for the treatment of vision disease or loss due to their autologous nature and capability to reconstruct the cornea. However, the potential problems that are related to both cell support standardization and graft longevity require further exploration. Clinically, the rejection of limbal allograft(s) is the most critical concern in limbal transplants and, therefore, improvement of the immunosuppressive regimens should be regularly reviewed. The stem cellebased therapeutic approaches for different retinal disorders and diseases using MSCs and other stem cell types have intriguing potential, but this important field is still in its early steps. There are many studies in progress, and the results are highly anticipated, with encouraging and promising improvements in visual function.

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Impending research tends to enhance cellular expansion and find variable sources of stem cells. The transplantation of LSCs can recover both vision and quality of life in patients who suffer from the ocular surface disorders associated with LSCD, and overall, the use of analogous tissues gives the best consequences. The expansion of autologous stem cellebased therapies in corneal diseases associated with LESC damage requires the optimization of LESC cultures and/or standardization of conducted limbal discrimination of iPSCs. Furthermore, in unilateral LSCD cases, the contralateral healthy eye could be used as a source for harvesting stem cells. Patients with bilateral disease could be treated using allogeneic tissue expanded ex vivo, requiring systemic immunosuppressive therapy. Both hESCs and iPSCs that are specified for the therapy of particular patients are a beneficial tool and might be promising efficient therapies for conditions that are currently considered incurable. The most important challenges for using these stem cell therapies are precisely diminishing the possibilities of tumorigenicity and targeting differentiation. In addition, it should always be remembered that sight-threatening vitreoretinal complications might develop after the intravitreal and subretinal applications of stem cell therapies. Furthermore, the chronic and complex disease process is a major limiting factor for applications of stem cellebased therapies in many eye disease patients. More preclinical research and clinical trials, with expanded follow-up durations, are still needed.

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Tucker, B.A., Park, I.H., Qi, S.D., Klassen, H.J., Jiang, C., Yao, J., Redenti, S., Daley, G.Q., Young, M.J., 2011. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice. PLoS One 6, 18992. Uchino, Y., Goto, E., Takano, Y., Dogru, M., et al., 2006. Long-standing bullous keratopathy is associated with peripheral conjunctivalization and limbal deficiency. Ophthalmology 113 (7), 1098e1101. https://doi.org/10.1016/j.ophtha.2006.01.034. Ueno, H., Kurokawa, M.S., Kayama, M., Homma, R., Kumagai, Y., Masuda, C., Takada, E., Tsubota, K., Ueno, S., Suzuki, N., December 2007. Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells. Cornea 26 (10), 1220e1227. Uy, H.S., Chan, P.S., Cruz, F.M., 2013. Stem cell therapy: a novel approach for vision restoration in retinitis pigmentosa. Med. Hypothesis Discov. Innov. Ophthalmol. 2, 52e55. Vazirani, J., Ali, M.H., Sharma, N., Gupta, N., Mittal, V., Atallah, M., et al., 2016. Autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency: multicentre results. Br. J. Ophthalmol. 100 (10), 1416e1420. https://doi.org/10.1136/bjophthalmol-2015307348. Vazirani, J., Basu, S., Sangwan, V., 2013. Successful simple limbal epithelial transplantation (SLET) in lime injury-induced limbal stem cell deficiency with ocular surface granuloma. BMJ Case Rep. 1e3. https://doi.org/10.1136/bcr-2013-009405. Vazirani, J., Lal, I., Sangwan, V., 2015. Customised simple limbal epithelial transplantation for recurrent limbal stem cell deficiency. BMJ Case Rep. 1e3. https://doi.org/10.1136/bcr-2015209429. Wagoner, M.D., 1997. Chemical injuries of the eye: current concepts in pathophysiology and therapy. Surv. Ophthalmol. 41, 275e313. Watanabe, A., Yamada, Y., Yamanaka, S., 2013. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 20120292. Whiting, P., Kerby, J., Coffey, P., da Cruz, L., McKernan, R., 2015. Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic. Philos. Trans. R. Soc. Lond. B Biol. Sci. 370, 20140375. Yang, X., Qu, L., Wang, X., Zhao, M., et al., 2007. Plasticity of epidermal adult stem cells derived from adult goat ear skin. Mol. Reprod. Dev. 74 (3), 386e396. Yin, J., Jurkunas, U., 2018. Limbal stem cell transplantation and complications. Semin Ophthalmol 33 (1), 134e141. https://doi.org/10.1080/08820538.2017.1353834. Yoon, J.J., Ismail, S., Sherwin, T., September 2014. Limbal stem cells: central concepts of corneal epithelial homeostasis. World J. Stem Cells 26 (6(4), 391e403. ISSN 1948-0210. Zarbin, M., 2016. Cell-based therapy for degenerative retinal disease. Trends Mol. Med. 22, 115e134. Zhang, W., Yang, W., Liu, X., Zhang, L., Huang, W., Zhang, Y., 2014. Rapidly constructed scaffold-free embryonic stem cell sheets for ocular surface reconstruction. Scanning 36, 286e292. Zhao, Y., Ma, L., 2015. Systematic review and meta-analysis on transplantation of ex vivo cultivated limbal epithelial stem cell on amniotic membrane in limbal stem cell deficiency. Cornea 34 (5), 592e600. https://doi.org/10.1097/ICO.0000000000000398. Zhao, T., Zhang, Z.,N., Rong, Z., Xu, Y., 2011. Immunogenicity of induced pluripotent stem cells. Nature 13 (474), 212e215.

78 Mesenchymal Stem Cells in Human Health and Diseases Zhong, X., Gutierrez, C., Xue, T., Hampton, C., Vergara, M.N., Cao, L.H., Peters, A., Park, T.S., Zambidis, E.T., Meyer, J.S., et al., 2014. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat. Commun. 5, 4047. Zhou, S.Y., Zhang, C., Baradaran, E., Chuck, R.S., 2010. Human corneal basal epithelial cells express an embryonic stem cell marker OCT4. Curr. Eye Res. 35 (11), 978e985.

Further reading Cheng, J., Zhai, H., Wang, J., et al., 2017. Long-term outcome of allogeneic cultivated limbal epithelial transplantation for symblepharon caused by severe ocular burns. BMC Ophthalmol. 17, 8.

Chapter 5

Applications of the stem cell secretome in regenerative medicine Ba´rbara Mendes-Pinheiro1, 2, Ana Marote1, 2, Cla´udia R. Marques1, 2, Fa´bio G. Teixeira1, 2, Jorge Cibra˜o Ribeiro1, 2, Anto´nio J. Salgado1, 2 1

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; 2ICVS/3B’s e PT Government Associate Laboratory, Braga/Guimara˜es, Portugal

Introduction The potential of stem cells in regenerative medicine The last 10 years have witnessed huge advances in the field of stem cell (SC) therapy and regenerative medicine. There is a great deal of excitement about the potential promise of SC treatments, particularly in the repair of the central nervous system (CNS). In fact, the CNS is a promising target considering the wide spectrum of human brain diseases and the overall lack of effective therapeutic approaches for most of them. The low regeneration potential of the CNS further emphasizes this complexity (Goldman, 2016). Although endogenous SCs have been found in specific niches of the adult human brain (Ernst et al., 2014; Urban and Guillemot, 2014), little evidence exists about the contribution of these cells to structural repair following disease or insult. Thus, cell therapy has been proposed as an attractive option, and SCs have emerged as a frontline regenerative medicine source due to their remarkable potential to self-renew, proliferate, and differentiate into a variety of cell phenotypes (Edgar et al., 2013; Mahla, 2016). SCs are the key elements for all tissue and organ systems of the body, and mediate distinct roles in disease progression, disease development, and tissue repair processes (Mahla, 2016). Based on their differentiation potential, these cells can be classified as follows: totipotent SCs, which are able to create an entire organism; pluripotent SCs, which give rise to all cell types from the three embryonic germ layers (mesoderm, ectoderm, and endoderm); and multipotent cells, which can yield a more restricted subset of cell lineages (Fortier, 2005). Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00005-0 Copyright © 2020 Elsevier Inc. All rights reserved.

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On the basis of developmental and regenerative applications, SCs can be categorized as embryonic SCs (ESCs), induced pluripotent SCs (iPSCs), and adult SCs (Vizoso et al., 2017; Mahla, 2016). The most plastic of all SCs, ESCs, are derived from the inner cell mass of the blastocyst, are pluripotent in their nature, and have unlimited differentiation potential (Thomson et al., 1998). The diverse lineage commitment potential represents ESCs as an ideal model for regenerative therapeutics of disease (Dogan, 2018). Nonetheless, many issues need to be addressed before clinical translation regarding immunogenicity and neoplastic transformation, as well as ethical considerations (Dogan, 2018; Mahla, 2016). Interestingly, in 2006 Takahashi and Yamanaka demonstrated that pluripotency could be induced in adult cells through genetic incorporation of a defined set of core transcription factorsdOct3/4, Sox2, Klf4, and c-Mycdgiving rise to iPSCs (Takahashi and Yamanaka, 2006). This study has shed light on the use of these cells as a potential alternative for ESCs, representing an advance in personalized treatment based on an autologous cell source (Hallett et al., 2015). Adult SCs (also known as somatic or tissue SCs) are populations of undifferentiated cells that are found in specific regions throughout the majority of postnatal life with the capacity to differentiate into tissue-specific cell types (Hsu and Fuchs, 2012). These cells are usually maintained in a quiescent state and, when activated, they proliferate to repair damage and to maintain tissue homeostasis (Goodell et al., 2015). It is not clear whether all organs contain dedicated tissue-specific SCs, with hematopoietic, neural, gastrointestinal, epidermal, hepatic, and mesenchymal tissues being some examples of where adult SCs can be found (Goodell et al., 2015; Hsu and Fuchs, 2012). Tissuespecific SCs are multipotent and have less self-renewal ability in comparison with ESCs. Nevertheless, adult SCs have emerged as promising therapeutic candidates, since they carry no ethical concerns, show less tumorigenicity, and allow the opportunity of autologous transplantation (Mariano et al., 2015). Nonetheless, while the original hypothesis underlying SC regenerative therapies was based on functional recovery as a consequence of cellular differentiation and replacement, it has become evident that other mechanisms are at play. A paradigm shift has emerged suggesting that these cells might exert their beneficial effects through their secreted substancesdthe secretome (Baraniak and McDevitt, 2010; Drago et al., 2013). The present chapter will discuss the current understanding of the SC secretome, emphasizing the secretome composition, modulation, and characterization, as well as its possible application in different CNS pathologies, namely stroke, traumatic brain injury (TBI), Parkinson disease (PD), and spinal cord injury (SCI).

The concept of the secretome The concept that cell-based therapies were the future of regenerative medicine started to change after the work of Gnecchi et al., in 2005 (Gnecchi et al., 2005).

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His group was one of the first showing that paracrine factors released from mesenchymal SCs (MSCs) could promote tissue regeneration. This paracrine hypothesis has inspired an alternative outlook on the use of the cell secretome in the most diverse areas of regenerative medicine, including regenerative neurology. The secretome is defined as a set of factors/molecules actively or passively released by a cell, tissue, or organism into the extracellular space (Skalnikova et al., 2011). These factors include, among others, soluble proteins (e.g., cytokines, chemokines, and growth factors), lipids, and extracellular vesicles (EVs) (Fig. 5.1; Baraniak and McDevitt, 2010; Vizoso et al., 2017). The secretome of individual cells and tissues is specific, and it changes in response to fluctuations in physiological states or pathological conditions (Vizoso et al., 2017). Nevertheless, several studies have been designating SCs as trophic mediators of regenerative processes. Indeed, it has been demonstrated that different SC populations can secrete important trophic molecules, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF),

FIGURE 5.1 Stem cell (SC) secretome and strategies for its modulation. SCs can secrete potent combinations of bioactive molecules, including growth factors, cytokines, lipids, and extracellular vesicles (i.e., exosomes, microvesicles, and apoptotic bodies). Several approaches have been explored to maintain, expand, differentiate, and increase the number of factors secreted by SCs. The culture conditions can be changed in different manners to evoke a variety of biological responses, by using three-dimensional culture systems with or without biomaterials under static (e.g., spheroids) and dynamic conditions (e.g., bioreactors), as well as subjecting SCs to a controlled amount of a stimulus for a defined period of time, such as hypoxic preconditioning and proinflammatory cytokine exposure.

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glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor-2 (FGF2), insulin-like growth factor-1 (IGF1), vascular endothelial growth factor (VEGF), among others, that are capable of promoting cell survival, proliferation, and differentiation; neurite and axonal outgrowth; and immunomodulation of microglial cells (Salgado et al., 2015; Drago et al., 2013). Moreover, these factors are likely to be responsible for the prevention of neuronal programmed cell death and glial scar formation in different neurological disorders (Bacigaluppi et al., 2009; Chu et al., 2004; Lu et al., 2003; Carletti et al., 2011; Ryu et al., 2004; Ziv et al., 2006; Redmond et al., 2007). SCs can also protect other cells from damaging oxygen free radicals through the production of antioxidants and antiapoptotic molecules (Baraniak and McDevitt, 2010). In addition to soluble paracrine factors, findings have shown that EVs are also an important component of the secretome. EVs were primarily considered cellular debris or a way to excrete unneeded products from cells. However, since 2010, it has been recognized that they perform crucial physiological roles in intercellular communication (Riazifar et al., 2017). EV-mediated signals can be transmitted by the different biomolecule categoriesdprotein, lipids, nucleic acids, and sugarsdand the unique package of this information provides both protection and simultaneous delivery of multiple different messengers even to distant sites of the vesicular origin (Yanez-Mo et al., 2015). Robust data indicate that EVs have protective properties similar to those of their cellular counterparts that condition and reprogram the surrounding microenvironment, influencing a variety of endogenous responses, particularly in injured tissues (Koniusz et al., 2016). It has been shown that EVs can affect other cells via transfer of genetic cargo, transfer of receptors, and ultimately initiating pathways (Momen-Heravi et al., 2013). They can modify cell fate, function, and plasticity, and have the capacity to modulate the immune system and facilitate tissue regeneration (Koniusz et al., 2016). EVs are generally divided into three main types: exosomes produced from intracellular endosomes, microvesicles that bud directly from the plasma membrane, and apoptotic bodies released during apoptosis (Riazifar et al., 2017). Exosomes are the best-characterized species of EVs as of this writing, and MSC-derived exosomes have been the most widely studied in the context of the CNS, showing therapeutic benefits in the treatment of neurological and neurodegenerative disorders (Marote et al., 2016). For example, it has been demonstrated that MSC-generated exosomes promote neurite outgrowth and enhance neurite remodeling, neurogenesis, and angiogenesis, and therefore improve functional recovery in models of stroke and TBI, as reviewed by Marote et al. (Marote et al., 2016). Moreover, it is important to recall that they may suppress the immune response, which in turn may provide novel therapeutic approaches for treating CNS disorders like PD and SCI (Koniusz et al., 2016). Taking all these into account, the use of the SC secretome in regenerative medicine holds several advantages over more conventional stem cellebased

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applications regarding manufacturing, storage, handling, and their potential as a ready-to-go biologic product (Vizoso et al., 2017). Storage can be done for long periods without loss of product potency and quality (Bermudez et al. 2015, 2016). The time and cost of expansion and maintenance of cultured SCs could be significantly reduced, and secretome therapies could be immediately administered for the treatment of acute conditions like cerebral ischemia and trauma (Vizoso et al., 2017). Moreover, production in large quantities is possible under controlled laboratory conditions, and the biological product could be modified to desired cell-specific effects (Vizoso et al., 2017; McKee and Chaudhry, 2017). Importantly, the use of secretome derivatives could bypass potential issues associated with cell transplantation, including the number of available cells for transplantation and their survival after this procedure, immune compatibility, tumorigenicity, and infection transmission (Tran and Damaser, 2015). Nevertheless, regulatory requirements for manufacturing and quality control will be necessary to establish the safety and efficacy profile of these products (Vizoso et al., 2017).

Modulation of the stem cell secretome profile Since the realization that the beneficial effects of SCs may be due to the release of bioactive molecules, and not attributed only to SC differentiation, novel methods have been investigated for the modulation of the paracrine/ secretory actions of these cells to enhance therapeutic efficacy. A variety of approaches, focused on in vitro preconditioning and the use of dynamic 3D cultures as illustrated in Fig. 5.1, have been studied to increase the number of secreted factors and the duration of this secretion upon SC transplantation or to directly influence the SC secretome. Although diverse studies have examined the secreted factor production of different cell types, most of them focus on MSCs due to their widespread preclinical use for tissue regeneration. Therefore, most of the concepts discussed in this topic emphasize MSC culture conditions modulation and their impact on trophic factors secretion.

Three-dimensional cultures and biomaterials Cells naturally reside in a 3D niche. Therefore, attempts have been made to design 3D culture methods that better recapitulate physiologic microenvironments by recapitulating the spatial organization of the cell. Approaches have included the use of multicellular aggregates, prefabricated scaffolds, and different types of biomaterials that facilitate greater cellecell contact and interaction with the extracellular matrix (ECM). This allows cells to adapt their native morphology, which may influence signaling activity and ultimately may be a qualitative improvement in their secretome (McKee and Chaudhry, 2017; Qazi et al., 2017; Silva et al., 2013). One of the simplest methods of 3D culture was achieved by the formation of multicellular aggregates or spheroids (McKee and Chaudhry, 2017).

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Spheroids are either self-assembling or forced to grow as cell clusters through a variety of aggregation techniques such as hanging drop, rotating culture, or low-adhesion culture plates in suspension culture (Lewis et al., 2016; Keller, 1995; Wang et al., 2016). Depending on the research objective and the method used, it is possible to obtain spheroids of any dimension, and generally they are characterized by an external proliferating zone; an internal quiescent zone caused by limited distribution of oxygen, nutrients, and metabolites; and a necrotic core, resembling the cellular heterogeneity (Zanoni et al., 2016; Edmondson et al., 2014). Compared with cells cultured on a flat surface, they more closely mimic the complex scenario of tissues and organs where each cell interacts with nearby cells and with the ECM in the absence of additional substrates (Edmondson et al., 2014). In different studies, spheroid culture has been employed for the maintenance and expansion of MSCs, with spheroid-grown MSCs displaying an undifferentiated morphology and enhanced differentiation potential via increased ECM deposition, as well as increased clonal growth and multipotency (Wang et al., 2009; Baraniak and McDevitt, 2010; Li et al., 2015). Moreover, the 3D spheroids could modulate the secretome of MSCs, increasing the paracrine secretion of neurotrophic, angiogenic, antioxidative, and antiinflammatory factors, both in vitro and in vivo after transplantation (Xu et al., 2016; Redondo-Castro et al., 2018a; Berg et al., 2015; Laranjeira et al., 2015). Nonetheless, large-scale expansion of SCs using this method is difficult due to the inability to control aggregate size that leads to agglomeration, necrosis/apoptosis in spheroids, and inhibition of cell proliferation (Bartosh et al., 2010). This limitation could be exceeded, for example, by the incorporation of biomaterials (McKee and Chaudhry, 2017). In line with that, another drawback of secretome administration per se is that biomolecules may quickly lose their bioactivity due to short half-lives (Qazi et al., 2017). At the same time, the transplantation of SCs, either systemically or locally at the site of injury, is related to rapid cell death, accumulation in nontargeted tissues, and diffusion of cells away from the site of the injury (Qazi et al., 2017). Using biomaterials that provide a temporary matrix that can facilitate cell attachment, viability, and growth (Qazi et al., 2017) can consequently allow an improved and long-lasting secretome production. A wide range of biomaterials with different structures, chemistries, and mechanical properties have been explored, and hydrogels have stood out for CNS-related applications (Qazi et al., 2017; Nisbet et al., 2008). Hydrogels can be classified as either natural (e.g., collagen, hyaluronic acid, fibrin) or synthetic (e.g., polyethylene glycol, polyacrylamide) in origin (Huang et al., 2012). They are 3D polymer networks with high percentages of water, which are similar to native ECMs. This beneficial property, together with high oxygen and nutrient permeability, and low interfacial tensions, makes them more advantageous over alternative scaffolds (Huang et al., 2012; Nisbet et al., 2008). Moreover, hydrogels can

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easily adapt to any defect shape, and they can be modified to include neurotrophins for the control of neuronal cell adhesion, proliferation, and axonal extension, as well as supporting the integration of cells in vivo (McKee and Chaudhry, 2017; Nisbet et al., 2008). However, while a great deal of research effort has focused on using biomaterials to direct SC fate, there is a lack of knowledge on how biomaterials can modulate the SC secretome (Qazi et al., 2017; Silva et al., 2013). Nevertheless, some studies, mainly conducted with MSCs, showed that, indeed, 3D scaffold hydrogels can influence the paracrine activity of these cells. For instance, Silva and colleagues (Silva et al., 2013) showed that grafting fibronectin-based peptide within the hydrogel in which the cells were cultured positively influenced their proliferation and morphology. Furthermore, it affected the MSC secretome, allowing it to better support the survival and differentiation of hippocampal neurons. Moreover, other studies demonstrated that collagen hydrogels promoted the secretion of neurotrophic factors, particularly NGF and BDNF (Lee et al., 2014), and increased the levels of angiogenic factors (Rustad et al., 2012). Similarly, conditioned medium (CM) derived from hydrogel-embedded MSCs could induce stronger antioxidant and neuroprotective responses (Chierchia et al., 2017). Nevertheless, it is important to emphasize that 3D systems are very complex, since a wide number of parameters need to be consider. It is important to be careful in choosing the type of 3D culture models, the source of cells, and the type of biomaterials, considering the disease context and the desired outcome.

Preconditioning of stem cells Priming or preconditioning acts as a sublethal event that can trigger an adaptive response and increased recovery after injury or damage (Cunningham et al., 2018). The beneficial effect of preconditioning was first demonstrated in the 1980s by Murry and colleagues (Murry et al., 1986). They showed that treating healthy myocardium with intermittent brief ischemia followed by reperfusion protected the heart from ischemic episodes. Since then, a variety of preconditioning strategies, like hypoxic exposure, have been investigated to improve SC survival and to increase production of desired trophic factors, thereby augmenting cell paracrine actions (Sart et al., 2014; Baraniak and McDevitt, 2010). In vitro cell culture is traditionally performed at 21% O2 (normoxia), the level in the ambient atmospheric air. However, in the human body, these cells are exposed to much lower concentrations of oxygen, since the levels of inhaled O2 progressively decrease as it reaches the various internal organs and tissues (Sart et al., 2014; Yu et al., 2013). Moreover, oxygen levels of 21% have been linked to DNA damage, leading to genomic instability and cellular senescence, which could alter the metabolic efficiency of the cells

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(Jagannathan et al., 2016). In line with this, some studies have shown that hypoxic culture conditions enhance the regenerative potential of SCs, including MSCs and neural SCs (NSCs), reducing senescence, increasing the levels of proliferation, and maintaining their differentiation properties (Teixeira et al., 2015b; Roemeling-van Rhijn et al., 2013; De Filippis and Delia, 2011; Tsai et al., 2011). Moreover, it has been demonstrated that hypoxia increases the expression of several growth factors such as VEGF, FGF2, BDNF, and hepatocyte growth factor (HGF), factors known to play an important role in cell survival and recruitment, neuroprotection, and angiogenesis (Chuang et al., 2012; Liu et al., 2013; Chang et al., 2013; Wei et al., 2012). In addition to this, it is known that proinflammatory cytokines are necessary for the activation of certain SC populations. Thus, it has been postulated that SCs can be preconditioned with these cytokines to augment paracrine modulation of the host immune response (Baraniak and McDevitt, 2010). For example, MSCs are known for their immunomodulatory properties, and to enhance this characteristic, they can be primed with inflammatory mediators such as interleukin-1 (IL-1), tumor necrosis factor a (TNFa), interferon-g, or combination of these (Cunningham et al., 2018). In response to these priming stimuli, MSCs secrete higher concentrations of immunomodulatory mediators, like prostaglandin E2 and granulocyte-colony stimulating factor (G-CSF), and upregulate adhesion molecule expression (Cunningham et al., 2018). This leads to increased promotion of endogenous repair mechanisms, including angiogenesis (Cunningham et al., 2018), which consequently could be important in the context of some CNS diseases like stroke. Moreover, priming with IL-1a leads the MSC secretome toward a more antiinflammatory phenotype, which decreases secretion of TNFa and IL-6 from inflamed microglia (Redondo-Castro et al., 2017). Therefore, cytokine preconditioning methodologies may also work for other stem and progenitor cell populations and could have utility in modulating the SC secretome through enhanced paracrine actions. Alternatively, preconditioning can also be achieved using other preconditioning mediators, heat shock proteins, or pharmacological agents. However, characterization of enhanced trophic factor production by various SC populations after exposure to these agents has yet to be rigorously examined, especially in the context of CNS-related therapies.

Bioreactors Current SC production bioprocesses use the conventional static culture systems (i.e., tissue culture flasks) for their expansion. They are cost efficient and easy to operate; however, large quantities of homogeneous high-quality cells are needed for clinical applications, which is not feasible using the traditional methods (McKee and Chaudhry, 2017; Panchalingam et al., 2015). Dealing with large quantities of culture flasks not only is very labor intensive,

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but also tends to result in flask-to-flask variability and increases the chance of contamination with external agents (Panchalingam et al., 2015). Early simple 3D culture models were based on static methods; however, the design and use of bioreactors is increasingly being integrated into 3D culture systems (Haycock, 2011). Bioreactors are devices in which biological and biochemical processes develop under monitored and controlled environmental conditions, including temperature, pH, medium flow rate, oxygen concentrations, nutrient supply, and removal of metabolites and waste products (Haycock, 2011; Martin et al., 2004). There are several types of bioreactors available, such as spinner flasks, rotating wall vessels, direct perfusion bioreactors, and mechanical force bioreactors, to name a few (Haycock, 2011; Panchalingam et al., 2015). Each system has its own specific features and benefits, their use being dependent on the application requirements (Panchalingam et al., 2015). The spinner flask bioreactors are the most commonly employed in culture procedures due to their low cost, ease of setup, and ability to provide continuous agitation of suspended cells by varying the rate of stirring (Ismadi et al., 2014). This method results in hydrodynamic shear stress, mixing the cells with oxygen and nutrients, and leads to a more homogeneous environment (King and Miller, 2007). On the other hand, excessive agitation could lead to cell death, and because of that, rotating wall vessels were developed for cell constructs that need to grow under low shear stress, by rotating horizontally, allowing for culture mixing without an internal stirring mechanism (McKee and Chaudhry, 2017; Mazzoleni et al., 2009). Alternatively, direct perfusion systems are suitable for large-scale cell expansion, since they allow continuous exchange of nutrients and waste (McKee and Chaudhry, 2017; Alvarez-Barreto et al., 2007). The major advantage is the ability to seed cells directly into the scaffold under flow conditions, which usually allows for a high seeding efficiency. The control of medium flow thereafter enables cell adhesion and growth, where a high mass transfer rate is typically achieved throughout the entire construct (Haycock, 2011). Last, mechanical force bioreactors exploit the mechanism by which tissues respond to force during growth. They mimic tissue physiology, using compressive and tensile forces, and are increasingly being used with tissue-engineered constructs for cell differentiation (Haycock, 2011; McKee and Chaudhry, 2017). These techniques can be modified through the use of biomaterials (e.g., microcarriers and microcapsules) that provide, for instance, large surface areas for cell growth at high densities and the augmentation of the diffusion of nutrients, oxygen, or growth factors essential for cell growth (McKee and Chaudhry, 2017). Most of the studies have been focused on the use of bioreactors for the expansion and/or differentiation of different SCs. However, a few studies have addressed the use of these systems to modulate the paracrine signaling of SCs for CNS purposes. Nevertheless, it has already been shown that bioreactors are able to modulate the behavior of MSCs. Using spinner flasks functionalized

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with microcarriers, Hupfeld and colleagues (Hupfeld et al., 2014) showed that these cells secrete higher concentrations of VEGF, FGF2, monocyte chemoattractant protein-1, and stromal cell-derived factor-1 (SDF-1) compared with static culture conditions. Furthermore, other studies conducted by our group showed that computer-controlled stirred bioreactors enhance the neuroregulatory profile of the MSC secretome, which induces an increased differentiation of neuronal cells (Teixeira et al., 2016) (but also leads to an improvement of motor performance and neuronal structure recovery in a PD rat model (Teixeira et al., 2017). Overall, it is important to produce SCs for therapeutic applications in a well-defined manner under good process control that can be operated in a closed system according to good manufacturing practice. Furthermore, it is logical to hypothesize that these systems could act as modulators of the SC secretome, which may be extremely valuable for cell-free therapies in the CNS.

Application of the stem cell secretome in CNS disorders Stroke Stroke is a major global health problem and is caused by the interruption of blood supply to the brain, which leads to the lack of oxygen and nutrients in the affected regions (Xing et al., 2012). Initial symptoms include weakness or numbness of the face, arm, or leg and difficulties in speech. According to the World Health Organization, this cardiovascular disease accounts for 6.7 million deaths annually and its limited therapeutic options lead to a significant number of people living with disability worldwide (Feigin et al., 2014; World Health Organization, 2014). Indeed, 70% of the victims are no longer able to get back to active life and 30% need assistance with self-care (Moskowitz et al., 2010). The major modifiable risk factors for stroke are high blood pressure and tobacco use, whereas age is the most powerful nonmodifiable risk, it being estimated that after 55 years of age the risk of stroke doubles every decade (Moskowitz et al., 2010; Ovbiagele and Nguyen-Huynh, 2011). Therefore, the disease burden is expected to increase with the aging population. Approximately 85% of the cases are classified as ischemic strokes, which are due to a thromboembolic occlusion of a major cerebral artery. A transient ischemic attack might also occur when the occlusion is temporary. Less frequently, but more lethal, hemorrhagic stroke occurs upon rupture of a weakened blood vessel due to aneurysm, arteriovenous malformation, or uncontrolled hypertension. During ischemic stroke, occlusion of a major cerebral artery leads to secondary thrombosis in the downstream microvessels, which is followed by dysfunction of cerebral endothelial cells, pericytes, and astrocytes, leading to the disruption of the bloodebrain barrier (BBB) and ischemic cell damage (Zhang and Chopp, 2016). This downstream

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microvascular thrombosis continues for hours and correlates with the progression of neuronal damage from reversible to irreversible, highlighting the narrow therapeutic window of stroke (Zhang and Chopp, 2016). Tissue plasminogen activator is the gold standard treatment, applied within 4.5 h of stroke onset to promote the dissolution of the clot (Kwiatkowski et al., 1999). Endovascular thrombectomy has extended the therapeutic window to 12 h (Campbell et al., 2015). These therapeutic options are able to reestablish tissue reperfusion in the ischemic regions, preserve vascular integrity, and thus minimize neuronal death (Zhang and Chopp, 2016). However, if administered beyond a critical time point, the restoration of cerebral blood flow cannot rescue irreversibly damaged brain cells, leading to long-term disability in stroke survivors. Therefore, there is a need to develop therapies that enhance functional recovery in these patients by promoting angiogenesis and neurogenesis and modulating inflammatory responses. SCs have been put forward as promising therapeutic tools to promote brain repair and improve functional recovery in preclinical models of stroke. Cell-based therapies relying on local and systemic administration of SCs have been shown to exert their therapeutic effect through the secretion of important paracrine factors, motivating the development of secretome-based therapies. NSCs derived from human umbilical cord cells have been shown to secrete trophic factors when locally delivered in the corpus callosum in a rat model of lacunar stroke, which factors potentiate neurogenesis through the upregulation of trophic factor expression in the recipient even after graft death (Jablonska et al., 2016). A 2017 study also identified the paracrine therapeutic potential of iPSC-derived NSCs upon epidural transplantation 7 days after permanent middle cerebral artery occlusion (MCAO) in adult rats (Lee et al., 2017). Cell-transplanted rats exhibited a significantly reduced lesion volume that correlated with functional improvements in grip strength and use of the left paretic forelimb. At the cellular level, treated groups presented attenuated infiltration of Cluster of Differentiation 68 (CD68)-immunoreactive resident microglia, astrogliosis, and apoptosis, as well as enhanced angiogenesis and white mater tract integrity (Lee et al., 2017). In vitro, using a transmembrane coculture system, oxygen and glucosee deprived (OGD) cortical cells exposed to iPSCeNSC paracrine factors showed superior neuronal survival and reduced astrogliosis in a direct comparison with iPSC-, bone marrow-derived MSC (BM-MSC)e, and Wharton’s jelly-derived MSC (WJ-MSC)eexposed cells. In further molecular analysis, the authors identified a cocktail of factors, including BMP7, CXCL14, FGF8, FGF9, and IGFBP2, as possible mediators of these effects (Lee et al., 2017). Intravenously administered rat adipose-tissue SCs (ASCs) have also been shown to promote recovery in a rat model of subcortical ischemic stroke through paracrine mechanisms, since donor cells were implanted only in peripheral organs (kidney, liver, lung, and spleen) rather than in the injured brain area, 1 day after being injected into rats (Otero-Ortega et al., 2015).

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Nevertheless, treated animals exhibited improved functional recovery, reduced lesion area, and increased oligodendrocyte proliferation. Importantly, the treatment also induced higher connectivity of the white matter, which is mainly affected in subcortical and lacunar strokes, which account for up to 25% of the ischemic strokes in humans (Otero-Ortega et al., 2015). These studies highlight the paracrine potential of SC-based therapies in stroke treatment, although placing a question mark on the need for performing cell transplantation. Consequently, cell-transplantation-free therapies based on CM or their vesicular components have been tested as new approaches for stroke treatment. Choosing the most effective source of SCs for secretome collection is one of the aspects to weigh in the designing of this kind of approach. Hsieh and colleagues (Hsieh et al., 2013) demonstrated that WJ-MSCs secretome outweighs BM-MSCs secretome in promoting angiogenesis, neuronal differentiation, and neuroprotection of primary cortical cells after OGD insult. In another study, daily intranasal administration of human umbilical cord SC CM, starting 24 h after MCAO in rats, promoted functional recovery and enhanced BBB integrity, possibly by modulating the expression of vascular remodeling genes (Zhao et al., 2015). Tsai and colleagues (Tsai et al., 2014) further explored the effectiveness of BM-MSC CM obtained from normal healthy rats and cerebral ischemia rats. The intravenous (i.v.) administration of either CM immediately after blood reperfusion in MCAO rats remarkably improves functional recovery, enhances neurogenesis, and attenuates microglia infiltration, therefore showing similar outcomes between both donors (Tsai et al., 2014). Even though cerebral ischemia by itself did not lead to BM-MSC secretory differences, a 2018 study has shown that cigarette smoking, a major risk factor for stroke, negatively affects the secretome of ASCs, by compromising their vasculogenic activity (Barwinska et al., 2018). Cytokine analysis further evidenced lower amounts of HGF and SDF-1 and the presence of angiostatic/proinflammatory factor activin A in the CM of ASCs isolated from cigarette smoking donors (Barwinska et al., 2018). In addition to source and donor variability, CM regenerative properties can also be modulated by the culture conditions of SCs before secretome collection. In a 2018 study, Redondo-Castro and colleagues (Redondo-Castro et al., 2018b) have shown that culturing human BM-MSCs as 3D spheroids significantly increased the secretion of IL-1 receptor antagonist, VEGF, and G-CSF, the last being further increased by priming with IL-1. Nevertheless, this increased antiinflammatory phenotype was not translated into an antiinflammatory effect of MSCs on lipopolysaccharide-mediated cytokine release in BV2 cells in coculture. Hypoxic preconditioning is another promising modulation approach. Jiang and colleagues (Jiang et al., 2018) have addressed this effect in a rat model of stroke, showing that intravenously administered hypoxic CM, 12 h after MCAO and every 2 days for 28 days, significantly enhanced the therapeutic effects displayed by

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normoxic CM, namely reduction of lesion volume, increased neuronal survival, decreased apoptosis, and increased angiogenesis. This increased regenerative potential might be explained by the overexpression of cytokines implicated in angiogenesis and neurogenesis (Jiang et al., 2018). The vesicular components of the secretome have been more intensively studied as therapeutic agents for stroke. Chopp’s group (Xin et al., 2013a) provided the first evidence of the therapeutic potential of MSC-based exosomes. In this study, exosomes derived from rat BM-MSCs intravenously injected 24 h after injury improved functional recovery of MCAO rats and promoted increased neurite remodeling, neurogenesis, and angiogenesis in the ischemic boundary zone (Xin et al., 2013a). A subsequent study by Doeppner and colleagues (Doeppner et al., 2015) demonstrated therapeutic equivalence of exosomes and MSCs in a mouse model of MCAO receiving systemic (i.v.) and multiple (1, 3, and 5 days postinjury) treatment administrations. The authors reported similar effectiveness in the reduction of motor coordination impairment, comparable long-term neuroprotection, and increased angioneurogenesis, although they failed to show modulation of cerebral immune cell infiltration (Doeppner et al., 2015). Intracerebroventricular administration of BM-MSC EVs has also been shown to restore functional integrity of synaptic transmission and plasticity, leading to improved spatial learning and memory in a mouse model of transient global ischemia, partly attributed to suppression of COX-2 pathogenicity in the hippocampus (Deng et al., 2017). Studies from Otero-Ortega and colleagues (Otero-Ortega et al., 2018) have also shown the potential of ASC-derived exosomes to promote functional recovery after intracerebral hemorrhage when intravenously infused 24 h after surgery. The authors further reported a reduced lesion area, improved fiber tract integrity, axonal sprouting, and white matter repair markers 28 days after stroke. Importantly, 24 h after administration exosomes were found not only in the brain, but also in the peripheral organs such as the lung, liver, and spleen. Proteomic analysis of the exosomes identified a high number of proteins associated with synaptic transmission, neuronal differentiation, neurite outgrowth, apoptosis, and angiogenesis that could have mediated the exosomeelicited responses. A different research group has also identified another possible signaling pathway responsible for increased neuronal survival and proliferation of exosomes. El Bassit and colleagues (El Bassit et al., 2017) demonstrated the presence of metastasis-associated lung adenocarcinoma transcript 1, a long noncoding RNA in human ASC-derived exosomes, which mediates PKCdII splicing, thereby increasing neuronal survival (El Bassit et al., 2017). In addition to proteins, microRNA (miR) content in exosomes has been shown to play a major role in EV-based therapies. In line with previous studies demonstrating the role of miR-133b in mediating MSC therapeutic effects in a rat model of stroke (Xin et al., 2013b), Chopp’s group further investigated whether exosomes isolated from MSCs overexpressing miR-133b (Ex-miR-133bþ)

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could enhance exosomes’ therapeutic potential (Xin et al., 2017b). In vivo studies have shown that Ex-miR-133bþ intraarterial injection significantly increased functional improvement and neurite remodeling/brain plasticity in the ischemic boundary area compared with naive exosome treatment. In vitro studies provided a mechanistic insight into Ex-miR-133bþ action, showing that Ex-miR-133bþ significantly increased the amount of exosomes released by OGD-exposed astrocytes, which in turn increases neurite branching and elongation of cultured cortical embryonic rat neurons. Following a similar approach, the same group reported enhanced neuroplasticity and functional recovery after stroke in an MCAO rat model that received an i.v. administration of miR-17e92 cluster-enriched exosomes (Xin et al., 2017a). Possible downstream targets of the miR-17e92 cluster include PTEN, which is involved in neurite remodeling, cell proliferation, and differentiation via the PI3K/Akt/mTOR signaling pathway (Xin et al., 2017a). Along with the modulation of miR content, EV regenerative properties have also been shown to be enhanced by treating MSCs with normal and stroke-injured brain extracts. In a rat model of MCAO, a single injection of microvesicles derived from normal and stroke-injured brain extractetreated human ASCs 48 h after injury induced functional amelioration, reduced brain lesioned area, and improved neurogenesis and modulation of inflammation in ischemic tissue (Lee et al., 2016). A growing amount of studies have revealed the therapeutic potential of cell-transplantation-free strategies, based on complete CM or on its EVs for the treatment of stroke (Fig. 5.2). Systemic administration of these SC paracrine effectors potentiates brain repair mainly by enhancing neurogenesis, white matter tract integrity, and angiogenesis as well as modulation of inflammation in the affected regions.

Traumatic brain injury TBI is defined as an “alteration in brain function, or other evidence of brain pathology, caused by an external force” (Menon et al., 2010). The severity of the damage is determined by the nature of the external force (direct impact, rapid acceleration or deceleration, a penetrating object, or blast waves from an explosion), as well as its direction, intensity, and duration (Maas et al., 2008). TBI is a complex continuous process, in which the primary damage is followed by a secondary injury. Both stages may lead to physical, cognitive, and emotional deficits with a severity that leads either to hospitalization or to death (Hasan et al., 2017; Xiong et al., 2015). Annually, around 10 million people worldwide suffer from this condition (Hyder et al., 2007), which affects not only athletes or military personnel, but also the general population, especially young (less than 24 years of age) and elderly (more than 65 years of age) people (Bruns and Hauser, 2003). In the sequence of the primary damage, normal local blood flow is interrupted. Therefore, an ischemic cascade begins with the accumulation of lactic

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FIGURE 5.2 Stem cell (SC) secretome-based therapy for CNS repair. The trophic action of SCs has been increasingly accepted as a new concept for the regeneration of distinct CNS damage, including stroke, traumatic brain injury, Parkinson disease, and spinal cord injury. This is carried out by a wide panel of biomolecules involved in activation/modulation of some endogenous processes such as the promotion of neurogenesis and angiogenesis and the reduction of inflammation, contributing in this way to neuroprotection and regeneration in different disease contexts.

acid, due to a metabolic switch to anaerobic respiration (Jalloh et al., 2015). Given that this adaptation does not ensure cellular energy homeostasis, ATP reservoirs are consumed, followed by the failure of ATP-dependent ion pumps, leading to an increase in membrane permeability (Werner and Engelhard, 2007). Afterward, terminal membrane depolarization and release of glutamate result in perturbations in the homeostasis of various ions (Hinzman et al., 2012). For instance, calcium uptake increases the levels of this ion inside the mitochondria, leading to the generation of reactive oxygen species and additional damage (Prins et al., 2013). Subsequently, the inflammatory response is triggered, generating the recruitment and migration of leukocytes and microglia to the site of injury and the release of pro- and antiinflammatory cytokines, as well as oxygen radicals, nitric oxide, proteases, and other factors that contribute to aggravating neuronal loss (Corrigan et al., 2016). Therefore, inflammation can be regarded as beneficial, as it contributes to local regeneration, or prejudicial, due to the induction of further damage (Dekmak et al., 2018). In a later stage, a glial scar is eventually generated. By surrounding the

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site of injury, it prevents the passage of toxic molecules to healthy tissues, thereby protecting the cells that are still viable. However, the formation of this physical barrier can have an inhibitory effect on axonal regrowth and regeneration (Silver and Miller, 2004). Currently, the available therapeutic approaches lack the capacity to protect cells that were not disrupted by the primary injury and to induce neurological recovery by promoting critical processes such as angiogenesis, neurogenesis, or oligodendrogenesis. They act only by attenuating the consequences of the primary insult and minimizing the complications linked with the secondary injury. Furthermore, early rehabilitation contributes to improvements in residual function, quality of life, and independence (Xiong et al., 2013; Xiong et al., 2015). For several years, research has been focused on the development of neuroprotective agents, and since the 1990s more than 30 clinical trials for potential treatments for this condition have been initiated, as of this writing, and almost all were unsuccessful (Xiong et al., 2015). Attending to its epidemiology and pathophysiology, TBI represents a major public health problem and an economic burden, demanding the development of efficient therapeutic approaches capable of improving functional recovery. SC therapies based on the use of different cell populations, such as neural precursor cells (NPCs), ESCs, iPSCs and MSCs, have been considered promising tools for the treatment of brain injury. Preclinical studies have been featuring the potential of MSCs to promote internal protective and reparative mechanisms following brain injury in different experimental models of TBI. The interplay of transplanted MSCs with the lesioned microenvironment leads to both functional and structural neurological recovery (Bonilla Horcajo et al., 2018; Guan et al., 2013; Pischiutta et al., 2014) as well as brain metabolism improvement (Guan et al., 2013). Darkazalli et al. (Darkazalli et al., 2017) also showed that intravenous delivery of human BM-MSCs normalized the expression of 49% of all genes disrupted by TBI. In a clinical trial, 40 people with sequelae of TBI received umbilical cord MSC (UC-MSC) transplants via lumbar puncture. The transplantation of these cells significantly improved balance, motor performance, self-care capacity, mobility, locomotion, and communication (Wang et al., 2013). The benefits behind the transplantation of MSCs could be mediated by differentiation into neural cell types (Anbari et al., 2014). Indeed, these cells have the capacity to home or migrate toward the injury site, where they could replace lost cells (Hasan et al., 2017). Nevertheless, these phenomena are not frequent, because only a small portion of MSCs survive the transplantation and from those that survive, few are able to differentiate (Maltman et al., 2011). So, in addition to their differentiation potential, the therapeutic benefits of MSCs for brain remodeling and functional recovery after TBI are mainly attributed to neuroprotection and immunomodulatory actions mediated by the secretion of paracrine factors (Galindo et al., 2011; Kappy et al., 2018; Kim et al., 2010; Tajiri et al., 2014).

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When MSCs are administered directly to the brain they can exert their therapeutic properties by either one or both mechanisms. For intravenously administered MSCs, studies suggest that they may act in an indirect way, through their secretome, given that few cells reach the brain (Mahmood et al., 2001; Menge et al., 2012). Pischiutta et al. (Pischiutta et al., 2016) performed i.v. and intracerebroventricular injections of human amniotic MSCs in a controlled cortical impact mouse model of TBI. Even though MSCs were detected in the brain of only animals that received intracerebroventricular injection, the treatment with this cell population provided long-term protection of sensorimotor function regardless of the method of delivery. This suggests that the effects of human amniotic MSCs were mediated by the secretion of paracrine factors. In fact, when only the secretome from various populations of MSCs was administered in in vitro and in vivo models of TBI, results showed that in addition to exerting neuroprotective actions, it stimulated cell viability and migration in vitro (Baez-Jurado et al., 2018a,b; Chuang et al., 2012; Torrente et al., 2014). In an attempt to understand the molecular mechanisms underlying the benefits of the secretome from MSCs observed in the context of TBI, Menge et al. (Menge et al., 2012) found that the administration of MSCs increased the gene and protein expression of the factor tissue inhibitor of matrix metalloproteinases-3 (TIMP3). When this protein was intravenously delivered, the authors observed the reduction of BBB permeability through promotion of cerebral endothelial adherent junction integrity. Given that the knockdown of TIMP3 in MSCs ceases the capacity of MSCs to reduce BBB permeability, this study suggests that the effects of human MSCs on the BBB in TBI may be attributed to the secretion of the soluble factor TIMP3. Moreover, the same group showed that treatment with TIMP3 led to neuroprotective effects dependent on the activation of the Akt/mTORC1 pathway and attenuation of hippocampal-dependent deficits (Gibb et al., 2015). Another protein present in the secretome of BM-MSCs, namely Wnt3a, seems to have a role in the benefits of the secretome in TBI (Zhao et al., 2016). Indeed, when administered through an i.v. route alone, Wnt3a recapitulated the neuroprotective effects seen with the administration of BM-MSCs and led to an improvement in neurocognition. The potential of exosomes has also been explored in the context of brain trauma (Marote et al., 2016). For instance, Zhang et al. showed that the systemic administration of exosomes in rats after TBI led to functional recovery through, at least in part, the promotion of angiogenesis and neurogenesis and the reduction of neuroinflammation (Zhang et al. 2016, 2017). Given the capacity of MSCs to adapt and grow in different culture conditions, studies have been focusing on the modulation of the MSC secretome to potentiate its therapeutic benefits (Hoban et al., 2015; Liu et al., 2013; Teixeira et al., 2016). For instance, Chang et al. (Chang et al., 2013) showed that the i.v. administration of the secretome from BM-MSCs cultured under normoxic or hypoxic conditions improved functional recovery and

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neurogenesis and reduced brain damage and apoptosis. However, the secretome from cells cultured under hypoxic conditions led to better outcomes in this TBI animal model, regarding functional recovery, neurogenesis, and brain damage, compared with the secretome from MSCs cultured under normoxia. Another group adopted a different strategy for the modulation of the secretome from MSCs for application in TBI. By genetically engineering MSCs to overexpress CXCR4, the accumulation of these cells after systemic delivery at the periphery of the lesion was enhanced, as well as the secretion of cytokines and growth factors, which provided stimulation of vasculogenesis and neuroprotection (Wang et al., 2015). Overall, studies using the secretome from MSCs to treat TBI validated the capacity of the secreted factors from MSCs to induce neuroprotection, reduce neuroinflammation, and stimulate angiogenesis and neurogenesis, which translated to functional recovery or neurocognition improvement (Fig. 5.2). Therefore, these studies point toward the potential of the secretome from MSCs as a cell-transplantation-free therapy for TBI.

Parkinson disease PD is the second most common neurodegenerative disorder worldwide (Poewe et al., 2017). In fact, the prevalence of PD is 1 in 100 people over the age of 50 (Balestrino and Martinez-Martin, 2017), and the diagnosis is usually made in the sixth or seventh decade of life. Nevertheless, there are rare cases in which the disease is found in people in their 40s (Zuo et al., 2013), being classified as a young onsetdthe juvenile PD. Still, PD is more common in men than in women (Van Den Eeden et al., 2003), and it has been suggested that a protective effect of female sex hormones, a sex-associated genetic mechanism, or sex-specific differences in exposure to environmental risk factors might be the explanation for the male prevalence (Poewe et al., 2017). Clinically, the diagnosis of PD is based on the identification of cardinal features affecting the motor system, namely, bradykinesia (slowness in the execution of voluntary movements), postural instability (a tendency to fall even in the absence of weakness or cerebellar balance disturbance), muscular rigidity (stiffness), and tremor at rest, with an asymmetric onset, which becomes bilateral with time (Poewe et al., 2017). These motor deficits are the result of a progressive degeneration of dopaminergic neurons in the nigrostriatal pathway at the level of the substantia nigra pars compacta (SNpc) (Langston, 2006), which leads to a dramatic reduction in dopamine (DA) being released within the striatum (Lees et al., 2009). As reviewed in 2018 by Teixeira and colleagues (Teixeira et al., 2018), current PD treatment relies on the alleviation of its symptomatic impairments through the use of pharmacological strategies such as levodopa, DA agonists (e.g., ropinirole or pramipexole), and monoamine oxidase B (e.g., rasagiline or selegiline) and catechol-O-methyltransferase (e.g., entacapone or tolcapone)

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inhibitors, to compensate for the deficits of DA in the nigrostriatal dopaminergic pathway (Dong et al., 2016; Lindholm et al., 2016; Vijverman and Fox, 2014). Although efficacious, studies have demonstrated that they can elicit undesirable side effects, a fact that may limit their use over long periods of time (Onofrj et al., 2008). In addition to all these pharmacological treatments, surgical interventions, such as deep brain stimulation have also been used as a strategy for the treatment of PD (Okun, 2012). However, the apparent clinical recovery appears to be not effective in long-term approaches, and thus PD progression is not avoided (Krack et al., 2017). Therefore, based on such limitations, the SC secretome has been proposed as a promising therapeutic tool for the treatment of CNS neurodegenerative disorders, including PD (Drago et al., 2013). NPCs and MSCs are being used as promising SC sources with therapeutic effects on PD, due to its trophic capability, potentiating DA neuronal survival and protection, as well as increasing PD motor function (Drago et al., 2013). Concerning NPCs, studies have demonstrated that these SCs are a source of bioactive agents (e.g., growth factors, cytokines, and vesicles) (Drago et al., 2013). In fact, the secretome of NPCs is believed to be important in the modulation of several biological processes, such as cell survival, proliferation, and differentiation; immunomodulation; antiapoptosis; and stimulation of tissue-adjacent cells (Pluchino and Cossetti, 2013; Skalnikova et al., 2011; Cossetti et al., 2012), suggesting them to be neurotrophic factoresecreting cells (Drago et al., 2013). Indeed, several studies have shown that after transplantation of NPCs, important neurotrophic factors such as GDNF, BDNF, SC factor, and IGF, were increased, thereby supporting NPC-mediated effects (Drago et al., 2013; Ourednik et al., 2002; Yasuhara et al., 2006; Ebert et al., 2008). Our group has demonstrated, for the first time, that the sole injection of the human NPC secretome modulated DA neuronal cell survival, leading to PD motor deficit amelioration, and improved outcomes compared with cell transplantation approaches (Mendes-Pinheiro et al., 2018). Using a nontargeted proteomics-based analysis (e.g., mass spectrometry), we have found that, in addition to the well-known classical neurotrophic factors, NPCs are able to secrete important neuroregulatory candidates, such as 14-3-3 proteins, PEDF, galectin-1, cystatin C, clusterin, glial-derived nexin, semaphorin-7A, and cadherin-2, described as important modulators of neuronal migration, differentiation, and neuroprotection mechanisms both in vitro and in vivo (Fraga et al., 2013; Chen et al., 2007; Yabe et al., 2010; Sakaguchi and Okano, 2012; Kajitani et al., 2009; Gauthier et al., 2011; Wicher et al., 2008; Farmer et al., 1990; Pasterkamp et al., 2003; Zuo et al., 2013; Gao et al., 2001). Of those factors, PEDF has been described as a promising candidate for the treatment of PD (Falk et al., 2010; Yabe et al., 2010). For instance, Falk and colleagues (Falk et al., 2009) demonstrated that PEDF is not only neurotrophic but also acts as a neuroprotective factor, able to increase the survival and

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protection of primary midbrain cultures in different in vitro models of PD. Such neuroprotective actions of PEDF have been linked to its capacity to modulate signaling pathways such as the nuclear factor NF-kB signaling cascade, allowing NF-kB to act as a transcription factor that induces the expression of genes/factors that are critical to DA neuronal protection and survival, namely BDNF and GDNF (Falk et al., 2010). Regarding the MSC secretome, Crigler and colleagues (Crigler et al., 2006) were the first group to show that MSCs could modulate neuronal cell survival and neurite outgrowth, through an active secretion of neurotrophic factors such as BDNF and NGF. In fact, the secretome of MSCs has already demonstrated promising outcomes, either by a direct secretion of bioactive molecules in situ, after MSC transplantation, or by its sole injection in the form of CM (Teixeira et al., 2013). In fact, our lab has demonstrated that the injection of the MSC secretome led to an increase in neuronal densities to similar levels compared with those promoted by MSC cell transplantation (Teixeira et al., 2015a). In the context of PD, the MSC secretome has also shown promising prospects. Indeed, Sadan and colleagues (Sadan et al., 2009), using BM-MSCs as neurotrophic factoresecreting cells, observed a significant decrease in the amphetamine-induced rotation test, as well as in the loss of tyrosine hydroxylase (TH)eimmunoreactive nerve terminals compared with the untreated MSC group, correlating these effects with an active secretion of BDNF and GDNF (Sadan et al., 2009). In 2017, Teixeira and colleagues (Teixeira et al., 2017) demonstrated that after a local injection of the BM-MSC secretome into the SNpc and striatum, there was a partial reversion of the PD histological deficits (increasing TH-positive cells and fibers in the SNpc and striatum), which was positively correlated with evident gains in the animals’ motor performance, in which PEDF was found to be at least partly involved. More recently, by using bioengineering approaches, Chierchia and colleagues (Chierchia et al., 2017) demonstrated the feasibility of a biomaterial-based approach coupling MSC neuroprotective action to injectable hydrogels, showing that they may increase the controlled release of the MSC secretome in relevant models of PD. In addition, similar to the protein faction, the vesicular fraction of the MSC secretome is also being indicated as a promising therapeutic tool for CNS disorders, including PD (Marote et al., 2016). For instance, Jarmalaviciute and colleagues (Jarmalaviciute et al., 2015) have demonstrated that in vitro, MSC-secreted exosomes rescued DA neurons from 6-OHDA-induced apoptosis, thereby providing a potential regenerative treatment for this disorder. Altogether, these data strongly suggest that the MSC secretome may in fact be a promising therapeutic tool to replace SC transplantation strategies, and be a new route for the treatment of PD (Blandini et al., 2010) (Fig. 5.2). Actually, genetic modification of MSCs to specifically release trophic factors such as GDNF into the striatum and SNpc has remarkably demonstrated long-term amelioration of PD (Moloney et al., 2010).

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Spinal cord injury SCI is a disabling and devastating neurological outcome that results from spinal column fractures. The annual incidence of SCI has been estimated at approximately 40 cases per million population in the United States, and varies from 13.9 to 19.4 per million population in European countries. SCI is more frequent in young people and children ages 5e44 years in the developed world, and in most cases occurs due to preventable causes such as road traffic crashes, falls, or violence (Stephan et al., 2015). Briefly, any form of trauma, disease, or degeneration to the spinal cord could lead to a debilitating lifetime condition, usually with permanent sequels to patients. Commonly these include sensation loss and deficits in motor function (tetraplegia or paraplegia). Consequently, patients and their relatives are affected at the personal, economic, and social levels, leading to an increased number of depression and anxiety cases (Le and Dorstyn, 2016; Williams and Murray, 2015; Wolf et al., 2012). Primary injury is caused by blood vessel damage, axon disruption, and neuronal death due to the mechanical trauma applied to the spinal cord. Subsequent expansion of the edema to the adjacent areas, fluid accumulation, and ischemia further contribute to the worsening of the lesion (Tator and Fehlings, 1991). Moreover, these deleterious effects are exacerbated by a cascade of events resulting from the release of several compounds from the disrupted cells, including Na2þ and Ca2þ toxicity (Agrawal and Fehlings, 1997), reactive oxygen species production (Oyinbo, 2011), glutamate excitotoxity (McAdoo et al., 1999; Xu et al., 2008), and inflammatory response (Jones et al., 2005; Schnell et al., 1999) accompanied by molecules that suppress axonal regeneration (Silver and Miller, 2004). In this area, several therapeutic approaches have been proposed, focused on remyelination, neuronal protection, excitotoxicity clearance, cell-replacement therapies, axonal regeneration, recruitment of endogenous host SCs, epidural stimulation, and brainemachine interfaces, as reviewed by Silva et al. (Silva et al., 2014). The use of secretomes from different cell sources to tackle SCI emerged during the 2010s. Secretome delivery is a promising cell-transplantation-free therapy, without the problems associated with cell-replacement strategies, such as low cell survival in the recipient spinal cord. In addition, a couple of authors have demonstrated that the positive outcomes of cell-replacement strategies come from the paracrine factors released by transplanted cells rather than their differentiation and replenishment of the cells lost after injury (Gomes et al., 2018; Quertainmont et al., 2012). A few studies have shown the potential of secretomes obtained from a wide range of cells and delivered intrathecally, systemically, or intraperitoneally in SCI animal models. CM from BM-MSCs delivered by an osmotic pump into the injury site is reported to promote locomotor recovery mainly by providing neuroprotection, which leads to a reduction of the cystic cavity and

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preservation of spinal tracts (Cantinieaux et al., 2013). A different group employed a similar strategy, injecting CM from the same source into the cerebral spinal fluid, and observed a similar outcome after treatment in rats, showing an increased locomotor score, reduction of the cystic cavity, and increased axon density in the lesion site (Kanekiyo et al., 2018). Using only the exosome fraction isolated from BM-MSC CM, Huang and colleagues (Huang et al., 2017) linked the systemic administration of this cellfree therapy to an improvement in the locomotor behavior, with reduction of inflammation and apoptosis and increased angiogenesis. The exosome fraction of BM-MSC CM injected intravenously 1 week after injury was also associated with M2 macrophages, which are associated with wound healing and tissue repair (Lankford et al., 2018). Another study demonstrated that olfactory ensheathing cell CMeinduced BM-MSCs survived in vivo, reduced the formation of SCI cavities, promoted the regeneration of nerve fibers, and enabled partial recovery of motor function in a rat model of SCI (Feng et al., 2017). More recently, in 2018, a group applied the BM-MSC secretome intrathecally for 13 days (4 days interval between each administration) and observed an improvement of motor function after lesion alongside increased tissue sparing, presence of GAP-43-positive axons (new outgrowing neurons), and modulation of the immune response in a rat model of SCI (Cizkova et al., 2018). In addition to MSCs, the CM collected from other cell sources has yielded promising regenerative effects for SCI treatment. For instance, improvement of locomotor performance was observed in rats that were injected intraperitoneally with the secretome from apoptotic human peripheral blood mononuclear cells. This is suggested to result from the suppression of secondary injury mechanisms, which include neuroprotection, reduction of M1 macrophages, white matter cavity reduction, and increased neoangiogenesis (Haider et al., 2015). The administration of endothelial progenitor cells and UC-MSC CM is also linked to neuroprotection and locomotor performance recovery (Wang et al., 2018; Yeng et al., 2016). Furthermore, administration of ESC CM intraperitoneally in mice is reported to shift macrophages into an M2 regenerative and antiinflammatory profile combined with locomotor recovery (Guo et al., 2016). Reduction of inflammatory markers and hind-limb motor improvement were also observed upon NSC CM administration intraperitoneally in mice (Cheng et al., 2017). The administration of secretome for SCI seems to be a reliable alternative to cell transplantation (Fig. 5.2). However, despite the positive results of this therapeutic approach a lot remains to be dissected, such as the molecules present in the secretome pool that are responsible for the improved outcomes (Beer et al., 2015; Pires et al., 2016).

Concluding remarks While the CNS has an incredible plasticity compared with other organ systems, it also has unique sensitivity to injury. Neural tissue engineering and cell-based therapies have been intensively explored for CNS repair strategies

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in different disease contexts. In line with this, SCs have emerged as a key element of regenerative medicine owing to their intrinsic characteristics. Interestingly, while SC therapies for the CNS have been conventionally driven by cell differentiation for the replacement of damaged tissues, advances in the field have brought attention to the SC paracrine mechanisms. In fact, it has been demonstrated that in many instances, the functional benefits of SCs are due in large part to the secreted biologically active components (e.g., growth factors, cytokines, vesicles), generally referred to as the secretome. However, beyond the great enthusiasm for new treatment perspectives, much investigative work is still in progress on the development of robust and customized SC secretome-based therapies for CNS repair. Moving this potential from the bench to the bedside requires research on important factors, such as (1) choosing the source of the richest secretome, paying attention to possible autologous versus allogeneic donor variability, (2) modulating secretome and/ or EV composition by changing the culture conditions or overexpressing specific molecules, and (3) selecting the regimen (route and dosage) of treatment administration that suits each patient’s condition. Furthermore, although candidate molecules are under investigation, further detailed studies are needed to carefully define which factors may be responsible for the SC secretome-mediated neuroprotective and regenerative properties. It will also be important to understand the mechanisms behind these beneficial effects, such as the elucidation of activation or inhibition of molecular pathways, as well as the temporal effects. It is important to remember that rigorous preclinical studies and innovative clinical trials should be designed to better understand the mechanisms of CNS diseases and ultimately develop safe, effective, and controlled approaches. By doing so, in a near future, it could be possible to rationally design new therapeutical strategies based on the use of the SC secretome.

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Wicher, G., Fex-Svenningsen, A., Velsecchi, I., Charnay, Y., Aldskogius, H., 2008. Extracellular clusterin promotes neuronal network complexity in vitro. Neuroreport 19, 1487e1491. Williams, R., Murray, A., 2015. Prevalence of depression after spinal cord injury: a meta-analysis. Arch. Phys. Med. Rehabil. 96, 133e140. Wolf, A.C., Tate, R.L., Lannin, N.A., Middleton, J., Lane-Brown, A., Cameron, I.D., 2012. The world health organization disability assessment scale, WHODAS II: reliability and validity in the measurement of activity and participation in a spinal cord injury population. J. Rehabil. Med. 44, 747e755. World Health Organization, 2014. Global Status Report on Noncommunicable Diseases. Xin, H., Katakowski, M., Wang, F., Qian, J.Y., Liu, X.S., Ali, M.M., Buller, B., Zhang, Z.G., Chopp, M., 2017a. MicroRNA cluster miR-17-92 cluster in exosomes enhance neuroplasticity and functional recovery after stroke in rats. Stroke 48, 747e753. https://doi.org/10.1161/ STROKEAHA.116.015204. Xin, H., Li, Y., Cui, Y., Yang, J.J., Zhang, Z.G., Chopp, M., 2013a. Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J. Cereb. Blood Flow Metab. 33, 1711e1715. https:// doi.org/10.1038/jcbfm.2013.152. Epub 2013 Aug 21. Xin, H., Li, Y., Liu, Z., Wang, X., Shang, X., Cui, Y., Zhang, Z.G., Chopp, M., 2013b. MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles. Stem Cells 31, 2737e2746. https://doi.org/10.1002/stem.1409. Xin, H., Wang, F., Li, Y., Lu, Q.E., Cheung, W.L., Zhang, Y., Zhang, Z.G., Chopp, M., 2017b. Secondary release of exosomes from astrocytes contributes to the increase in neural plasticity and improvement of functional recovery after stroke in rats treated with exosomes harvested from MicroRNA 133b-overexpressing multipotent mesenchymal stromal cells. Cell Transplant. 26, 243e257. https://doi.org/10.3727/096368916X693031. Epub 2016 Sep. 26. Xing, C., Arai, K., Lo, E.H., Hommel, M., 2012. Pathophysiologic cascades in ischemic stroke. Int. J. Stroke 7, 378e385. Xiong, Y., Mahmood, A., Chopp, M., 2013. Animal models of traumatic brain injury. Nat. Rev. Neurosci. 14, 128e142. Xiong, Y., Zhang, Y., Mahmood, A., Chopp, M., 2015. Investigational agents for treatment of traumatic brain injury. Expert Opin. Investig. Drugs 24, 743e760. Xu, G.Y., Liu, S., Hughes, M.G., McAdoo, D.J., 2008. Glutamate-induced losses of oligodendrocytes and neurons and activation of caspase-3 in the rat spinal cord. Neuroscience 153, 1034e1047. Xu, Y., Shi, T., Xu, A., Zhang, L., 2016. 3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney. J. Cell Mol. Med. 20, 1203e1213. Yabe, T., Sanagi, T., Yamada, H., 2010. The neuroprotective role of PEDF: implication for the therapy of neurological disorders. Curr. Mol. Med. 10, 259e266. Yanez-Mo, M., Siljander, P.R., Andreu, Z., Zavec, A.B., Borras, F.E., Buzas, E.I., Buzas, K., Casal, E., Cappello, F., Carvalho, J., Colas, E., Cordeiro-da Silva, A., Fais, S., FalconPerez, J.M., Ghobrial, I.M., Giebel, B., Gimona, M., Graner, M., Gursel, I., Gursel, M., Heegaard, N.H., Hendrix, A., Kierulf, P., Kokubun, K., Kosanovic, M., Kralj-Iglic, V., KramerAlbers, E.M., Laitinen, S., Lasser, C., Lener, T., Ligeti, E., Line, A., Lipps, G., Llorente, A., Lotvall, J., Mancek-Keber, M., Marcilla, A., Mittelbrunn, M., Nazarenko, I., Nolte-’t Hoen, E.N., Nyman, T.A., O’Driscoll, L., Olivan, M., Oliveira, C., Pallinger, E., Del Portillo, H.A., Reventos, J., Rigau, M., Rohde, E., Sammar, M., Sanchez-Madrid, F., Santarem, N., Schallmoser, K., Ostenfeld, M.S., Stoorvogel, W., Stukelj, R., Van der Grein, S.G.,

114 Mesenchymal Stem Cells in Human Health and Diseases Vasconcelos, M.H., Wauben, M.H., De Wever, O., 2015. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066. Yasuhara, T., Matsukawa, N., Hara, K., Yu, G., Xu, L., Maki, M., Kim, S.U., Borlongan, C.V., 2006. Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson’s disease. J. Neurosci. 26, 12497e12511. Yeng, C.H., Chen, P.J., Chang, H.K., Lo, W.Y., Wu, C.C., Chang, C.Y., Chou, C.H., Chen, S.H., 2016. Attenuating spinal cord injury by conditioned medium from human umbilical cord blood-derived CD34(þ) cells in rats. Taiwan. J. Obstet. Gynecol. 55, 85e93. Yu, S.P., Wei, Z., Wei, L., 2013. Preconditioning strategy in stem cell transplantation therapy. Transl. Stroke Res. 4, 76e88. Zanoni, M., Piccinini, F., Arienti, C., Zamagni, A., Santi, S., Polico, R., Bevilacqua, A., Tesei, A., 2016. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci. Rep. 6, 19103. Zhang, C., Wu, J.M., Liao, M., Wang, J.L., Xu, C.J., 2016. The ROCK/GGTase pathway are essential to the proliferation and differentiation of neural stem cells mediated by simvastatin. J. Mol. Neurosci. 60, 474e485. Zhang, Y., Chopp, M., Zheng, G.Z., Katakowski, M., Xin, H., Qu, C., Ali, M., Mahmood, A., Xiong, Y., 2017. Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury. Neurochem. Int. 111, 69e81. Zhang, Z.G., Chopp, M., 2016. Exosomes in stroke pathogenesis and therapy. J. Clin. Investig. 126, 1190e1197. https://doi.org/10.1172/JCI81133. Epub 2016 Apr 1. Zhao, Q., Hu, J., Xiang, J., Gu, Y., Jin, P., Hua, F., Zhang, Z., Liu, Y., Zan, K., Zu, J., Yang, X., Shi, H., Zhu, J., Xu, Y., Cui, G., Ye, X., 2015. Intranasal administration of human umbilical cord mesenchymal stem cells-conditioned medium enhances vascular remodeling after stroke. Brain Res. 1624, 489e496. https://doi.org/10.1016/j.brainres.2015.08.003. Epub 2015 Aug 13. Zhao, Y.H., Gibb, S.L., Zhao, J., Moore, A.N., Hylin, M.J., Menge, T., Xue, H.S., Baimukanova, G., Potter, D., Johnson, E.M., Holcomb, J.B., Cox, C.S., Dash, P.K., Pati, S., 2016. Wnt3a, a protein secreted by mesenchymal stem cells is neuroprotective and promotes neurocognitive recovery following traumatic brain injury. Stem Cells 34, 1263e1272. Ziv, Y., Avidan, H., Pluchino, S., Martino, G., Schwartz, M., 2006. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury. Proc. Natl. Acad. Sci. U.S.A. 103, 13174e13179. Zuo, T., Qin, J.Y., Chen, J., Shi, Z., Liu, M., Gao, X., Gao, D., 2013. Involvement of N-cadherin in the protective effect of glial cell line-derived neurotrophic factor on dopaminergic neuron damage. Int. J. Mol. Med. 31, 561e568.

Chapter 6

Innovation in induced mesenchymal stem cell uses in therapy Ahmed H.K. El-Hashash1, 2 1 The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; 2Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC

Introduction Mesenchymal stromal/stem cells (MSCs) are multipotent cells that were isolated for the first time from the bone marrow and can give rise to multiple cell lineages, such as cartilage, fat, fibroblasts, and bone. In addition to their multipotency and differentiation ability, MSCs are well known for tissue healing, repair, and regeneration due to their immunoregulatory functions and cytokine secretion ability. MSCs can be obtained from various fatal and adult tissues, including the umbilical cord tissue, Wharton’s jelly, umbilical cord blood, and bone marrow. MSCs are promising for the treatment of a wide range of human diseases, including stroke, peripheral ischemic diseases, myocardial infarction, other cardiovascular diseases, inflammatory airway disorders, pulmonary fibrosis, and liver diseases (Hsuan et al., 2016; Lee et al., 2016, 2018; Luo et al., 2017; Chuang et al., 2018; Fan et al., 2019; Yun and Lee, 2019). Induced pluripotent stem cells (iPSCs) have a remarkably high potential for both proliferation and differentiation and, therefore, have many advantages for tissue repair and regeneration. In addition, iPSCs are preferred for autologous transplantation due to their immunological safety (Cohen and Melton, 2011; Diederichs and Tuan, 2014). Importantly, iPSCs can be induced and converted into many different cell types in a controlled manner that enables obtaining a sufficient cell number. They are, therefore, a major source of the induced MSCs (iMSCs; Sabapathy and Kumar, 2016).

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Major sources of induced mesenchymal stromal/stem cells Human iPSCs are a well-established and characterized source for generating an unlimited number of lineage- and patient-specific MSCs due to their high proliferation and differentiation potentials. In addition, there no ethical concerns about deriving MSCs from iPSCs. Thus, the rapid progress in human iPSC research has led to the development of protocols for generating MSCs from human iPSCs by directed differentiation. MSCs derived from human iPSCs have features similar to those of mature MSCs at both functional and genetic levels (Jung et al., 2012; Ma et al., 2013; Okano et al., 2013; Kimbrel et al., 2014). However, developing an effective and reliable research protocol to produce mature adult MSCs from iPSCs is still challenging. The use of a culture medium containing typical MSC growth factors (e.g., basic fibroblast growth factor [bFGF]) or a high serum concentration after mesoderm induction is a well-established classical method for differentiating iPSCs into MSCs (Liu et al., 2012; Jung et al., 2012; Frobel et al., 2014; Steens et al., 2017; Roux et al., 2018). The formation of an embryoid body or treatment of cells with mesodermal-inductive factors, including bFGF, activin A/nodal, bone morphogenetic protein 4, and glycogen synthase kinase-3 inhibitors or WNT ligands, in a chemically defined monolayer system is usually used to induce mesodermal differentiation. The selected MSCs can then be isolated and expanded under chemically defined cell culture conditions, following their treatment with MSC-specific growth factors and/or sorting for cell surface markers that are specific for MSCs using either flow cytometry or immunomagnetic separation. Other well-developed and clinically compliant protocols for differentiating human iPSCs into MSCs have been reported. For example, some studies used three different iPSC lines (named as iPSC(iMR90)-5 and iPSC(iMR90)-4 cells, which are derived from IMR90 fibroblasts, as well as iPSC(foreskin) Clone1 cells, which are derived from foreskin fibroblasts). They grew these three different iPSC lines in culture on gelatin-coated plastic dishes in KnockOut-DMEM culture medium that was supplemented with 10% serum replacement, PDGF-AB, bFGF, and EGF to promote the outgrowth of MSCs (Lian et al., 2007, 2010). Later, they isolated the characteristic CD24
and CD105þ cells using flow cytometry (fluorescence-activated cell sorting) after 7 days in culture and expanded the colony, leading to the successful generation of MSCs that were therapeutically active and exhibited the classical MSC markers and characteristics, including prolonged self-renewal capacity in culture without altering their plasticity or detectable signs of replicative senescence (Lian et al., 2010). When transplanted into a mouse model, these iPSC-derived MSCs can attenuate severe hind-limb ischemia more effectively than the transplanted adult bone marrow MSCs (Lian et al., 2010). This is mostly because of the ability of transplanted iPSC-derived MSCs to significantly improve both the regeneration of muscle and vascular tissues and

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in vivo survival, as well as producing some endangered-cell-protecting trophic factors after ischemic injury (Lian et al., 2010). These characteristics of iPSCderived MSCs compared with adult bone marrow MSCs were also demonstrated in other studies, in which they clearly inhibited the proliferation of natural killer (NK) cells and cytolytic function and expressed more resistance to preactivated NK cells, leading to a better prevention of the allograft rejection (Giuliani et al., 2011). The higher proliferation capacity of iPSCderived MSCs compared with those derived from bone marrow is probably due to the differential ion channel expression, including the enhanced expression of the functional KCNH1-encoded human ether a`-go-go 1 potassium channel (Lian et al., 2010; Zhang et al., 2012). The overexpression of some genes or transcription factors that are related to MSCs ectopically in iPSCs is another well-developed method for iMSC generation (Steens et al., 2017). For example, the expression of some HOX genes that are specific for MSCs in human vascular wall (Klein et al., 2013) via lentiviral vectors is effective in reprogramming murine iPSCs directly into MSCs (Steens et al., 2017). These reprogrammed MSCs show well-identified MSC characteristics both in vivo and in culture (Steens et al., 2017). More studies are still needed to apply this direct reprogramming method in human iPSCs. Since the extracellular matrix protein type I collagen is known to activate the epithelial-to-mesenchymal transition (EMT) of epithelial cells (Medici and Nawshad, 2010), new research methods have used it or other effective biomaterial matrices to affect the fate of cultured cells by physicochemical stimulation. For example, human dermal fibroblast-derived HDFa-YK26 iPSCs can effectively and rapidly differentiate into MSCs if mimicking the structure of physiological collagen using fibrillar and thin type I collagen in culture (Liu et al., 2012). This led to the development of homogeneous spindle-shaped cell colonies when growing iPSCs in culture for 10 days (Liu et al., 2012). The small-molecule inhibitors, such as the transforming growth factor-b pathway inhibitor SB431542, can also be used for a rapid and onestep direction of iPSC differentiation into MSCs. Thus, growing iPSCs (MR90CL2 and ES4CL1 cells) in a culture medium containing SB431542 in vitro for 10 days leads to the formation of MSCs with typical MSC characteristics that conform to the International Society for Cell Therapy criteria for classifying MSCs (Chen et al., 2012). Notably, treating iPSCs with SB431542 inhibitor can trigger specific intrinsic and autocrine signaling mechanisms that result in EMT induction, directing the fate of treated cells to that of mesenchymal stromal cells and leading to an approximate increase of 75%e96% in MSC number (Chen et al., 2012). The generation of a high percentage (95%) of pure MSC cultures was further achieved from iPSC lines that arose from different somatic tissues (e.g., periodontal ligament, gingiva, and lung) by growing these iPSC lines continuously in specifically formulated MSC culture media that led to cell clones expressing MSC-associated markers, including CD90, CD73, CD105, CD166, and CD146 (Hynes et al., 2014).

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Interestingly, these iPSCeMSC-like cells can differentiate into several other cell types, including adipocyte cells, osteoblasts, and chondrocyte cells, in culture (Hynes et al., 2014). Further progress in the generation of MSCs that are highly identical and clinically compliant from human iPSCs was achieved by using a therapy-grade platelet lysate as a cell culture supplement and xeno-free supplements (e.g., no animal products and no coculturing with murine cells), which significantly increased the amount of MSCs in approximately 1 month. This was clearly demonstrated by using a combination of iPSCs that were reprogrammed from human foreskin fibroblasts, H9 human embryonic stem cells, and the medium supplement of platelet lysate to robustly produce pluripotent-derived MSCs through a two-stage protocol in approximately 1 month (Luzzani et al., 2015). The first stage included the induction of mesodermal differentiation by the dissociation of cultured pluripotent stem cell clusters, before plating some single-cell isolates on a basement membrane matrix that characteristically had a reduced growth factor content, together with both platelet lysate and B27 supplement (Chen et al., 2008), for 2 weeks in culture (Luzzani et al., 2015). Then, after their transition to a mesenchymal state, these cells were moved to stage 2, in which they grew in culture in a-MEM that was supplemented with 10% platelet lysate in plastic dishes for an additional 1e2 weeks (Luzzani et al., 2015). Remarkably, the derived platelet lysate and the thrombinactivated platelet releasate in plasma, together with human serum (in general), are apparently promising alternatives to the medium supplement fetal bovine serum for culturing MSCs, while the growth factor cocktail of HGF, TGF-b1, PDGF-AB/BB, IGF-1, and bFGF is essential for MSC proliferation (Auletta et al., 2011; Flemming et al., 2011; Fekete et al., 2012; Kinzebach et al., 2013; Iudicone et al., 2014). In 2017, it was reported that some specific growth factors or chemicals can be used for generating iMSCs from skin fibroblasts (Lai et al., 2017). Thus, primary human dermal fibroblasts can be chemically and efficiently converted into multipotent iMSCs using a well-defined cocktail of growth factors and small molecules in 6 days in culture (Lai et al., 2017). Using this protocol, iMSCs show higher clonogenicity and survival in culture than fibroblasts and demonstrate a suppression activity in lipopolysaccharide-mediated acute lung injury, like bone marrowederived MSCs. In addition, chemically converted iMSCs can differentiate into several other cell types, including adipocytes, osteoblasts, and chondrocytes, after a long-term expansion in regular MSC culture medium (Lai et al., 2017).

Induced mesenchymal stromal/stem cell applications in cell therapy Several studies show MSC therapy as a promising and effective strategy for the treatment of many severe human diseases, such as inflammatory bowel

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disease, due to their inherent antiinflammatory and immunomodulatory properties (Magro et al., 2013). However, some studies demonstrate a lack of long-lasting immunoregulatory functions in adult MSCs growing in vivo and in culture (Uccelli et al., 2008). In addition to their immunoregulatory functions, MSCs are multipotent and can secrete multiple cytokines to promote the healing of different tissues (Uccelli et al., 2008; Fitzsimmons et al., 2018; Lukomska et al., 2019). Due to these unique MSC properties, the effectiveness of MSCs in cell therapy is being tested in many clinical trials worldwide. However, as of this writing, autologous MSC therapy faces several challenges, including the donor age, cell source, and intrinsic viability of MSCs. The iPSC-derived MSCs (iMSCs) represent a potential solution to these defined problems, and their role in suppressing inflammation is well investigated in murine models (Lian et al., 2010; Sun et al., 2012; Lai et al., 2017). As discussed in previous sections, many research studies have demonstrated the successful derivation of functional MSCs from iPSCs (named as iMSCs; Lian et al., 2010; Giuliani et al., 2011; Zhang et al., 2011; Wei et al., 2012; Liu et al., 2012; Chen et al., 2012; Villa-Diaz et al., 2012; Guzzo et al., 2013). Many readily accessible adult tissues can act as a source for iMSCs that show a high proliferation potential (Lian et al., 2010). iMSCs can serve as an important source for cell therapy of a wide range of human diseases. Many preclinical experiments done in the 2010s demonstrate that iMSCs can effectively contribute to the repair and regeneration of several injured tissues, including liver, blood vessel, heart, periodontal tissue, chondrocyte, and skeletal muscle (Lian et al., 2010; Hynes et al., 2013; Moslem et al., 2013; Eberle et al., 2013; Jeong et al., 2014; Hu et al., 2016; Liang et al., 2017; Zhu et al., 2017). In addition, iMSCs can promote microbiome normalization and intestinal healing in a murine inflammatory bowel disease model, similar to adipose-derived MSCs (Sirikul et al., 2018). Studies suggest iMSCs as a new and effective source of therapeutically active MSCs. They can function as a powerful adult MSC replacement, and could be used successfully in drug screening, disease modeling, and many therapeutic applications (Sabapathy and Kumar, 2016; Zhao and Ikeya, 2018). Indeed, using iMSCs can help in bypassing the common cell therapyerelated immunological concerns (Amabile and Meissner, 2009). For example, MSCs were used for the prophylactic treatment of graft versus host disease (GVHD), which is a condition that might be fatal and happens after allogeneic transplantations and organ transplantations, due to their remarkable immunoregulatory properties (Le Blanc et al., 2008; Bartholomew et al., 2002; Koc et al., 2000; Ringden et al., 2006). Allogeneic transplantation is risky since it is a target for the circulating NK cells, which attack and eventually destroy the graft (Ljunggren and Karre, 1990; Kroemer et al., 2008). Notably, the cytotoxic activity of NK cells during this process can be reduced by cotransplanting MSCs, leading to GVHD prevention (Le Blanc et al., 2008; Koc et al., 2000; Ringden et al., 2006). Similarly, the cytolytic capability of NK cells can

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be clearly reduced in culture by human iMSCs, which are more potent than the bone marrowederived MSCs, suggesting iMSCs as an important therapy for preventing allograft rejection (Giuliani et al., 2011; Sabapathy and Kumar, 2016). However, the long-term research on the iMSC immunosuppressive activity still needs further exploration (Giuliani et al., 2011). In addition, iMSCs might be an effective therapeutic option for diabetic neuropathies, which are disorders with nerve damage that are associated with the diabetes mellitus disease, since they can ameliorate diabetic polyneuropathy disease in murine models (Himeno et al., 2013). The potential therapeutic effect of transplanted iMSCs on diabetic polyneuropathy disease is probably due to their secretion of some angiogenic/neurotrophic factors and more Schwanntype cell differentiation (Himeno et al., 2013). Periodontitis is an infection of the gums that leads to serious damage of the tooth-supporting bone and soft tissue, and periodontitis-derived periodontal abnormalities or defects could be potentially treated with iMSCs. In periodontitis animal (rat) models, for instance, iMSCs can facilitate the regeneration of periodontal tissues (Hynes et al., 2013). In addition, iMSCs that express tumor necrosis factor a-stimulated gene-6 (TSG6) were shown to reduce both the inflammation and the alveolar bone resorption in periodontitis rat models (Yang et al., 2014). Furthermore, human MSC therapy is promising for some important human diseases such as myocardial and limb ischemia (Kim et al., 2006; Trivedi et al., 2010), and many other human diseases (Sabapathy and Kumar, 2016). In addition, treatment with iMSCs could lead to the attenuation of limb ischemia in murine animal models that was more efficient than bone marrowederived MSC treatments, probably due to the long-term survival of transplanted iMSCs, for more than 5 weeks after transplantation (Lian et al., 2010). Remarkably, human iMSCs showed a high ability for continuous proliferation in culture (for 32 passages and more) without signs of cellular senescence (Wei et al., 2012). They also showed remarkable proangiogenic and wound-healing properties (Wei et al., 2012). Moreover, iMSCs can be successfully used for bone formation and regeneration in mice with calvarial abnormalities/defects, when derived on the synthetic polymeric coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) (Villa-Diaz et al., 2012). Interestingly, iMSCs can also be used successfully for studying and modeling rare human diseases such as Fanconi anemia, which is a rare and inherited human disease mainly affecting the bone marrow, leading to a decreased blood cell production and aplastic anemia (Liu et al., 2014). Similarly, HutchinsoneGilford progeria syndrome (HGPS) is another rare genetic human disorder, which leads to both rapid and premature aging in newborn children. Indeed, HGPS-derived iMSCs are both helpful and promising tools for investigating HGPS pathology at the molecular level (Zhang et al., 2011). Promising studies on the treatment of chronic renal insufficiency demonstrated the successful reprogramming of peripheral blood mononuclear cells (PBMCs)

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grown in culture into iMSCs using natural inducing factors (Pan et al., 2017). In these studies, both noninduced and induced PBMCs were transplanted into experimental animal models of chronic renal insufficiency showing a characteristic renal fibrosis, before evaluating the effects of these transplants on chronic renal insufficiency (Pan et al., 2017). The success of these studies provides a great hope for using iMSCs in treating chronic renal insufficiency. The proinflammatory cytokine interferon-g, a preactivating cytokine of MSC immunomodulatory properties, can make the targeted MSCs more detrimental and immunogenic, and therefore these cells are not feasible for clinical applications. In contrast, treatment of MSCs with interleukin-17A cytokine, as an alternative MSC preactivator, can lead to the enhancement of MSC therapeutic efficacy for different types of renal diseases by remarkably increasing the tolerance-promoting, immunosuppressive, and renoprotective properties of targeted MSCs (Sivanathan and Coates et al., 2018). Furthermore, most recent studies on inflammatory bowel disease provide evidence of increased intestinal healing and normalization of microbiomes using iMSCs in the murine model of inflammatory bowel disease (Soontararak et al., 2018). In these studies, the effects of iMSCs derived from iPSCs versus adipose-derived MSCs on inflammatory bowel disease and intestinal protection were compared using a mouse model of the disease. The two types of stem cells show equivalent effects on improving gut inflammation and other related abnormalities in treated mouse models (Soontararak et al., 2018). Notably, administered iMSCs can significantly enhance the proliferation of intestinal epithelial cells, Lgr5þ intestinal stem cell number, and intestinal angiogenesis, leading to the improvement of both intestinal healing and health (Soontararak et al., 2018). Similarly, clinical trials demonstrated that allogeneic chondrogenic iMSCs can effectively treat diseased joints in horses suffering from degenerative joint disease (Broeckx et al., 2019).

Conclusion Many studies have demonstrated the success of mesenchymal stem cell therapy in ameliorating a wide range of human diseases or injuries, including the cardiovascular diseases, autoimmune diseases, pulmonary diseases, and musculoskeletal diseases. However, adult MSC therapy of human diseases is still challenging, since a limited number of MSCs can be obtained from a single donor and a substantial cell quantity is required for their clinical therapeutic application. Other challenges include the limited adult MSC proliferation capacity, which may significantly constrain their capabilities of immunomodulation and secretion of bioactive factors. Significant progress has been achieved in the potential application of stem cells in the treatment of different human diseases based on the success in obtaining large amounts of iMSCs that are both therapeutically active and patient specific. iPSCs can be induced and converted into many different cell

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types in a controlled manner that enables obtaining a sufficient cell number and are a major source of iMSCs. iMSCs are a new stem cell population that can be produced by cellular reprogramming and shows the combined advantages of both MSCs and iPSCs and displays the characteristics of an MSC population. iPSC-derived iMSCs have a robust potential for both proliferation and differentiation and are a promising cell-based therapy for different immune-mediated human diseases and for tissue repair and regeneration. As of this writing, iMSCs’ scope is relatively limited to preclinical studies on tissue repair, regeneration, and engineering for treating different diseases. These preclinical studies have evaluated the utility of iMSCs that are derived from human iPSCs, as a powerful mesenchymal cell source with better proliferation, differentiation, and survival potentials than adult MSCs. In addition, iPSC-derived iMSCs have many better properties compared with adult MSCs, such as their cytokine profile, immunomodulation, secretion of paracrine factors, and microenvironment-modulating exosomes. More preclinical and clinical studies/trials are still needed to scale iMSCs toward routine clinical applications. Nevertheless, iMSCs are a promising cell-based therapy that is remarkably cost efficient and patient specific.

References Amabile, G., Meissner, A., 2009. Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol. Med. 15, 59e68. Auletta, J.J., Zale, E.A., Welter, J.F., Solchaga, L.A., 2011. Fibroblast growth factor-2 enhances expansion of human bone marrow-derived mesenchymal stromal cells without diminishing their immunosuppressive potential. Stem Cell. Int. 2011, 1e10. Bartholomew, A., Sturgeon, C., Siatskas, M.X., et al., 2002. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 30, 42e48. Broeckx, S.Y., Seys, B., Suls, M., Vandenberghe, A., Marie¨n, T., Adriaensen, E., Declercq, J., Van Hecke, L., Braun, G., Hellmann, K., Spaas, J.H., 2019. Equine allogeneic chondrogenic induced mesenchymal stem cells are an effective treatment for degenerative joint disease in horses. Stem Cells Dev. 28 (6), 410e422. Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B.A., Hell, J.W., 2008. NS21: re-defined and modified supplement B27 for neuronal cultures. J. Neurosci. Methods 171 (2), 239e247. Chen, Y.S., Pelekanos, R.A., Ellis, R.L., Horne, R., Wolvetang, E.G., Fisk, N.M., 2012. Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells. Stem Cells Transl. Med. 1 (2), 83e95. Chuang, H.-M., Shih, T.E., Lu, K.-Y., Tsai, S.-F., Harn, H.-J., Ho, L.-I., 2018. Mesenchymal stem cell therapy of pulmonary fibrosis: improvement with target combination. Cell Transplant. 27 (11), 1581e1587. Cohen, D.E., Melton, D., 2011. Turning straw into gold: directing cell fate for regenerative medicine. Nat. Rev. Genet. 12, 243e252. Diederichs, S., Tuan, R.S., 2014. Functional comparison of human-induced pluripotent stem cellderived mesenchymal cells and bone marrow-derived mesenchymal stromal cells from the same donor. Stem Cells Dev. 23, 1594e1610.

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124 Mesenchymal Stem Cells in Human Health and Diseases Kinzebach, S., Dietz, L., Kluter, H., Thierse, H.J., Bieback, K., 2013. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells. BMC Cell Biol. 14 (1), 48. Klein, D., Benchellal, M., Kleff, V., Jakob, H.G., Ergun, S., 2013. Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells. Sci. Rep. 3 (1), 2178. Koc, O.N., Gerson, S.L., Cooper, B.W., et al., 2000. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18, 307e316. Kroemer, A., Edtinger, K., Li, X.C., 2008. The innate natural killer cells in transplant rejection and tolerance induction. Curr. Opin. Organ Transplant. 13, 339e343. Lai, P.L., Lin, H., Chen, S.F., et al., 2017. Efficient generation of chemically induced mesenchymal stem cells from human dermal fibroblasts. Sci. Rep. 7, 44534. Le Blanc, K., Frassoni, F., Ball, L., et al., 2008. Mesenchymal stem cells for treatment of steroidresistant, severe, acute graft-versus- host disease: a phase II study. Lancet 371, 1579e1586. Lee, C.-W., Chen, Y.-F., Wu, H.-H., Lee, O.K., 2018. Historical perspectives and advances in mesenchymal stem cell research for the treatment of liver diseases. Gastroenterology 154 (1), 46e56. Lee, J.H., Ryu, J.M., Han, Y.S., Zia, M.F., Kwon, H.Y., Noh, H., Han, H.J., Lee, S.H., 2016. Fucoidan improves bioactivity and vasculogenic potential of mesenchymal stem cells in murine hind limb ischemia associated with chronic kidney disease. J. Mol. Cell. Cardiol. 97, 169e179. Lian, Q., Lye, E., Yeo, K.S., et al., 2007. Derivation of clinically compliant MSCs from CD105þ, CD24
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Ma, T., Xie, M., Laurent, T., Ding, S., 2013. Progress in the reprogramming of somatic cells. Circ. Res. 112 (3), 562e574. Magro, F., Langner, C., Driessen, A., et al., 2013. European consensus on the histopathology of inflammatory bowel disease. J. Crohns Colitis 7, 827e851. Medici, D., Nawshad, A., 2010. Type I collagen promotes epithelial-mesenchymal transition through ILK-dependent activation of NF-kB and LEF-1. Matrix Biol. 29 (3), 161e165. Moslem, M., Valojerdi, M.R., Pournasr, B., Muhammadnejad, A., Baharvand, H., 2013. Therapeutic potential of human induced pluripotent stem cell-derived mesenchymal stem cells in mice with lethal fulminant hepatic failure. Cell Transplant. 22, 1785e1799. Okano, H., Nakamura, M., Yoshida, K., et al., 2013. Steps toward safe cell therapy using induced pluripotent stem cells. Circ. Res. 112 (3), 523e533. Pan, X.-H., Zhou, J., Yao, X., Shu, J., Liu, J.-F., Yang, J.-Y., et al., 2017. Transplantation of induced mesenchymal stem cells for treating chronic renal insufficiency. PLoS One 12 (4), e0176273. Ringden, O., Uzunel, M., Rasmusson, I., et al., 2006. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 81, 1390e1397. Roux, C., Saviane, G., Pini, J., et al., 2018. Immunosuppressive mesenchymal stromal cells derived from human-induced pluripotent stem cells induce human regulatory T cells in vitro and in vivo. Front. Immunol. 8, 1991. Sabapathy, V., Kumar, S., 2016. hiPSC-derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine. J. Cell Mol. Med. 20 (8), 1571e1588. Sirikul, S., Lyndah, C., Valerie, J., et al., 2018. Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model. Stem Cells Transl. Med. 7, 456e467. Sivanathan, K.N., Coates, P.T., 2018. IL-17Aeinduced mesenchymal stem cells have promising therapeutic value for clinical translation. Kidney Int. 93 (4), 771e773. Soontararak, S., Chow, L., Johnson, V., Coy, J., Wheat, W., Regan, D., Dow, S., 2018. Mesenchymal stem cells (MSC) derived from induced pluripotent StemCells (iPSC) equivalent to adipose-derived MSC in promoting IntestinalHealing and microbiome normalization in mouse inflammatory bowel disease model. Stem Cells Transl. Med. 7 (6), 456e467. Steens, J., Zuk, M., Benchellal, M., et al., 2017. In vitro generation of vascular wall-resident multipotent stem cells of mesenchymal nature from murine induced pluripotent stem cells. Stem Cell Rep. 8 (4), 919e932. Sun, Y.Q., Deng, M.X., He, J., et al., 2012. Human pluripotent stem cell-derived mesenchymal stem cells prevent allergic airway inflammation in mice. Stem Cells 30, 2692e2699. Trivedi, P., Tray, N., Nguyen, T., et al., 2010. Mesenchymal stem cell therapy for treatment of cardiovascular disease: helping people sooner or later. Stem Cells Dev. 19, 1109e1120. Uccelli, A., Moretta, L., Pistoia, V., 2008. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8, 726e736. Villa-Diaz, L.G., Brown, S.E., Liu, Y., et al., 2012. Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells 30, 1174e1181. Wei, H., Tan, G., Manasi, Q.S., Kong, G., Yong, P., Koh, C., Ooi, T.H., Lim, S.Y., Wong, P., Gan, S.U., Shim, W., 2012. One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells. Stem Cell Res. 9, 87e100.

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Chapter 7

Role of mesenchymal stem cells in bone fracture repair and regeneration Yishan Chen, Junxin Lin, Yeke Yu, Xiaotian Du Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC

Introduction Bone is a unique tissue, responsible for exercising, supporting, and protecting the body. When bone fracture occurs, physiological repair programs can give a complete regeneration (Marzona and Pavolini, 2009). However, there are many conditions that cause healing delay and bone nonunion, such as inadequate blood supply, soft tissue injury, and mechanical instability. Currently, it is believed that autologous bone grafting can act as the standard treatment for these conditions, but a number of drawbacks (donor-site morbidity [Goulet et al., 1997], autologous bone limitation, and bone stock loss) make us turn to further investigations for other alternative approaches (Rosset et al., 2014). Cell therapy gives us another chance to obtain regenerative bone tissues. To promote bone repair, stem/progenitor cells are cultured, alone or on a scaffold, and supplied to the injury site, sometimes combined with cytokines. As a reliable multipotent cell resource (Rosset et al., 2014), mesenchymal stem cells (MSCs) have been widely used in tissue engineering. The guidelines that defined the conception of MSCs, issued by the International Society for Cell Therapy, are based on the following criteria (Dominici et al., 2006). First, cell adherence to the culture dish under standard culture conditions. Second, expression of some specific cell surface markers, including CD44, CD71, CD73, CD90, and CD105; lack of expression of other markers such as CD11b, CD14, CD19, CD34, CD45, CD79a, CD80, and CD86; and low expression of major histocompatibility complex (MHC) class I. Finally, cells are able to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro. In physical bone regeneration, MSCs may play a key role in the angiogenic activation phase, after the early inflammatory phase (Qin et al., 2014). Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00007-4 Copyright © 2020 Elsevier Inc. All rights reserved.

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MSCs can also migrate to injury sites and assist in the repair program. However, the molecular mechanisms behind these functions, such as the involved chemokine receptors with their ligands, remain unclear. This chapter covers the current understanding of MSC function in bone fracture healing, including the source of MSCs, internal and external factors that affect MSC homing, and bone tissue engineering with MSCs. We aim to summarize the clues to MSC-based therapy for future bone regeneration.

Sources of mesenchymal stem cells for bone regeneration MSC niches, which maintain tissue homeostasis and breed stem cell populations (Frenette et al., 2013), reside near the perivascular region, on the endosteal surfaces of trabecular bone, adjacent to the myofiber plasma membrane, or within the interfibrillary spaces (Via et al., 2012). MSCs have also been found in umbilical cord blood (Lee et al., 2004), dental tissues (Liu et al., 2015), and synovial fluid (Sekiya et al., 2012). Moreover, MSCs could also be differentiated from induced pluripotent stem cells (iPSCs; Chen et al., 2012). Although the sources of MSCs are widespread in the human body (Wang et al., 2013), an ideal stem cell source should be accessible through noninvasive methods, and cells should be expandable through in vitro culture. Also, the cells should survive and integrate well within the host bone tissue after transplantation, while showing no tumorigenicity. Bone marrow has been proved to be a more suitable source of cells for fracture repair (Freitas et al., 2017). Bone marrowederived stem cells (BMSCs) have been used for nonunion gap treatment in orthopedic surgery for many years. BMSCs were found to localize in areas of active bone formation within the endosteal callus in a mouse fracture model (Granero-Molto´ et al., 2009). But it is difficult to harvest MSCs from periosteum. MSCs of perivascular origin (pericytes) could be easily obtained because of the widespread vessel walls throughout the body. Some studies have investigated the roles of pericytes in bone healing (Ko¨nig et al., 2016; Tawonsawatruk et al., 2016). Pericytes have the ability to generate osteoblasts in vitro and successfully contribute to bone regeneration. Muscle-derived stem cells (MDSCs) have been shown to be qualified for regenerating bone in a critical size calvarial defect model (Gao et al., 2014); however, the harvest of MDSCs is difficult and the differentiation capacity of MDSCs is limited. Encouragingly, iPSCs offer an advantage over traditional MSCs, as they can be generated from almost any type of tissue in the human body, and display an unlimited growth capacity (Hynes et al., 2013). iPSC-derived MSCs (iPSC-MSCs) acquire an osteoblast phenotype and aid in bone regeneration without ectopic bone formation compared with BMSCs (Sheyn et al., 2016).

Mesenchymal stem cell homing in bone regeneration Bone fracture healing involves four overlapping events identified by their distinctive histological characteristics (Schindeler et al., 2008): inflammatory

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response, formation of cartilaginous callus, formation of bone callus, and bone union. During these four complex programs, a series of cellular events (migration, proliferation, and differentiation regulated by cytokines and growth factors) is required. Among the various cell types mobilized in bone regeneration, progenitors are recruited to the injury site on day 1 and will proliferate around day 3 (Dimitriou et al., 2005). They can also secrete and release cytokines to attract MSCs, stromal cells, and inflammatory cells to participate in the tissue repair (Chen et al., 2010). In the later phases, MSCs will differentiate into osteoblasts or chondrocytes for the preparation of bone tissue formation (Schindeler et al., 2008), which has been confirmed by numerous studies (Bruder et al., 1998; Gu et al., 2012; Zhu et al., 2012). In addition, it has been validated that the recruitment of MSCs and factors in the microenvironment around the injury site are effective for fracture repair (Giannoudis et al., 2009; Tsai et al., 2012; Zuo et al., 2013).

Molecular mechanisms affecting mesenchymal stem cell homing Normally, the circulating progenitor cells would migrate to the injured site of bone fracture (Kumagai et al., 2008; Ma et al., 2013). Research has partly unveiled the mechanisms for MSC homing to the injury, suggesting the vital roles of cytokines and chemokines, mostly as chemoattractants, during the fracture process. It has been proved that MSCs express many chemokine receptors (Honczarenko et al., 2006) and that chemokines mediate MSC migration in vitro and in vivo. Stromal cellederived factor 1 (SDF-1), also known as CXC ligand 12 (CXCL12), serves as a master regulator of CXC receptor 4 (CXCR4)-positive stem/progenitor cells, which could facilitate their differentiation into mature reparative cells (Rombouts and Ploemacher, 2003; Wynn et al., 2004). Although expressed on only a few MSCs’ surface (Wynn et al., 2004), CXCR4 contributes greatly in cell migration and recruitment (Granero-Molto´ et al., 2009; Kitaori et al., 2009). In another study, a CXCR4 antagonist, AMD3100, was injected to prove the essential role of SDF-1/CXCR4 signaling in fracture healing (Toupadakis et al., 2012). The migration of MSCs in vitro was probably mediated by SDF-1 in a dose-dependent manner (Chen et al., 2016). MSC sheets transplanted into the fracture site, along with the local administration of SDF-1, boosted new bone tissue and union formation (Pan et al., 2014). The relevance and interactions between SDF-1 and other cytokines vary. It was suggested that SDF-1 could act synergistically with monocyte chemoattractant protein 3 (MCP-3) to mediate the homing of MSCs from the systemic circulation in fracture repair (Shinohara et al., 2011). Meanwhile, MSC migration and differentiation could be improved by enhanced bone morphogenetic protein-2 (BMP-2) and SDF-1 expression (Granero-Molto´ et al., 2009).

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SDF-1 also facilitates autocrine and other paracrine factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), all of which inhibit cellular apoptosis (Giannelli et al., 2013). Moreover, CXCR4 is also regulated by SRY-related high-mobility group box 11 (SOX11) (Xu et al., 2015), which plays an important role in promoting MSC migration and ectopic bone formation. Transforming growth factor b (TGF-b) is expressed in many normal tissues, yet is most abundant in bone tissue and platelets. It is released by platelets, chondrocytes, osteoblasts, macrophages, and other inflammatory cells, and assists in callus formation in the early inflammatory phase of fracture healing. Being a chemotactic factor for BMSCs, TGF-b not only facilitates the proliferation of MSCs, but promotes that of preosteoblasts, chondrocytes, and osteoblasts. It also improves the expression of collagen, proteoglycans, osteopontin, osteonectin, alkaline phosphatase, and other extracellular proteins (Mendelson et al., 2011). Since there are many TGF-b-related receptors expressed by chondrogenic and osteogenic cells, TGF-b could play a major role in cartilage and endochondral bone formation (D’Amelio et al., 2010) and contribute to the whole process of fracture healing. Self-designed scaffolds spatially infused with TGF-b3-adsorbed collagen hydrogel were utilized to help repair rabbit humeral head defects (Buijs et al., 2007). Compared with spontaneous cell migration, 130% more cells were recruited to the regenerated articular cartilage by TGFb3, with both avascular cartilage and vascularized bone formation improved. Similarly, rabbit-derived MSCs cultured in a gene-activated biomimetic bone matrix expressing TGF-b1 could significantly increase osteogenic phenotype markers, and led to accelerated bone generation in vivo (Pan et al., 2014). However, the osteoinductive effect of TGF is relatively limited. Repeated or high-dose medication leads to a more significant effect, but may display side effects and difficulties in clinical application. During fracture repair, FGF could be synthesized by mesenchymal cells, osteoblasts, chondrocytes, monocytes, and macrophages. It possesses the capability to regulate cell migration, differentiation, and proliferation (Battula et al., 2007), involved in angiogenesis and wound healing by bonding to receptor tyrosine kinases. In the early phase of fracture healing, FGF could take part in vascular morphogenesis and mesenchymal cell mitosis. The use of exogenous bFGF can stimulate callus remodeling to restore the biomechanical properties rapidly (Chen et al., 2004). In addition, FGF can improve the mineral content of regenerated bone in a dose-dependent manner. A single FGF injection boosted cartilage formation by increasing the proliferation of chondrogenic precursors in the callus, but with no advantage in prompting chondrocyte maturation or cartilageebone tissue replacement (Ueno et al., 2011). NEL-like molecule-1 (NELL-1) is a craniosynostosis-associated molecule that regulates osteoblast differentiation (Aghaloo et al., 2007). Its osteoinductive capacity has been proved in several animal studies (Aghaloo et al., 2007; Lu et al., 2007; Siu et al., 2011; Zhang et al., 2010), but its role in long

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bone defect healing remains vague. Xue et al. (Xue et al., 2011) investigated the effects of NELL-1 on femoral distraction osteogenesis through a variety of in vivo analyses, suggesting increased osteocalcin and osteopontin with adenoviral NELL-1 transfection. In a rat model of femoral distraction osteogenesis, improved regeneration of good-quality bones and accelerated bone union were obtained with sustained delivery of NELL-1 via adenoviral transfection. BMSCs with modified BMP-2 and NELL-1 genes were shown to improve new bone formation and maturation, in both a maxillary sinus model and rapid distraction osteogenesis in tibial defect of rabbit (Xia et al., 2011; Zhu et al., 2011). Therefore, BMP-2 and NELL-1 may work synergistically on the osteogenic differentiation of BMSCs. Interestingly, compared with fulllength NELL-1, which is expressed in the embryonic period, the postnatally expressed N-terminal-truncated NELL-1 isoform (NELL-1570) stimulates osteogenic differentiation of more MSC-like populations. It also induces both significant calvarial defect regeneration and cell proliferation, being a potential choice for bone renovation (Pang et al., 2015). High-mobility group box 1 (HMGB1) protein is a nonhistone nucleosomal protein, and a novel cytokine taking part in the inflammation process. Research has also unveiled that it significantly induces MSC migration, while suppressing their proliferation in a dose-dependent manner (Fahmy-Garcia et al., 2018; Meng et al., 2008). In addition, HMGB1 stimulates MSCs to secrete diverse cytokines, and contributes in their osteogenic differentiation through the Ras/MAPK pathway (Feng et al., 2016). Connective tissue growth factor (also known as CCN2) is regarded as increasing MSC proliferation, migration, and aggregation and functioning in osteogenesis and chondrogenesis (Fahmy-Garcia et al., 2018). In S. FahmyGarcia’s study (Fahmy-Garcia et al., 2018), the osteogenic induction capacity of NELL-1, HMGB1, and CCN2 was side-by-side assessed and compared with that of BMP-2. Interestingly, the results showed that NELL-1, CCN2, and HMGB1 stimulated MSC migration, yet showed no effect in promoting MSC osteogenic differentiation or inhibiting preosteoblast differentiation and mineralization, while BMP-2 behaved just as the opposite. Blood platelet-derived growth factor (PDGF) serves as an effective chemotactic factor, showing a strong stimulatory effect on the proliferation and migration of MSCs and osteoblasts (Kodama et al., 2012). PDGF boosted the effects of mesenchymal cells in cartilage and bone formation from the early to middle stages of bone healing. In vivo trials unveiled that PDGF seems to influence MSC response relatively quickly in fracture patients, since there is a direct positive correlation between changes in MSCs and serum PDGF (Caplan, 1991). The combined application of PDGF and BMP accelerates bone defect repair, but the clinical utilization of PDGF in fracture treatment needs further exploration. Moreover, insulin-like growth factor, together with MSCs, can improve fracture healing, mostly through endochondral ossification (Ueno et al., 2011).

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Parathyroid hormone could also help repair bone defects by increasing the proliferation of MSCs while reducing senescence and apoptosis (Di et al., 2010). Fracture healing is not an isolated process; it consists of synergistic functioning of various factors that affect stem cell homing and promote differentiation. Research has proved that a low dose of the factors could provide better therapeutic effects. Bai et al. (Bai et al., 2013) unveiled that low concentrations of BMP-2, VEGF, and bFGF display dose- and time-dependent synergistic effects on the osteogenic differentiation of MSCs. Yet the appropriate time for applying the cytokines needs further exploration. In addition, according to Lee and colleagues, combining recombinant human BMP-2 (rhBMP-2) with epithelial growth factors such as epidermal growth factor, FGF, PDGF, or VEGF exhibits increased osteoinductivity, resulting in enhanced alkaline phosphatase level and activity, calcium accumulation, and new bone surface ratio, as well as trabecular number and thickness of cultured human MSCs (Chen et al., 2004). Moreover, a range of other molecules, including RANTES, MIP-1a, MCP-1, CCL25, and CXCL16, are recorded to be chemotactic for MSCs. Inflammatory cytokines such as tumor necrosis factor a (TNFa) recruit MSCs and facilitate endochondral ossification, yet in a manner dependent on the inflammation severity (Aghaloo et al., 2007). Not all cytokines or factors can promote MSC recruitment. According to Ode et al. (Ode et al., 2011), CD73/CD29 reduced migration of mechanically stimulated MSCs. Since sustained therapeutic effects of MSCs on bone fracture require mechanical stimulation in vivo, the appropriate mechanical microenvironment for MSCs needs to be better understood. Researchers are taking a close look at cellular characteristics and functional behavior of MSCs at the fracture site in response to mechanical loading, suggesting that mechanical embedding positively affects transplanted stem cells for better bone regeneration.

External factors affecting mesenchymal stem cell homing Some external factors also have effects on MSC homing. It was reported that mechanical stimulation affected the properties of callus and MSC migration. In one study (Weaver et al., 2010), axial displacement stimulation was introduced after systemic stem cell transplantation in SpragueeDawley rats, and could increase the mineral content and decrease the cartilage content in the fracture after 10 days. MSCs were still found after 48 days. Griffin et al. (Griffin et al., 2011) used degenerate wave (DW), capacitive coupling (CC), pulsed electromagnetic fields, and direct current (Tintut et al., 2003) to stimulate human BMSCs. Their study indicated that the stimulation of DW and CC affected cell intrusion and proliferation in vivo, and also improved the healing rate of fracture, perhaps by promoting MSC recruitment to the fracture site.

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Electrical stimulation also increases the maturation of callus by facilitating chondrocyte and osteoblast differentiation and matrix mineralization (Zhao et al., 2011), owing to the low oxygen tension with electrolysis and high pH in the microenvironment. Low-level laser irradiations may become a valid approach for MSC pretreatment, as they activate bone formation by promoting chondrocyte and osteoblast proliferation (Giannelli et al., 2013). Low-intensity pulsed ultrasound accelerates the process of endochondral bone formation (Tsai et al., 2012) by enhancing MSCecalcium phosphate composites and the bone mineral content/density and upregulating the expression of some bone proteins.

The application of mesenchymal stem cells in bone tissue regeneration Immunogenicity of mesenchymal stem cells It was believed that MSC transplantation was able to cross major histocompatibility barriers and create off-the-shelf therapies, owing to the hypoimmunogenic property of MSCs. MSCs do not express costimulatory molecules or MHC class II factors (Javazon et al., 2004) and also do secrete cytokines inhibiting B and T cell proliferation or monocyte maturation to suppress immune responses (Bernardo and Fibbe, 2013; Franc¸ois et al., 2012; Prockop, 2013). Thus, this resulted in a rapid rise in the therapeutic exploration of MHCunmatched allogeneic MSC transplantation (Ankrum et al., 2014). However, recent studies give contradictory results, suggesting that MSCs may not be completely immune privileged. It was confirmed that allogeneic MSC death would occur early after infusions (von Bahr et al., 2012). MSCs do cause immune responses, although with a slower rejection by hosts than other allogeneic cell types (Ankrum et al., 2014). The delay rate of rejection appeared to be strongly dependent on the balance between the immunogenic and the immunosuppressive factors expressed on MSCs (Ankrum et al., 2014). The comprehensive understanding of MSC immunogenicity becomes a challenge for allogeneic MSC therapy, but also encourages us to have new vision for modulating or prolonging MSC persistence.

The isolation and treatment approach of mesenchymal stem cells MSCs are rare in vivo, accounting for about 0.001%e0.01% of mononuclear cells in bone marrow; thus an efficient strategy for MSC enrichment is required (Griffin et al., 2011). Density gradient centrifugation with Ficoll and red blood cell (RBC) lysis buffer treatment have been widely used for MSC harvest and purification. It was shown that RBC lysis buffer treatment resulted in better biologically preserved characteristics of MSCs (Najar et al., 2014). The delivery methods of MSCs include direct injection, systemic infusion, tissue-engineered composite scaffolds, and scaffolds with cytokines. It is

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reported that systemic infusion through inhalation of MSCs may have inferior treatment outcomes because most infused MSCs are trapped in the lungs, while only a few migrate to the injury site (Gao et al., 2001; Schrepfer et al., 2007). Systemic infusion through intravenous delivery of BMSCs has been shown to promote bone regeneration by upregulating regulatory T cells (Liu et al., 2015). Thus, the ideal method of MSC delivery will depend on which mechanism is being utilized. The genetic engineering of MSCs using viralbased or nonviral vectors has been employed to overcome several problems in stem cell therapy, such as low survival rate and inefficiencies in differentiating into fully functional tissues (Hodgkinson et al., 2010).

Genetically modified mesenchymal stem cells for bone regeneration The use of growth factors and scaffold composites is a promising approach for MSC treatment; however, the long-term release of growth factors to promote MSC proliferation and maintenance is not satisfactory. Various studies have reported that osteoinductive growth factoremodified MSCs could result in successful bone induction in vivo. BMPs are the most studied growth factors for their use in the treatment of bone defects, and many of the BMPs have been shown to possess osteogenesis ability. BMP-2-transfected MSCs are applied to facilitate cranial bone regeneration (Vural et al., 2017) and bone formation in mouse hind limbs, as well as in the bony union of large-sized mouse radial defects (Gamradt et al., 2006). RhBMP-6-treated adipose-derived MSCs (ADSCs) and BMSCs exhibited greater osteogenic potential in vitro and in vivo compared with rhBMP-2 ADSCs and BMSCs (Mizrahi et al., 2013). Therefore, the genetically engineered growth factoreexpressing MSCs can be an alternative to promote MSC function. Nevertheless, a few studies have reported that the application of MSCs resulted in tumorigenicity in vivo (Tian et al., 2011; Wislet-Gendebien et al., 2012), even MSCs with short-term in vitro culture (Jeong et al., 2011). It should be noted that transfection and long-term in vitro culture could increase the tumorigenic potential of MSCs through alteration of genomic stability or the epigenetic landscape (Bentivegna et al., 2013; Izadpanah et al., 2008; Wagner, 2012). Thus, strict control of cell handling procedures is of great importance to minimize the risk of malignant transformation of MSCs.

Application of biological materials and growth factors in mesenchymal stem cell bone repair Bone cement and artificial bone transplantation are now widely used in clinical treatment. However, the following defects exist: the biocompatibility of organic bone cement is poor, the flexibility of inorganic bone cement is not

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strong enough and the cure rate is slow in vivo, and the source of artificial bone transplantation is limited and may give rise to excessive immune response. Tissue engineering is a viable way to solve these problems. The purpose of tissue-engineering scaffolds is to provide a three-dimensional (3D) scaffold for cell adhesion, proliferation, and differentiation and to address the shortcomings of the aforementioned conventional techniques. In the study of MSC bone repair, the pattern of “seed cell þ scaffold þ cytokine” is increasingly proving to be superior to the use of a single substance or the combination of two of them. Based on the source of material, scaffolds can be divided into natural material stent, artificial polymer material stent, and composites (Islam et al., 2015; Luo et al., 2015). New materials such as biodegradable microspheres and nanophase graphene are also progressing rapidly. Using the 3D printer to produce the scaffold material is developing rapidly in regenerative medicine. Rapid prototyping technology, including stereolithography, selective laser sintering, aerosol jet printing, and fused deposition modeling, can quickly and accurately generate complex 3D object models by means of a medical imaging system (CT), computer design software (CAD), and digital conversion system (Yuan et al., 2017). In clinical application, 3D data of bone injuries in patients can be obtained by nuclear magnetic resonance imaging. The bone tissue model and tissue scaffold will be made by the rapid prototyping technique. As an indispensable part of the differentiation medium, cytokines have attracted attention. Their role in the differentiation of MSCs varies with different growth factors, concentration of use, source of MSCs, and cell status. TGF-b, BMP, insulin-like growth factor, Wnt, Sox9, Hedgehog, FGF, NELL1, Notch, and other signaling molecules are believed to be involved in the regulation of MSC osteogenic differentiation (Almalki and Agrawal, 2016). In addition, other biological factors such as dexamethasone, ascorbic acid, b-glycerophosphate, etc., have been shown to be effective additives for osteogenesis and cartilage differentiation media. BMSCs combined with platelet-rich fibrin have also been shown to contribute to the bone repair process (Fernandes et al., 2016; Liu et al., 2017; Wang et al., 2017). It is worth mentioning that some researchers have shown that a higher concentration of traditional Chinese kidney medicine and herbal remedies can promote the osteogenic differentiation of BMSCs in animal experiments (Udalamaththa et al., 2016). In addition, macrophages play an important role in the regulation of bone repair and regeneration. In the coculture system, macrophages secrete BMP-2, OSM, exosomes, etc., to promote the osteogenic differentiation of MSCs, and TNFa to inhibit it (Chen et al., 2014). In other conditions, macrophageassociated inflammatory factors such as TNFa, IL-6, IL-1b, and OSM are also involved in MSC differentiation. Adding macrophages to the seed cells may lead to MSC osteogenesis promotion (Liu et al., 2015).

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Clinical consequences More studies have focused on the clinical application of artificial bone repair materials combined with MSCs. By modulating chemokineechemokine receptor interactions, MSCs may increase their ability to facilitate tissue repair and correct inherited disorders of mesenchymal tissues. Heng Zhu et al. showed that MSCs attenuated poly(lactic-co-glycolic acid) (PLGA)-induced inflammatory responses by inhibiting host dendritic cell maturation and function (Zhu et al., 2015), but more research needs to be done. Moreover, allogeneic MSC transplantation did not show significant rejection or adverse reactions, which may be related to the immunosuppressive effect of MSCs. For the repair of bone nonunion, closed surgery and open surgery combined with MSCs have been applied clinically. In the closed operation, BMSCs are injected into the nonunion site by local injection. The treatment method has the following advantages: simple, fast, low cost, and little trauma. However, despite closed surgery having a great advantage, the shortcomings are also obvious: it cannot eliminate the necrosis of bone and soft tissue, and after injection, it is difficult to confine the injected cells in the site of nonunion. If ossification occurs in the adjacent part, it may affect the treatment effect. In the open surgery combined with BMSCs for treatment of nonunion, the nonunion site can be fully revealed. BMSCs were transplanted into the nonunion site while clearing it, which has achieved good therapeutic effect. H.D. Ismail et al. explored the therapeutic potential of BMSCs combined with hydroxyapatite granules in nonunion of the long bone, and found that patients treated with this combination obtained functional and radiographic improvements (Ismail et al., 2016). To treat a bone nonunion in extreme clinical conditions, Dufrane et al. assessed a 3D human autologous scaffold-free osteogenic graft and demonstrated that this 3D graft could safely promote osteogenesis with no oncological side effects and minor donor site morbidity (Dufrane et al., 2015). Combining microsurgery with tissue-engineering bone construction is also an effective way to improve bone regeneration. Through local transfer and microsurgical vascular anastomosis, the new bone tissue is established and adequate blood is also provided at the same time.

Conclusion MSCs are widely spread in the human body, and can also be differentiated from iPSCs. It is believed that MSC homing in vivo plays a key role in bone regeneration, which is regulated by various cytokines and chemokines in the microenvironment. In addition, external factors, including mechanical stimulation, electric/electromagnetic fields, and laser irradiation, also have effects on MSC migration and recruitment. In transplantation treatment, MSCs were thought to be “immune privileged,” but more recent studies give contradictory results. MSCs are usually isolated from bone marrow, and can be injected

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locally into injury sites, intravenously, or supplied with scaffolds and cytokines. Nowadays, genetically modified MSCs and 3D scaffolds are developed to provide long-term release of growth factors, which can prolong MSC survival and maintain their regenerative function. In clinical applications, closed and open surgeries with MSCs have been applied in bone nonunion repair. Combining microsurgery with tissue-engineering bone construction is also an effective way to improve bone regeneration. In this chapter, we draw a conclusion on the current understanding of the role of MSCs in fracture healing and bone regeneration. Overall, the scientific research and clinical consequences provide clues to the biological mechanism study and future technique development.

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142 Mesenchymal Stem Cells in Human Health and Diseases Tsai, M.T., Lin, D.J., Huang, S., Lin, H.T., Chang, W.H., 2012. Osteogenic differentiation is synergistically influenced by osteoinductive treatment and direct cell-cell contact between murine osteoblasts and mesenchymal stem cells. Int. Orthop. 36, 199e205. Udalamaththa, V.L., Jayasinghe, C.D., Udagama, P.V., 2016. Potential role of herbal remedies in stem cell therapy: proliferation and differentiation of human mesenchymal stromal cells. Stem Cell Res. Ther. 7 (1), 110. Ueno, M., Urabe, K., Naruse, K., Uchida, K., Minehara, H., Yamamoto, T., Steck, R., Gregory, L., Wullschleger, M.E., Schuetz, M.A., et al., 2011. Influence of internal fixator stiffness on murine fracture healing: two types of fracture healing lead to two distinct cellular events and FGF-2 expressions. Exp. Anim. 60, 79e87. Via, A.G., Frizziero, A., Oliva, F., 2012. Biological properties of mesenchymal stem cells from different sources. Muscles Ligaments Tendons J. 2, 154e162. von Bahr, L., Batsis, I., Moll, G., Ha¨gg, M., Szakos, A., Sundberg, B., Uzunel, M., Ringden, O., Le Blanc, K., 2012. Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem Cells 30 (7), 1575e1578. Vural, A.C., Odabas, S., Korkusuz, P., et al., 2017. Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells. Artif. Cells Nanomed. Biotechnol. 45 (3), 544e550. Wagner, W., 2012. Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation. Stem Cell Res. Ther. 3 (6), 54. Wang, X., Li, G., Guo, J., et al., 2017. Hybrid composites of mesenchymal stem cell sheets, hydroxyapatite, and platelet-rich fibrin granules for bone regeneration in a rabbit calvarial critical-size defect model. Exp. Ther. Med. 13 (5), 1891e1899. Wang, X., Wang, Y., Gou, W., Lu, Q., Peng, J., Lu, S., 2013. Role of mesenchymal stem cells in bone regeneration and fracture repair: a review. Int. Orthop. 37 (12), 2491e2498. Weaver, A.S., Su, Y.P., Begun, D.L., Miller, J.D., Alford, A.I., Goldstein, S.A., 2010. The effects of axial displacement on fracture callus morphology and MSC homing depend on the timing of application. Bone 47 (1), 41e48. Wislet-Gendebien, S., Poulet, C., Neirinckx, V., et al., 2012. In vivo tumorigenesis was observed after injection of in vitro expanded neural crest stem cells isolated from adult bone marrow. PLoS One 7 (10), e46425. Wynn, R.F., Hart, C.A., Corradi-Perini, C., O’Neill, L., Evans, C.A., Wraith, J.E., Fairbairn, L.J., Bellantuono, I., 2004. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 104, 2643e2645. Xia, L., Xu, Y., Chang, Q., Sun, X., Zeng, D., Zhang, W., Zhang, X., Zhang, Z., Jiang, X., 2011. Maxillary sinus floor elevation using BMP-2 and Nell-1 gene-modified bone marrow stromal cells and TCP in rabbits. Calcif. Tissue Int. 89, 53e64. Xu, L., Huang, S., Hou, Y., Liu, Y., Ni, M., Meng, F., Wang, K., Rui, Y., Jiang, X., Li, G., 2015. Sox11-modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs. FASEB J. 29, 1143e1152. Xue, J., Peng, J., Yuan, M., Wang, A., Zhang, L., Liu, S., Fan, M., Wang, Y., Xu, W., Ting, K., et al., 2011. NELL1 promotes high-quality bone regeneration in rat femoral distraction osteogenesis model. Bone 48, 485e495. Yuan, B., Zhou, S.Y., Chen, X.S., 2017. Rapid prototyping technology and its application in bone tissue engineering. J Zhejiang Univ. Sci. B 18 (4), 303e315. Zhang, X., Zara, J., Siu, R.K., Ting, K., Soo, C., 2010. The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration. J. Dent. Res. 89, 865e878.

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Chapter 8

Tendon stem cells and their interaction with microenvironments Yangwu Chen, Xiao Chen, Zi Yin Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, PRC

Introduction Adult stem cells are those undifferentiated cells that exist in various differentiated tissues. Under specific conditions, adult stem cells either self-renew or differentiate into terminally functional cells in accordance with procedures, thus maintaining dynamic homeostasis of tissues and organs. They usually lie dormant in peacetime and would be activated when faced with pathological lesion or exogenous induction. Since Bi et al. (2007) identified a specific stem cell population in the tendon, researchers in this field have made great progress in the study of these cells. But indeed, the isolated cells show some extent of heterogeneity that is not completely in conformity with the definition of stem cells, indicating there is an ingredient of progenitor cells in the newfound cells. As a result, this isolated cell population is called tendon stem/progenitor cells (TSPCs). Like other stem cells, the behavior of TSPCs is closely connected with the dynamic changes in the cell niche. In other words, complying with external signal fluctuation, TSPCs would adjust themselves and make corresponding physiological or pathological function changes. In this section, we will discuss the characterization of TSPCs and focus on the interaction between TSPCs and the microenvironment.

Isolation and culture of tendon stem/progenitor cells TSPCs have been isolated and cultured from adult human (Bi et al., 2007), fetal human (Hu et al., 2016b), mouse (Bi et al., 2007), rat (Rui et al., 2010), and rabbit (Zhang and Wang, 2010a) tendons. The methods are largely identical but with minor differences. As shown in Fig. 8.1, first, the peritendinous Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00008-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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FIGURE 8.1 Isolation and culture of tendon stem/progenitor cells from the Achilles tendon and tail tendon of mice.

tissues are removed and the tendon sheath is stripped off to obtain tendon tissues. Then the tendon tissues are cut into small pieces and digested with collagenase type 1 to hydrolyze the extracellular matrix (ECM). Afterward, the resulting cells are planted in the culture medium at a suitably low density.

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TSPCs will remain quiescent for several days before entering the stage of rapid proliferation to form cell colonies. Finally, these primary cells are collected for the following experiments. Screening by this low-density planting method can minimize the contamination by tenocytes. Bi et al. (2007) isolated TSPCs from human and mouse tendon tissue and further verified that this group of cells could self-renew and differentiate into tenocytes, chondrocytes, osteocytes, and adipocytes under respective induction conditions. Furthermore, both allogeneic or xenogeneic transplantation of TSPCs could form tendon-like tissue. This evidence implies that TSPCs have multipotent capability and undergo spontaneous tenogenic differentiation after transplantation.

Characterization of tendon stem/progenitor cells TSPCs are capable of expressing stem cell markers, including Sca1, Nanog, nucleostemin, Oct4, Ssea4, c-Myc, and Sox2 (Lui, 2015). These markers are frequently used to evaluate the differentiation degree of TSPCs in in vitro culture, and they can also help to distinguish TSPCs from tenocytes. It is worth noting that many of them are nuclear proteins that restrict their application in the purification of TSPCs, and only marker proteins such as the Ssea family expressed on the surface of the cytomembrane may have potential in this regard (Lui and Chan, 2011). In addition, TSPCs express other specific markers, like CD44, CD90.2, and CD146, but do not express hematopoietic cell markers CD34 and CD45 or the endothelial cell markers CD106, CD144, and Flk1 (Bi et al., 2007; Lui, 2015). TSPCs that do not express CD105 are more prone to differentiate into osteocytes and bring about degenerative lesions than the CD105-positive cells in injured tendons (Asai et al., 2014). Nestin is a biomarker of proliferating cells at the developmental stages of various tissues (Wiese et al., 2004). By single-cell quantitative gene analysis, researchers identified a subpopulation of nestin-positive TSPCs and observed that nestin is essential for the phenotype maintenance of TSPCs, while the absence of nestin would significantly affect cell fate decisions (Yin et al., 2016). A study using the combination of bromodeoxyuridine staining with labeling for nucleostemin in rat Achilles tendon showed that the distal tendon has a higher amount of stem cells than the remainder (Runesson et al., 2013). In addition, methods such as the iododeoxyuridine label retention can be utilized to help distinguish TSPCs from tenocytes (Tan et al., 2013). Colocalization of label-retaining cells with different markers contributes to in vivo identification of TSPCs and their roles in the repair of tendon injury (Tan et al., 2013). Bone marrowederived mesenchymal stem cells (BMSCs) have strong stem cell characteristics and are another cell type frequently used in the tendon regeneration strategy. Compared with BMSCs, TSPCs express higher levels of scleraxis (Scx) and tenomodulin (see later) (Bi et al., 2007). This suggests that these stem progenitor cells resident in tendon tissues are more prepared to differentiate into tenocytes, suggesting TSPCs are a preferable source of seed

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cells in tissue engineering. TSPCs have more typical stem cell morphology and structure, including smaller cell bodies and larger nuclei, than terminally differentiated tenocytes; look more like cobblestone; and proliferate faster in culture (Zhang and Wang, 2010a).

Culture conditions TSPCs lose their phenotype with time and passaging in in vitro culture. Mimicking in vivo cell living conditions, a hypoxic environment in vitro helps maintain the stemness of TSPCs (Lee et al., 2012; Zhang and Wang, 2013a). TSPCs cultured under low oxygen conditions express higher levels of stem cell markers (Zhang and Wang, 2013a). With small-molecule screening assays, researchers found that retinoic acid receptor agonists potently increase the expression of nuclear Scx (Webb et al., 2016). They confirmed that retinoic acid receptor agonists inhibit spontaneous differentiation of TSPCs. The effect is also reversible, with the withdrawal of this compound, thus providing an efficient way to maintain stemness of TSPCs during expansion in in vitro culture (Webb et al., 2016).

Cell passage and senescence After repeated passaging, TSPCs at late passages (P20 and P30) are inclined to differentiate into osteoblasts and have a weaker ability for chondrogenesis and adipogenesis than those at early and middle passages (P5 and P10) under induction conditions (Tan et al., 2012). In line with expectations, the expression of tendon-related genes is also decreased in TSPCs at late passages (Tan et al., 2012). Furthermore, there is significant upregulation of b-galactosidase activity and downregulated expression of CD90 and CD73, indicating that TSPC aging increases with passage time. TSPCs isolated from older individuals also express a lower level of stem cell markers such as Oct4, nucleostemin, Sca1, and Ssea1. However, these TSPCs exhibit stronger adipogenesis ability (Kohler et al., 2013; Zhang and Wang, 2015; Zhou et al., 2010). This suggests that cell senescence in in vitro passage cultivation is not entirely consistent with cell senescence that occurs in the natural aging model. The senescent cell is accompanied by upregulation of p16, reduced cell adhesion and migration, and slow turnover of actin filaments (Kohler et al., 2013). Rho-associated coiled-coil protein kinase (ROCK) plays a major role in cell senescence, and its downstream signaling regulates stress fiber formation (Kohler et al., 2013). MicroRNA (miR)-135a binds to ROCK and is downregulated in aged TSPCs (Chen et al., 2015). Furthermore, miR135a overexpression in young TSPCs can suppress cell senescence and promote TSPC proliferation, migration, and tenogenesis (Chen et al., 2015). In another experiment, researchers found that ephrin receptors (EphA4) are downregulated in older TSPCs, and rescue experiments significantly increased

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TSPC proliferation, migration, and actin turnover (Popov et al., 2015). It is believed the understanding of these age-related data will provide potential targets for future tissue regeneration research. At this writing, the situation is that tendon repair after injury is not satisfactory. With the promise of regenerating defective organs or tissues, stem cell therapy receives considerable attention and high expectations. Current stem cellebased therapies predominantly refer to cell transplantation involved with cell isolation, sorting, culture expansion, and transplantation. Nevertheless, how to modulate the outcome of TSPCs in tissue regeneration engineering remains a challenge. Next, we will introduce several approaches that have been applied to regulate the behavior of TSPCs in vitro and in vivo.

Growth factors Treating TSPCs with growth factors also facilitates in vitro TSPC phenotype maintenance. Growth factors are a group of naturally occurring cytokines that regulate a variety of cellular processes such as growth, proliferation, and differentiation. Current attempts focus on the application of growth factors to induce the differentiation of cells to a specific lineage. The main growth factors that affect the growth and differentiation of tendon tissue include connective tissue growth factor (CTGF) (Lee et al., 2015; Sharma et al., 2011), basic fibroblast growth factor (Molloy et al., 2003; Oryan and Moshiri 2014), transforming growth factor-b (TGFb) (Molloy et al., 2003), growth and differentiation factor (GDF) (Hogan et al., 2011), and insulin-like growth factor-1 (IGF1) (Molloy et al., 2003). Here we mainly discuss two currently quite debatable growth factors: CTGF and prostaglandin E2 (PGE2). CTGF is highly expressed at the early stage of tendon repair (Chen et al., 2008). It was associated with the formation of fibrosis in a range of pathological conditions (George and Tsutsumi 2007), so that CTGF was once regarded as a positive factor in tendon scar formation after injury. But a 2015 study showed that delivery of CTGF to the injury site can enrich the endogenous TSPCs, followed by tenogenic differentiation via FAKeERK1/2 signaling, ultimately promoting better regeneration of transected rat tendon (Lee et al., 2015). And pretreatment of TSPCs with CTGF and ascorbic acid before cell transplantation similarly promotes the rate and quality of tendon repair after injury (Lui et al., 2016). The contradictory conclusion indicates that our existing cognition is limited. In addition, an experimental study reports that GDF-5 initiates the transition of TSPCs into tenocytes, TGFb1 supplementation induces differentiation along multiple pathways, and IGF1 could preserve the multipotency of TSPCs (Holladay et al., 2016). PGE2 is a primary mediator of pain and acute inflammation. By inducing osteogenic differentiation of TSPCs via bone morphogenetic protein-2 (BMP-2) (Zhang and Wang, 2012), PGE2 treatment results in degenerative changes in the tendon characterized by tissue calcification (Liu et al., 2014). Moreover, PGE2

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can give rise to lipid accumulation in the tendon, because IGF1 is also increased when TSPCs are treated with PGE2 (Liu et al., 2014). Together with BMP-2, IGF1 prominently enhances the adipogenic differentiation of TSPCs via the cAMP/PKA/CEBPd pathway (Liu et al., 2014). Still, different concentrations of PGE2 may have different influences on tendon tissues (Zhang and Wang, 2014). Although mechanical loading induces high expression of PGE2 (Rui et al., 2011), a foundation level of PGE2 is detected in the normal mouse tendon (Zhang and Wang, 2010b). In vitro, cell proliferation is impaired and tenogenic differentiation is weakened when TSPCs are treated with high concentrations of PGE2 (>1 ng/mL), but low levels of PGE2 (