Frontiers in Stem Cell and Regenerative Medicine Research [1 ed.] 9781608059942, 9781608059959

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Frontiers in Stem Cell and Regenerative Medicine Research (Volume 1) Editors

Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK &

Shazia Anjum Department of Chemistry Cholistan Institute of Desert Studies The Islamia University of Bahawalpur Pakistan

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CONTENTS Preface

i

List of Contributors

ii

CHAPTERS 1.

Safety Assessment of Mesenchymal Stem Cells in Musculoskeletal Implantation

3

Greg Asatrian, Angel Pan, Michelle A. Scott and Aaron W. James

2.

Strategies to Improve Immune Reconstitution After Haematopoietic Stem Cell Transplantation

30

Guy Klamer, Shlvie Shen, Ning Xu, Tracy A. O’Brien and Alla Dolnikov

3.

Alternative Biomaterial Substrates for Human Embryonic Stem Cell Culture

65

Deepak Kumar, Ying Yang and Nicholas R. Forsyth

4.

Frontiers in Regenerative Medicine for Cornea and Ocular Surfaces

92

Maria P. De Miguel, Ricardo P. Casaroli-Marano, Nuria Nieto-Nicolau, Eva M. MartínezConesa, Jorge L. Alió del Barrio, Jorge L. Alió, Sherezade Fuentes and Francisco ArnalichMontiel

Subject Index

139

The designed cover image is created by Bentham Science and Bentham Science holds the copyrights for the image.

i

PREFACE Research on stem cells is progressing with leaps and bounds, transforming the face of medicine as it will be practiced tomorrow. This is reflected in the award of a Nobel Prize to Gurdon and Yamanaka for their seminal contributions in stem cells research. The first volume of ‘Frontiers in Stem Cell and Regenerative Medicine Research’ features reviews written by experts in important areas of stem cells and regenerative medicine. In the opening chapter James et al. describe the safety assessment of mesenchymal stem cells (MSC) in musculoskeletal implantation that can bridge the gap between translation from animals to humans. Current practices and techniques are briefly presented across three common types of MSC differentiation: bone, cartilage, and muscle tissue. The authors have emphasized the importance of in depth safety assessments in MSC implantation. The review explores the current laboratory practices for the safety assessments of MSC implantation from several angles such as histopathology, cytopathology and cytogenetics. Dolnikov et al. in chapter 2 shed light on the prevalent strategies to improve immune reconstitution after haematopoietic stem cell transplantation. Immunologic reconstitution is critically important for the successful outcome of haematopoietic stem cell transplantation. This is because chemotherapy and pre-transplant conditioning impairs thymic function. This can lead to delayed T-cell regeneration, increased risk of opportunistic infections and relapse in cases of leukaemia. Administration of γ-chain cytokines such as IL-2, IL-7 and IL-15 can promote immune reconstitution. Moreover, cell therapy appears to be a promising therapeutic approach to improve immune reconstitution after transplantation. Forsyth et al. in the next chapter discuss the use of alternative biomaterial substrates for human embryonic stem cell culture. These present exciting opportunities for tissue engineering and regenerative medicine. The authors emphasise the use of nanofibrous substrates as an alternative tool for the expansion and differentiation of embryonic stem cells. In future, such technologies could promote the use of hESC-derived cells for clinical applications Finally Miguel et al. discuss the applications of regenerative medicine for repair of damaged cornea and ocular surfaces. The novel techniques that have been presented will help to treat patients with autologous grafts, and can prevent the use of precious corneal stem cells. Both limbal and extraocular stem cells have been tested clinically. These techniques for tissue engineering of functional corneal equivalents represent a new and fascinating way to treat corneal diseases. We would like to express our sincere thanks to the editorial staff of Bentham Science Publishers, particularly Ms. Maria Baig and Mr. mahmood Alam for their constant help and support. Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK

Shazia Anjum Department of Chemistry Cholistan Institute of Desert Studies The Islamia University of Bahawalpur Pakistan

ii

List of Contributors Aaron W. James

Department of Pathology and Laboratory Medicine; University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, California 90095, USA

Angel Pan

Department of Pathology and Laboratory Medicine; University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, California 90095, USA

Deepak Kumar

Institute of Science and Technology in Medicine, University of Keele, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, UK

Alla Dolnikov

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia

Eva M. MartínezConesa

Transplant Services Foundation (TSF) at Hospital Clinic de Barcelona, Spain

Francisco ArnalichMontiel

Ophthalmology Department, Ramon y Cajal Hospital, Madrid, Spain

Greg Asatrian

Department of Pathology and Laboratory Medicine; University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, California 90095, USA

Jorge L. Alió

Vissum Ophthalmological Institute and Miguel Hernandez University, Alicante, Spain

Jorge L. Alió del Barrio

Ophthalmology Department, Ramon y Cajal Hospital, Madrid, Spain

Guy Klamer

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia

Maria P. De Miguel

Cell Engineering Laboratory, IdiPAZ, La Paz Hospital Research Institute, Madrid, Spain

Michelle A. Scott

Department of Pathology and Laboratory Medicine; University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, California 90095, USA

Nicholas R. Forsyth

Institute of Science and Technology in Medicine, University of Keele, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, UK

iii

Nuria Nieto-Nicolau

Transplant Services Foundation (TSF) at Hospital Clinic de Barcelona, Spain

Tracy A. O’Brien

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia

Ricardo P. CasaroliMarano

Transplant Services Foundation (TSF) at Hospital Clinic de Barcelona, Spain and Department of Surgery, School of Medicine at University of Barcelona, Barcelona, Spain

Sylvie Shen

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia

Sherezade Fuentes

Cell Engineering Laboratory, IdiPAZ, La Paz Hospital Research Institute, Madrid, Spain

Ning Xu

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia

Ying Yang

Institute of Science and Technology in Medicine, University of Keele, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, UK

Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 1, 2015, 3-29

3

CHAPTER 1 Safety Assessment of Mesenchymal Stem Cells in Musculoskeletal Implantation Greg Asatrian, Angel Pan, Michelle A. Scott and Aaron W. James* Department of Pathology and Laboratory Medicine; University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, California 90095, USA Abstract: There has been a rapid increase in our understanding in the isolation, culture and application of mesenchymal stem cells (MSC). Despite an increased understanding of MSC biology and new avenues for in vivo application - the standardization of laboratory practices for MSC is lacking. One particular example is in the examination of safety issues in MSC in vivo implantation. The following review will explore the current laboratory practices for the safety assessments of MSC implantation, from diverse viewpoints of such as histopathology, cytopathology, and cytogenetics. A snapshot of current practices and techniques is presented across three common types of MSC differentiation: bone, cartilage, and muscle tissue. Overall, we uncovered a relative lack of investigation of safety issues in MSC implantation. For example, cell proliferation and local inflammation were only assessed in less than one third of manuscripts. Additionally, the average length of study was less than two weeks, a short period limited in its detection of adverse outcomes. The present review uncovers a relative paucity of papers that place emphasis on safety outcomes for animal studies. Given the potential role of MSC in sarcomagenesis and other tumorigenesis, the routine performance safety assays for MSC mediated tissue engineering studies will facilitate the future translation to clinical use. Finally, we provide a set of practical preliminary suggestions is presented for safety assessments in MSC implantation models. In summary, in order to bridge the gap in translation from animal to human, increased practice and routinization of safety assessments in MSC implantation will be beneficial.

Keywords: Bone engineering, cartilage engineering, chondrogenesis, mesenchymal stem cells, MSC, muscle engineering, myogenesis, osteogenesis, safety, sarcomagenesis, stem cells, tissue engineering, toxicity, tumorigenesis INTRODUCTION The use of stem cells in tissue engineering has grown exponentially in the past *Corresponding author to Aaron W. James: Department of Pathology & Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, 10833 Le Conte Ave., Rm. A3-251 CHS, Los Angeles, California 90095, USA; Tel: (415) 860-2815; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds) All rights reserved-© 2015 Bentham Science Publishers

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decades. The application of pluripotent stem cells (embryonic and induced pluripotent stem cells), has shown great promise as they possess unlimited differentiation potential, however numerous investigators have reported the adverse safety consequence of teratoma formation with pluripotent stem cell use [1, 2]. Therefore, recent studies have sought to investigate the use of mesenchymal, multipotent, stem cells (MSC) for tissue regeneration. While many laboratory groups have convincingly shown that MSC can restore form and even function of injured mesenchymal tissues, the issue of safety is less commonly explored. Sporadic concerns of MSC safety have been reported throughout the literature. For example, culture propagation of MSC (as with many primary cell types) introduces the risk of genetic instability and tumorigenicity [3]. These findings are commonly observed in murine cells, and may or may not similarly be observed in cells of human derivation. Multiple independent investigators have found that long-term culture of murine bone marrow mesenchymal stem cells (BMSC) can lead to malignant transformation [4-6]. This is attributable to the gross chromosomal alterations that are observed in the MSC after two - three passages [7]. Typically, these transformations induce sarcoma formations, which are neoplasms of mesenchymal origin. Interestingly, sarcomas share several surface markers with MSC, including expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled-4), and CD271 [8, 9]. Recently, a close correlation has been observed in vitro whereby BMSC expressing increased levels of CD34, and dysregulated Notch, Hedgehog and Wnt signaling, induce sarcoma formation [10]. Moreover, even short-term culture of mouse BMSC has been reported to result in chromosomal modification and malignant transformation, leading to in vivo sarcoma formation [11]. As early as passage four, genetically unmodified MSC were observed to undergo chromosomal mutations, and induce sarcoma formation when transplanted in vivo. Likewise, another research group found that spontaneous transformation among rat BMSC occurred as early as passage 3, although no tumorigenic potential was found in this study [12]. Human MSC have previously been reported to undergo similar spontaneous transformation after long-term culture, although this finding has been later retracted citing possible cell contamination [13]. A second group was not able to

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reproduce these findings in human MSC, and so the relative risk of malignant transformation among human MSC remains unknown [14]. The in vivo use of MSC, however, is by no means without risk of sarcoma formation. The basic biological cell origins of sarcomas remain uncertain, and MSC and/or perivascular stem cells (PSC) themselves remain a candidate [15]. As previously mentioned, recent studies have indicated that advanced soft-tissue sarcomas bear immunophenotypic similarities to corresponding mesenchymal precursors [8, 9]. Such an immunophenotypic similarity is also observed in pericytes, a cell population surrounding blood vessels, which have been shown to exhibit MSClike potential [9, 16]. Additionally, these MSC-based sarcomas are associated with drug resistance and metastasis, and therefore may be attributable to frequent relapses seen in sarcoma patients [17-19]. Provided the evidence linking MSC and sarcomas, researchers are currently developing MSC-based sarcoma models to better understand the underlying mechanisms of initiation and progression of mesenchymal tumors [20]. What remains uncertain however is whether malignant tumors adopt a phenotype similar to that of MSC, or conversely if MSC themselves are in fact the cellular origin of sarcomas. The role of MSC in other types of tumor progression, including carcinoma progression, may also be an indirect phenomenon [21, 22]. It has been reported that MSC localize where cytokines such as chemokine (C-C motif) ligand 25 (CCL25) and hepatocyte-growth factor (HGF) are secreted and are present in high concentrations [23, 24]. Carcinomas secrete high concentrations of these cytokines, and this explains a mechanism by which MSC migrate toward sites of carcinoma growth [25, 26]. In addition, evidence suggests that MSC play a role in tumor neovasculogenesis. Tumor-elaborated VEGF (vascular endothelial growth factor) resulted in paracrine stimulation of capillary-like structure formation [27, 28]. Furthermore, studies have implicated BMSC not only in tumor proliferation and neovasculogenesis, but also in the metastasis of primary breast cancer to bone [29]. This potential role of MSC in carcinoma metastasis has also been observed in other neoplasms including colon [30] and ovarian cancer [31]. Thus and in summary, MSC are known to actively migrate to sites of carcinoma growth, induce tumor neovascularization, and also may play a role in tumor metastasis.

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In a 2006 review, Gould Halme and Kessler highlighted key safety and efficacy regulations in existence that the Food and Drug Administration (FDA) has outlined [32]. As a class of therapeutic agents, stem cell-based products meet the definition of numerous regulated products including: biologic product, drug, device, xenograft product, and human cells, tissues and cellular and tissue-based products. Moreover, such biologics are also subject to Public Health Safety Act, Section 351, regulating the licensure of such products. As such the authors raised key questions which must be answered prior to FDA submission for clinical studies. Questions such as, but not limited to, whether the product can infect the recipient with transmittable diseases from the donor, and whether the cells are pure and efficacious. Lastly, the authors brought light to the fact that as stem cells possess self-renewal potential, they also bear the risk of tumorigenesis. Although Gould Halme and Kessler provided an excellent review of FDA regulations of stem cell based therapies, a list of routine preliminary suggestions on how to properly assay for neoplastic growth was absent. As an apparent paucity is present regarding the basic safety of human MSC for tissue engineering, we seek to 1) provide an overview of the what is known and unknown regarding the safety of MSC transplantation, 2) take a ‘snapshot’ of the current practices in evaluation of safety parameters for MSC use, and 3) give a practical set of preliminary suggestions for the evaluation of safety parameters in preclinical studies. We focused on experimental studies that examined the local application of MSC in three common tissue engineering settings: the formation / regeneration of bone, cartilage and muscle tissue. Seventy-five recently published studies were analyzed, in order to assess the frequency with which basic safety assessments were made. Overall, we discovered a relative infrequency of investigation of safety issues in MSC implantation. Finally, we provide recommendation for a set of facile and inexpensive methods for assessment of basic safety issues. Increased practice and routinization of safety assessments will increase our knowledgebase of the risks in MSC use, but also help bridge the gap in translation from animal to human studies. MATERIALS AND METHODS A literature review was performed using PubMed, for any study pertaining to the in vivo application of MSC for the formation of bone, cartilage or muscle tissue.

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The following PubMed search terms were used: “mesenchymal stem cell” AND “in vivo” AND “bone” OR “cartilage” OR “muscle.” As the field of stem cell biology is rapidly evolving, the inclusion of the most recent articles 25 articles pertaining to each tissue type (i.e. bone, cartilage and muscle) were determined to provide a reflection of the most current research practices most recent research findings. Exclusion criteria included the use of any differentiated cell type (osteoblasts, myocytes, or chondrocytes), in vitro study only, or intravenous rather than local implantation. Recorded aspects of the study design included the cell type (site and species of derivation), the animal model type (species, immune status, site of implantation, study length). Recorded safety assessments included inflammation, cellular proliferation, presence of cytologic or cytogenetic abnormalities, and tumor formation. The percentage of articles that assessed each safety outcome was calculated, as well as the details of each finding. RESULTS Safety Assessments in MSC Osteogenesis Studies Taken as a whole, the studies examining the in vivo application of MSC for osteogenic differentiation have large similarities in study design (Fig. 1) [33-57]. Nearly all investigators study bone marrow mesenchymal stem cells (BMSC, 88%) of either human (40%) or murine origin (44%) (Fig. 1a, b). The majority of experiments were performed in mouse or rat (76%), while the minority were performed in larger animals including rabbit, sheep and dog (24%) (Fig. 1c). MSC application was split relatively evenly between ectopic (48%) and orthotopic (bone defect, 52%) models (Fig. 1f). Ectopic bone models were most often of a subcutaneous location (36% of total), while the second most common was an intramuscular location (12% of total) (Fig. 1e). The mean length of study time was 2 months (median length of study: 2 months). In 24% of studies (6/25), an attempt was made to distinguish the donor from the transplanted cells, either via fluorescent labels prior to implantation, or by immunohistochemical detection of species specific proteins. Among studies involving MSC mediated bone formation, safety assessments were evaluated both infrequently and non-uniformly, being investigated in only 76% of

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Figure 1: Recent studies in MSC bone formation. Articles range 01/2012 to 06/2012. (a, b) Cell types used, stratified by site of derivation and species and presented as percentage of total articles. (c-e) Animals used, stratified by (c) species, (d) location of MSC implantation, and (e) ectopic versus orthotopic implantation site and presented as percentage of total articles. (f) Percentage of articles assessing any safety parameters, and proliferation, cytology, inflammation, cytogenetics and tumor formation, specifically. Please note that data only indicates if the assessment was performed, rather than the specific findings. (See Table 1 for details).

articles (19/25) (Fig. 1f). Cellular proliferation was among the most commonly assessed marker of safety (examined in 44% of articles, 11/25). However proliferation was used more commonly as a proxy for stem cell engraftment, survival or persistence, rather than for safety. Cell proliferation was assessed most

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commonly by BrdU incorporation or MTT assays, and was noted to be increased among MSC treated samples in 73% of those studies in which proliferation was examined (8/11). Cytologic detail was recorded in 52% of studies (13/25). Normal fibroblastic and osteoblastic cell morphology was most often observed, while other benign findings included multinucleated giant cells or osteoclasts. No cases of cytologic atypia were identified. The presence of inflammation was assessed in 28% of studies (7/25), assessed only by routine H&E staining. No studies reported any increase in inflammatory cell infiltration, or other microscopic findings. Of note for the evaluation of inflammation, 36% of studies used immunocompromised animals (9/25). Cytogenetic and tumor formation were rarely assessed, in only 1/25 articles for each. Overall, basic assessments of safety of MSC implantation were not examined on a routine basis (depending on the marker from 4-52% of studies examined). Of those studies that did assess a marker of safety, none reported an abnormal finding. A comprehensive breakdown of each rubric of safety examined by article is presented in Table 1. Safety Assessments in MSC Chondrogenesis Studies Studies examining the in vivo application of MSC for chondrogenesis also have similarities to bone studies, in respect to stem cells studied and safety assays performed (Fig. 2) [58-82]. Like studies in bone tissue engineering, the majority of studies were performed in bone marrow mesenchymal stem cells (BMSC, 92%) of either human (28%) or rabbit origin (40%) (Fig. 2a, b). The majority of experiments are performed in rabbit (54%) or murine (23%) models, while the minority were performed in larger animals including pig, sheep, and goat (23%) (Fig. 2c). MSC application was most often in femoral condylar defects (84%), while ectopic cartilage formation was studied most commonly in a subcutaneous model (12%) (Fig. 2d). The mean length of study time was 2.9 months (median length of study: 2 months). 44% (11/25) of studies attempted to distinguish the donor from the transplanted cells, either via pre-implantation fluorescent labeling, donor-host gender mismatch, or species specific immunohistochemistry.

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Table 1: Review of studies in MSC bone formation and safety assessments Cell Type First Author (PMID#)

Publication Cell Species Year TypeA

Kang (23000587)

2012

Elbackly (22849574)

Animal Model

Safety Assessments Performed

ImmunoIncreased Increased Cytologic Cytogenetic Tumor Sorted Anatomic Study Proliferation Inflammation Species suppression Abnormalties Formation Abnormalities (Y/N) Location Period (Y/N) (Y/N) (Y/N) (Y/N) (Y/N) (Y/N)

ASC, BMSC

Y

Dog

N

Radial Defect

5 mo.

Y

N

Y

N

N

2012

Rabbit BMSC

N

Rabbit

N

Ulnar Defect

2 mo.

Y

N

Y

N

N

Wen (22841430)

2012

Human BMSC

N

Rat

N

Calvarial Defect

2 mo.

Y

N

Y

N

N

Wang (22807243)

2012

Rat

BMSC

N

Rabbit

N

Femoral Defect

3 mo.

Y

Y

N

N

N

Florcyzk (22767533)

2012

Rat

BMSC

N

Rat

N

Gluteus Muscle

2 mo.

N

Y

N

N

N

Domev (22731654)

2012

Human BMSC

Y

Mouse

Y

Dorsal Subcutis

1 mo.

Y

N

Y

Y

N

Li (22729020)

2012

Human BMSC

N

Dog

N

Femoral Muscle

3 mo.

N

Y

N

N

N

Mizrahi (22717741)

2012

Pig

BMSC

Y

Mouse

Y

Thigh Muscle

1 mo.

N

N

N

N

N

Wang (22700033)

2012

Rat

BMSC

Y

Rat

N

Femoral Defect

2 mo.

Y

N

Y

N

N

Lu (22698726)

2012

Human BMSC

N

Mouse

Y

Dorsal Subcutis

1 mo.

N

N

N

N

N

Zeitouni (22553253)

2012

Human BMSC

N

Mouse

N

Calvarial Defect

1 mo.

N

Y

Y

N

N

Jin (22538727)

2012

BMSC

Y

Rat

N

Calvarial Defect

1 mo.

N

N

N

N

N

Seebach (22507568)

2012

Human BMSC

Y

Rat

Y

Femoral Defect

2 mo.

N

Y

N

N

Y

Dog

Rat

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Table 1: contd… Osugi (22443121)

2012

Rat

BMSC

N

Rat

N

Calvarial Defect

2 mo.

Y

Y

N

N

N

Park (22420633)

2012

Human

ABC

Y

Mouse

Y

Dorsal Subcutis

2 mo.

N

N

Y

N

N

Carroll (22403399)

2012

Mouse BMSC

N

Mouse

N

Femoral Defect

2 mo.

N

N

N

N

N

Kumar (22342795)

2012

Rat

BMSC

N

Mouse

N

Tibial Defect

2 mo.

Y

N

N

N

N

Hu (22333987)

2012

Rat

BMSC

N

Rabbit

N

Femoral Defect

3 mo.

N

N

Y

N

N

Li (22331603)

2012

Rat

BMSC

N

Rat

N

Calvarial Defect

2 mo.

N

N

Y

N

N

Kang (22313966)

2012

Dog

ASC

N

Dog

N

Dorsal Subcutis

3 mo.

Y

N

Y

N

N

Barhanpukar (22293197)

2012

Human BMSC

Y

Mouse

Y

Dorsal Subcutis

2 mo.

N

N

N

N

N

Cheng (22287558)

2012

Human BMSC

N

Mouse

N

Dorsal Subcutis

1 mo.

Y

N

Y

N

N

Visser (22261233)

2012

Rat

BMSC

N

Rat

N

Dorsal Subcutis

1 mo.

N

N

N

N

N

Gomide (22260840)

2012

Rat

BMSC

Y

Rat

N

Dorsal Subcutis

2 mo.

Y

Y

Y

N

N

Ye (22250840)

2012

Human BMSC

Y

Mouse

Y

Dorsal Subcutis

2 mo.

N

N

Y

N

N

A: Bone Marrow Stromal Cell (BMSC), Adipose Stromal Cell (ASC), Alveolar Bone Derived Stromal Cell (ABC) Search Terms: Mesenchymal Stem Cells, Bone, In Vivo

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Figure 2: Recent studies in MSC cartilage formation. Articles range 03/2010 to 06/2012. (a, b) Cell types used, stratified by site of derivation and species. (c, d) Animal models used, stratified by (c) species, and (d) location of MSC implantation. (e) Percentage of articles assessing safety parameters. Please note that data only indicates if the assessment was performed, rather than the specific findings. (See Table 2 for a complete listing of articles).

The lack of routine safety assessments for studies involving MSC chondrogenesis was equally apparent, being assayed in 80% of articles (20/25) (Fig. 2e). Cellular proliferation was assessed in 36% of articles (9/25). Again, cell proliferation was also considered as a proxy for stem cell engraftment or persistence. Cell proliferation was noted to be increased with MSC transplantation in 78% of those

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studies in which proliferation was examined (7/9). Cytologic morphology was assessed in 40% of studies (10/25). Morphologic assessments most often yielded fibroblastic and more rounded typical chondrocytic cells, while no cytologic atypia was reported. The degree of inflammation was assessed in 48% of studies (12/25), assessed only by routine H&E staining. All studies reported either an absence of inflammation or a reduced inflammatory response with MSC implantation, as assessed by gross examination, routine histology, and rarely by lymphocyte immunostaining. Of note for the evaluation of inflammation, 28% of the studies used immunocompromised animals (7/25). Cytogenetic and tumor formation were rarely assessed, in only 8% and 4% of articles, respectively. No abnormal findings in either assessment were reported. Overall and in summary, basic assessments of safety of MSC implantation were not examined on a routine basis (depending on the marker from 4-48% of studies examined). Of those studies that did assess a marker of safety, none reported an abnormal finding. A comprehensive breakdown of each rubric of safety examined by article is presented in Table 2. Safety Assessments in MSC Myogenesis Studies Unlike efforts in cartilage and bone regeneration, those examining the in vivo application of MSC for myogenic differentiation have disparate targets of investigation (Fig. 3) [11, 56, 83-105]. The majority of studies were performed in bone marrow mesenchymal stem cells (BMSC, 85%), while other cell types included adipose derived stem cells (ASCs) and synovial membrane derived stem cells (15%). Cell origin was most often of murine (64%) or human derivation (28%) (Fig. 3a, ̀b). The great majority of experiments were performed in murine models (92%), while the minority were performed in larger animals including rabbit and dog (8%) (Fig. 3c). The type of regenerated muscle was diverse, including cardiac muscle (48%), skeletal muscle (36%), and smooth muscle (16%). Overall, the vast majority of studies examined intramuscular muscle regeneration (92%), while a minority studied ectopic myogenesis (8%) (Fig. 3d). The mean length of study time was 0.92 months (median length of study: 1 month). In 48% (12/25) of studies, an attempt was made to distinguish the donor from transplanted cells, either via MSC fluorescent labeling prior to implantation, or species specific immunohistochemistry.

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Table 2: Review of studies in MSC cartilage formation and safety assessments Cell Type First Publication Author Species Year (#PMID) Xie (22818985) Zhang (22788986) Lee (22765885) Lee (22491215) Fernandez (22406527) Huang (22384246) Tay (21917609) Yang (22133004) Acosta (21910592) Chang (21630328) Marquass (21527412) Dashtdar (21445989) Aulin (21394931)

Cell TypeA

Animal Model ImmunoSorted Species suppression (Y/N) (Y/N)

LocationA

Safety Assessments Performed Increased Increased Cytologic Study Cytogenetic ProliferationInflammation Abnormalities Period Abnormalties (Y/N) (Y/N) (Y/N) (Y/N)

Tumor Formation (Y/N)

2012

Rabbit

BMSC

Y

Rabbit

N

FCD

2 mo.

Y

N

N

Y

N

2012

Human

BMSC

N

Rat

N

FCD

1 mo.

N

Y

Y

N

N

2012

Rabbit

Synovial Membrane Derived Stem Cell

N

Rabbit

N

FCD

1 mo.

Y

Y

Y

N

N

2012

Human

BMSC

N

Rat

Y

FCD

1 mo.

Y

N

Y

N

N

2012

Rabbit

BMSC

N

Rabbit

N

FCD

1 mo.

N

Y

N

N

N

2012

Human

BMSC

N

Mouse

Y

Flank Subcutis

2 mo.

Y

N

N

N

Y

2011

Rabbit

BMSC

N

Rabbit

Y

FCD

3 wks.

N

N

N

N

N

2011

Human

BMSC

N

Mouse

N

Dorsal Subcutis 4 mo.

N

N

N

N

N

2011

Pig

BMSC

N

Pig

N

2 mo.

N

Y

N

Y

N

2011

Pig

BMSC

N

Pig

N

FCD

6 mo.

N

N

N

N

N

2011

Sheep

BMSC

N

Sheep

N

FCD

1 yr.

N

Y

N

N

N

2011

Rabbit

BMSC

N

Rabbit

N

FCD

3 wks.

N

N

N

N

N

2011

Rat

BMSC

N

Rabbit

N

FCD

1 mo.

N

Y

Y

N

N

Vertebral Disc

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Table 2: contd… Cao (21377725) Shaflee (21887742) Zhu (21362490) Park (21122912) Chen (20972620) Pec (20927712) Miller (20851201) Wang (20822812) McCarty (20721323) Shimaya (20633668) Zscharnack

2011

Rabbit

BMSC

N

Rabbit

N

FCD

3 mo.

Y

N

N

N

N

2011

Rabbit

BMSC

N

Rabbit

N

FCD

3 mo.

Y

N

N

N

N

2011

Goat

BMSC

N

Goat

N

FCD

6 mo.

N

Y

N

N

N

2011

Human

BMSC

N

N

FCD

1 mo.

Y

Y

Y

N

N

2011

Rabbit

BMSC

N

Rabbit

N

FCD

6 mo.

Y

N

Y

N

N

2010

Rat

BMSC

N

Rabbit

N

FCD

3 mo.

Y

Y

Y

N

N

2010

Rabbit

BMSC

N

Rabbit

N

FCD

3 mo.

N

Y

N

N

N

2010

Rabbit

BMSC

N

Rabbit

N

FCD

3 mo.

N

Y

N

N

N

2010

Sheep

BMSC

N

Sheep

N

Tibial Condylar 1 mo. Defect

N

N

Y

N

N

2010

Human

BMSC

N

Rabbit

N

FCD

1 mo.

N

N

Y

N

N

2010

Sheep

BMSC

N

Sheep

N

FCD

4 mo.

N

Y

Y

N

N

2010

Human

ASC

N

Mouse

N

Dorsal Subcutis 4 mo.

N

N

N

N

N

Mouse; Rabbit

(20508078) Jung (20489452)

A: Bone Marrow Stromal Cell (BMSC), Adipose Derived Stromal Cell (ASC), Femoral Condylar Defect (FCD) Search Terms: Mesenchymal Stem Cells, Cartilage, In Vivo

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Figure 3: Recent studies in MSC muscle formation. Articles range 10/2009 to 07/2012. (a, b) Cell type used, stratified by site of derivation and species. (c-e) Animals used, stratified by (c) species, (d) location of MSC implantation, and (e) ectopic versus orthotopic implantation site. (f) Percentage of articles assessing safety parameters. Please note that data only indicates if the assessment was performed, rather than the specific findings. (See Table 3 for details).

Of all areas of MSC application discussed so far, safety assessments in myogenesis were least often assessed (76% of articles, or 18/25) (Fig. 3e). For example, cellular proliferation was assessed in only 16% of articles (4/25), and sporadically observed to be increased with MSC transplantation in 2/4 studies.

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Next, cytologic morphology was assessed in 48% of studies (12/25). In one instance, cytologic features of malignancy were identified, including hypercellularity, nucleoli, cytologic atypia with increased mitotic figures [11]. The degree of inflammation was assessed in 24% of studies (6/25), usually in the context of iatrogenic muscle injury. No increase in inflammation was observed with MSC implantation. Of note for the evaluation of inflammation, 20% of the studies used immunocompromised animals. Cytogenetic and tumor formation were rarely assessed, in only 8% and 4% articles, respectively. In the same study showing samples with cytologic atypia, multiple chromosome abnormalities were identified [11]. Overall and in summary, basic assessments of safety of MSC implantation were not examined on a routine basis (depending on the marker from 4-48% of studies examined). Abnormal findings were reported in 4% of studies in which assessments were performed (1/25). This study found hypercellularity, malignant cytologic atypia, and sarcoma formation. A comprehensive breakdown of each rubric of safety examined by article is presented in Table 3. All findings in aggregate are shown in Fig. 4, incorporating all 75 articles in each of the three domains of MSC mediated osteogenesis, chondrogenesis and myogenesis. Overall, at least one safety study was performed in 76% of articles, (57 of 75). Cellular proliferation, cytology, and inflammation were the safety characteristics most commonly assessed, in 32%, 46.7, and 33.3% of articles, respectively. Cytogenetics and tumor formation were infrequently assessed, ranging from 4 - 6.7% of articles.

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Table 3: Review of studies in MSC muscle formation and safety assessments. Cell Type

Animal Model

ImmunoFirst Author Publication Cell Sorted Species Species suppression A Type (Y/N) (PMID) Year (Y/N) Jiang (22777537) Winkler (22761111) Zheng (22544360) Von Roth (22361839) Xing (22240454)

Pinheiro

De la GarzaRodea

Li

Gao (21828931) Numasawa (21755575) Shinmura (21732492)

Cytologic Cytogenetic Tumor Abnormalities Abnormalties (Y/N) Formation (Y/N) (Y/N)

(Y/N)

(Y/N)

2 wks. Skeletal

N

N

N

N

N

Y

Rat

N

GM

2012

Rat BMSC

Y

Rat

N

Soleous Muscle

1 mo.

Skeletal

N

N

N

N

N

2012

Rat BMSC

Y

Rat

N

IM

1 mo.

Cardiac

N

N

N

N

N

2012

Rat BMSC

N

Rat

N

Soleous Muscle

1 mo.

Skeletal

Y

N

Y

N

N

2012

Rat BMSC

N

Rat

N

1 mo.

Cardiac

N

N

Y

N

N

2012

Dog BMSC

N

Dog

N

Extensor Carpi Ulnaris TAM

1 mo.

Skeletal

Y

N

Y

N

N

N

Mouse

N

GM

1 mo.

Skeletal

Y

N

Y

N

N

N

Mouse

Y

3 mo.

Cardiac

N

Y

N

N

N

Cardiac Scar Tissue 2 wks. Skeletal

N

Y

Y

N

N

2012

Mouse ASC

2012

Human BMSC

ASC

(21669036)

(21667244)

Increased Inflammation

Rat BMSC

(21934652) (21874281)

Increased Proliferation

Study Muscle Period Type

2012

NitaharaKasahara

LocationA

Safety Assessments Performed

SMC

Abdominal Muscle

Tibialis Anterior Muscle

2012

Human BMSC

N

Rat

N

2011

Mouse BMSC

N

Mouse

N

IM

1 wk.

Cardiac

N

N

Y

Y

N

2011

Rat BMSC

N

Rat

N

Cardiac Muscle

2 wks.

Cardiac

N

N

N

N

N

2011

Human BMSC

N

Rat

N

IM

2 wks.

Cardiac

N

N

N

N

N

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Table 3: contd… Jeong (21493893) Tang (21345805) Ling (21344679) Cho (21233455) Qiu (21091881) Song (21173226) Huang (21098445) Behfar (20723802) De la GarzaRodea

2011

Rat BMSC

N

Mouse

N

Thigh Muscle

1 wk.

Smooth

N

Y

Y

Y

Y

2011

Human BMSC

N

Rat

N

Cardiac Muscle

1 mo.

Cardiac

N

N

N

N

N

2011

Rat BMSC

N

Rat

N

IM

1 mo.

Cardiac

N

N

Y

N

N

2011

Mouse BMSC

N

Mouse

N

IM

1 wk.

Cardiac

N

N

N

N

N

2011

Rat BMSC

N

Rat

N

1 mo.

Smooth

N

N

Y

N

N

2010

Rat BMSC

N

Rat

N

IM

2 wks.

Cardiac

N

N

Y

N

N

2010

Rat BMSC

Y

Rat

N

IM

3 wks.

Cardiac

N

Y

N

N

N

2010

Human BMSC

Y

Mouse

Y

IM

1 wk.

Cardiac

N

Y

N

N

N

2010

Human BMSC

Y

Mouse

Y

TAM

4 mo.

Skeletal

Y

N

N

N

N

2010

Mouse

Y

Mouse

N

TAM

1 mo.

Skeletal

N

N

Y

N

N

Y

Rat

N

2 wks.

Smooth

N

N

Y

N

N

1 wk.

Skeletal

N

Y

N

N

N

1 mo.

Smooth

N

N

N

N

N

Corpora Cavern-osum

(20719081) Leroux (20551912) Kinebuchi (20202003) Meng (20034794) AgaheeAfshar

2010

BMSC

Rat BMSC

2010

Human SMC

Y

Mouse

N

2009

Rabbit BMSC

Y

Rabbit

N

(19966609)

Urethral Sphincter Injury Tibial Cryodamaged Muscle External Anal Sphincter Injury

A: Bone Marrow Stromal Cell (BMSC), Adipose Stromal Cell (ASC), Synovial Membrane Stem Cells (SMC), Gastrocnemius Muscle (GM), Infarcted Myocardium (IM), Tibialis Anterior Muscle (TAM) Search Terms: Mesenchymal Stem Cells, Muscle

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Figure 4: Characterization of all recent studies in aggregate. (a, b) Cell type used, stratified by site of derivation and species. (c-e) Animal model used, stratified by (c) species, and (d) ectopic versus orthotopic implantation site. (e) The percentage of articles that did or did not assess safety parameters. Please note that data only indicates if the assessment was performed, rather than the specific findings.

DISCUSSION Our findings reveal that across areas of study in MSC-based tissue engineering, basic assessments of safety are not frequently performed. Although human MSC have not been documented to undergo malignant transformation and in vivo tumor formation, murine MSC have been shown to undergo malignant transformation with even short culture times [11]. This tumorigenic potential of culturepropagated MSC makes a striking contrast to the relative infrequency in which safety parameters are currently assessed.

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This incongruency is concerning based on our study, in which we found an increased rate of cell proliferation and sporadic frank tumor formation in the small sampling of the recent literature. For example, mild to moderately increased cellular proliferation was observed in only 23% of articles (17/75) out of the overall 32% of articles (24/75) that assessed proliferation. However this is a nonspecific finding, and the majority of these studies did not include an account of the morphologic details of the proliferating MSC. Cytologic atypia, cytogenetic abnormalities or frank tumor formation was observed in only 1.3% (1/75) of articles. While low, our review suggests that a good number of atypical findings may not have been reported, simply owing to the low rate at which they were assessed. Performing routine safety assays in preclinical studies will not only deepen our understanding of MSC biology, but also bridge the translational gap from bench to bedside application. The primary purpose of each of the 75 studies evaluated was not assessing the safety, but rather the efficacy of MSC mediated tissue engineering. Therefore, a rigorous analysis of safety outcomes from each study would be neither expected nor cost-effective. However, what can be done on a routine basis to collectively increase our understanding of the risks of MSC transplantation? Three key areas of safety assessments can be examined with minimal or no extra expense and a small amount of extra effort: cellular proliferation, cytology, and inflammation (Fig. 5). An assessment of cell proliferation can start with examining for hypercellular areas, and can be followed by a search for mitotic figures or even formal mitotic count (number of mitotic figures per high power fields). Special stains including Ki67 (MKI67, present during all phases of the cell cycle) or PCNA (Proliferating Cell Nuclear Antigen, present during DNA synthesis) can give a more easily interpretable indication of proliferation. These stains can be quantitatively expressed as the fraction of positive cells (labeling index), or simply to qualitatively and geographically identify areas of high proliferative activity. Areas of high proliferation can then be examined cytologically, for inflammation, active tissue formation, or potential cytologic atypia. Examination of cytologic detail can be performed on H&E staining and can simply examine the degree of atypia present. As sarcomas are the most common tumor type arising from MSC, cytologic features of sarcomas can be reviewed and their absence

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remarked upon [91, 93, 102, 106]. Frank invasion into surrounding can be a clear tip off for malignancy, but can be difficult to appreciate with tissue distortion of a wound environment. Interdisciplinary collaboration with clinical or research pathologists may also aid in the interpretation of histopathological findings. Finally, inflammation can be easily divided as composed of neutrophils (or polymorphonuclear) and indicating acute inflammation, or composed of mononuclear cells (lymphocytes, plasma cells, macrophages) and indicating chronic inflammation. The composition, timing and distribution of inflammation can suggest whether it is post-surgical change, or a specific reaction to implanted MSC or other foreign material. Comparison to cell-free control grafts may also help determine an etiology for any inflammation observed.

Figure 5: Recommendation Safety Parameters. Key areas of safety, cellular proliferation, cytology and inflammation are recommended to be assessed when performing studies pertaining to mesenchymal stem cells. Cellular Proliferation may be performed by assays for hypercellular areas, the presence of mitotic figures, as well as cell cycle markers Ki67 and PCNA. To evaluate cytology, H&E staining may be performed to assess cellular morphology. Lastly for inflammation, a composition for mononuclear and polymorphonuclear should be performed.

Finally, there are a few ways in which our review design may have included both sampling and reporting bias. First, the search term “mesenchymal stem cell” was chosen as a common key word used in our target studies, but may have led to both the inappropriate inclusion and exclusion certain studies. For example, many studies claiming to use “MSC” do not formally prove the identity and multipotential differentiation of cells used - items which were likewise not verified in this review. Similarly, there are many synonyms for MSC in current use, including tissue specific terms which would not have been included within this review. As another example of bias, MSC mediated tumorigenesis may have inherent reporting bias, where an unusual and dramatic outcome may be over reported in the literature (the 1 of 75 studies we encountered).

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Nevertheless, basic safety issues are relatively infrequently examined in the current literature regarding MSC mediated tissue engineering. As previously mentioned, several studies have shown a distinct link between MSC and cancer, by means of tumor initiation, progression, and metastasis. Additionally, it has been reported that sarcomas express phenotypic similarity to MSC, suggesting malignant tumors either adopt a phenotype similar to that of MSC or that MSC themselves are the origin of sarcomas. Thus, in order to bridge the gap of new scientific findings from animals to humans, increased practice and routinization of safety assessments in MSC implantation would be beneficial. ACKNOWLEDGEMENTS We would like to thank Ms. Le Chang, Ms. Georgina Ang, and Mr. Todd Rackohn for their helpful assistance. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

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Gao XR, Tan YZ, Wang HJ. Overexpression of Csx/Nkx2.5 and GATA-4 enhances the efficacy of mesenchymal stem cell transplantation after myocardial infarction. Circ J 2011; 75(11): 2683-2691. Numasawa Y, Kimura T, Miyoshi S, et al. Treatment of human mesenchymal stem cells with angiotensin receptor blocker improved efficiency of cardiomyogenic transdifferentiation and improved cardiac function via angiogenesis. Stem Cells 2011; 29(9): 1405-1414. Shinmura D, Togashi I, Miyoshi S, et al. Pretreatment of human mesenchymal stem cells with pioglitazone improved efficiency of cardiomyogenic transdifferentiation and cardiac function. Stem Cells 2011; 29(2): 357-366. Tang JM, Wang JN, Zhang L, et al. VEGF/SDF-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart. Cardiovasc Res 2011; 91(3): 402-411. Ling SK, Wang R, Dai ZQ, et al. Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction. Biotechnol Prog 2011; 27(2): 473-482. Cho J, Zhai P, Maejima Y, Sadoshima J. Myocardial injection with GSK-3betaoverexpressing bone marrow-derived mesenchymal stem cells attenuates cardiac dysfunction after myocardial infarction. Circ Res 2011; 108(4): 478-489. Qiu X, Lin H, Wang Y, et al. Intracavernous transplantation of bone marrow-derived mesenchymal stem cells restores erectile function of streptozocin-induced diabetic rats. J Sex Med 2011; 8(2): 427-436. Song H, Hwang HJ, Chang W, et al. Cardiomyocytes from phorbol myristate acetateactivated mesenchymal stem cells restore electromechanical function in infarcted rat hearts. Proc Natl Acad Sci U S A 2011; 108(1): 296-301. Huang XP, Sun Z, Miyagi Y, et al. Differentiation of allogeneic mesenchymal stem cells induces immunogenicity and limits their long-term benefits for myocardial repair. Circulation 2010; 122(23): 2419-2429. Behfar A, Yamada S, Crespo-Diaz R, et al. Guided cardiopoiesis enhances therapeutic benefit of bone marrow human mesenchymal stem cells in chronic myocardial infarction. J Am Coll Cardiol 2010; 56(9): 721-734. de la Garza-Rodea AS, van der Velde I, Boersma H, et al. Long-term contribution of human bone marrow mesenchymal stromal cells to skeletal muscle regeneration in mice. Cell Transplant 2011; 20(2): 217-231. Leroux L, Descamps B, Tojais NF, et al. Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4dependent pathway. Mol Ther 2010; 18(8): 1545-1552. Kinebuchi Y, Aizawa N, Imamura T, et al. Autologous bone-marrow-derived mesenchymal stem cell transplantation into injured rat urethral sphincter. Int J Urol 2010; 17(4): 359-368. Meng J, Adkin CF, Arechavala-Gomeza V, et al. The contribution of human synovial stem cells to skeletal muscle regeneration. Neuromuscul Disord 2010; 20(1): 6-15. Aghaee-Afshar M, Rezazadehkermani M, Asadi A, et al. Potential of human umbilical cord matrix and rabbit bone marrow-derived mesenchymal stem cells in repair of surgically incised rabbit external anal sphincter. Dis Colon Rectum 2009; 52(10): 1753-1761. Fletcher CDM, Unni KK, Mertens F. Pathology and genetics of tumours of soft tissue and bone. World Health Organization Classification of Tumors. Lyon: IARCPress 2002.

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CHAPTER 2 Strategies to Improve Immune Reconstitution Haematopoietic Stem Cell Transplantation

After

Guy Klamer1,2, Shlvie Shen1,2, Ning Xu1,2, Tracy A. O’Brien1,2 and Alla Dolnikov1,2,* 1

Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Institute, University of New South Wales, Sydney, NSW, Australia and 2 Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia Abstract: Immunologic reconstitution is a critical component for successful outcome of haematopoietic stem cell transplantation. Chemotherapy and pre-transplant conditioning impairs thymic function leading to delayed T-cell regeneration and the increased risk of opportunistic infections and leukaemia relapse. Immune reconstitution can be promoted through administration of common γ-chain cytokines such as IL-2, IL-7 and IL-15. Prevention of thymic involution achieved by administration of keratinocyte growth factor, growth hormone and sex hormone inhibition has also been shown to improve immune reconstitution. Additionally, cell therapy that includes adoptive transfer of ex vivo generated T-cells or T-cell precursors, T-cells specific for viral or tumour antigens and, natural killer (NK) cells appears to be a promising therapeutic approach to improve immune reconstitution after transplantation. Pharmacological modulation of signalling pathways, such as Wnt and Notch, play an important role during different stages of Tcell development. Activation of Wnt signalling using small molecule inhibition of GSK3β was shown to promote post-transplant T-cell regeneration in pre-clinical models. The use of pharmaceutical agents to accelerate T-cell reconstitution and boost T-cell-mediated immunity in recipients of haematopoietic stem cell grafts warrants further investigation.

Keywords: Haematopoeitic stem cell transplantation, immune reconstitution, Tcell, thymus, Wnt signaling. INTRODUCTION Haematopoeitic Stem Cell Transplantation (HSCT) is used in the treatment of *Corresponding author Ally Dolnikon: Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia; School of Women and Children's Health, University of New South Wales, NSW, Australia; Tel: 61293821879; Fax: 61293820372; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds) All rights reserved-© 2015 Bentham Science Publishers

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haematological and non-haematological disorders. In 1956, the American physician Edward Donnell Thomas performed the first successful bone marrow transplant (BMT) between genetically identical twins (syngeneic transplant) to remove the chance of graft and host-directed rejection that is caused by disparities in minor and major histocompatibility gene complexes [1]. By the 1960s enough was known about the HLA gene complex and tissue typing to perform the first successful allogeneic transplant using a non-identical donor. In 1968, the first allogeneic transplant between siblings was performed [2], and in 1969 E. Donnell Thomas performed the first BMT in a leukaemia patient using a graft from a nonidentical relative [3]. Professor Thomas’s pioneering work was rewarded with a Nobel Prize in Physiology or Medicine in 1990. In 1973, the first successful nonrelated allogeneic BMT was performed in New York’s Memorial Sloan Kettering Cancer Center on a 5 years old patient with severe combined immunodeficiency disease (SCID) [4]. In 1981, the first successful autologous HSCT using peripheral blood (PB) stem cells was performed in London [5], and in 1988 the first HSCT using umbilical cord blood (UCB) was performed in Paris on a patient with Fanconi’s anaemia [6]. The Worldwide Network for Blood and Marrow Transplantation has announced that 1 million HSCTs have been performed globally with “Europe providing 53%, the Americas 31%, Australasia 14% and Eastern-Mediterranean and Africa 2% to the total HSCT number” [7]. Autologous HSCT (auto-HSCT) is the infusion of a patient with his or her own mobilized peripheral blood stem cells that are isolated before transplant conditioning. Auto-HSCT is primarily used to treat solid tumours and some haematological malignancies such as multiple myeloma, Hodgkin and nonHodgkin lymphoma, acute myelogenous leukaemia (AML) and chronic lymphocytic leukaemia (CLL) [8, 9]. If a HSCT engrafts successfully, neutrophil and platelet recovery is observed within the first 3 weeks of transplant [10-12], however, B-cell and T-cell reconstitution can take between 6-12 months [13-15]. Delayed immune reconstitution results in a high risk of infection and disease relapse after HSCT. Infections and disease relapse account for approximately 8% and 73% of mortality in recipients of auto-HSCT, respectively. Chronic or refractory disease is associated with lower rates of survival compared to early stage disease [16]. Allogeneic HSCT (allo-HSCT) is the infusion of donor stem

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cell grafts into patients as a curative therapy for high-risk disease. In cancer patients, the allo-HSCT eliminates residual cancer cells that survive chemotherapy and transplant conditioning through adaptive immune responses mediated by donor T-cells present in the graft. This process is known as the Graft-versusTumour (GVT) or Graft-versus-Leukaemia (GVL) effect and is the primary reason why allo-HSCT is employed in preference over auto-HSCT in certain disease settings [17, 18]. Unfortunately, genetic disparities in minor histocompatibility antigens (mi-HA) and major histocompatibility (MHC) antigens that are required for initiation of cancer-directed adaptive immune responses also result in host-directed adaptive immune responses termed Graftversus-Host Disease (GVHD) that occurs in up to 80% of non-T-cell depleted allo-HSCT recipients to a certain degree after myeloablative conditioning [19]. Tissue typing is used to determine the level of MHC gene matching between donor and host, and administration of immunosuppressive drugs is used for GVHD prophylaxis, the latter of which also delays immune responses to infections and interferes with GVL responses [19, 20]. A treatment modality that permits GVL while minimizing GVHD has not been discovered. Potential strategies to induce GVL without GVHD include transplantation of haploidentical Natural Killer cells that have been shown to induce GVL in AML patients without causing GVHD [21-23]. Additionally, infusion of virus and cancer specific T-cells are being trialed as a means to improve immunity of transplant recipients (NCT00840853, NCT01430390). Donor PB, UCB and BM are all used as cell sources for allo-HSCT and although PB is the most commonly adopted, UCB HSCT is becoming increasingly common due to the emergence of international cord blood banks and advances in donor graft selection [24]. Advantages of UCB HSCT include a greater degree of tolerance for MHC gene disparity, weakened GVHD, lower risk of viral transmission, non-invasive procurement, lower donor attrition and rapid speed of availability [13]. Disadvantages of UCB include restricted stem cell doses that permit its use in a limited number of patients and slower immune reconstitution [13, 24]. Strategies such as ex vivo HSC expansion, dual cord blood transplantation, intra-bone injection and agents to improve stem cell homing to BM have been investigated to improve the outcome of UCB HSCT [24]. The

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limited stem cell dose in UCB has typically restricted its use to children, however, the introduction of dual cord blood transplantation has seen it performed in adolescents and adults. The selection of one or two cord blood units is primarily determined by the cell dose per recipient kilogram in context of MHC matching. Typically, a single unit will be used if the cell dose is ≥ 3x107/kg and a dual cord blood transplant will be employed it the cell dose falls below this value [24, 25]. Therefore, double UCB HSCT is the only treatment modality for a large proportion of patients requiring UCB HSCT. In 2011, an estimated 600.000 cryopreserved UCB units were being stored in UCB banks, and over 20.000 UCB units had been distributed around the world for the treatment of adult and paediatric haematological diseases [24]. Prior to transplant, patients receive a conditioning regimen that is either myeloablative (MA) or non-myeloablative (NMA) depending on the chemotherapy and/or total body irradiation dose. MA or NMA is chosen depending on disease type, patient co-morbidity and age. MA conditioning, which is applied to induce remission and ablate host immunity that would act to reject the graft, causes a significant amount of morbidity in patients [26]. NMA conditioning, also known as reduced intensity conditioning (RIC), is applied to induce host susceptibility to donor cell engraftment. Compared to MA, the detriment of NMA is the higher cancer burden present after conditioning that increases the risk of relapse [27, 28]. In patients with co-morbidity or in elderly patients, non-relapse mortality rates due to the toxicity of MA conditioning is high, resulting in NMA being the only feasible treatment modality [29-31]. Benefits of NMA conditioning include reduced rates of post-transplant infections due to residual host immunity after conditioning, and weakened acute GVHD due to reduced damage of gastrointestinal epithelial cells that release proinflammatory cytokines and bacterial endotoxins that exacerbate GVHD [32, 33]. NMA also attenuates donor T-cell-mediated GVL reactions that are promoted by host epithelial cell secretions [31]. New studies have demonstrated a benefit to upfront combination autologous and reduced intensity conditioning allogeneic (auto/RICallo) HSCT for multiple myeloma with improved progression free survival (PFS), overall survival (OS) and relapse rates achieved compared to upfront autologous HSCT [29]. The selection of conditioning regimen, HSCT

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source, and the level of MHC matching vary according to patient age, disease type and disease status. At the Sydney Children’s Hospital, a retrospective analysis of 136 acute lymphoblastic leukaemia (ALL) patients that received an allo-HSCT over the past 25 years revealed that OS and event-free survival (EFS) has significantly improved due to reduced transplant related mortality (TRM) (OS: 41.8% vs. 78.9%, P < 0.0001; EFS: 31.6% vs. 64.8%, P < 0.0027; TRM: 30% vs. 5%, P < 0.0004; relapse static, P = 0.07) [26]. This was despite a significant increase in the number of allo-HSCTs performed in recent times. Significant reduction of TRM was observed in recipients of matched sibling grafts and UCB grafts. The retrospective audit concluded that improved survival in the patients undergoing UCB HSCT was due to improved MHC gene matching, higher nucleated cell doses, significantly faster neutrophil engraftment, and improved supportive care. Patient survival can be improved if relapse rates can be reduced in recipients of allo-HSCT. Thus, we must focus on strategies that are able to reduce minimal residual disease and promote GVL effects without worsening GVHD outcome and severity. IMMUNE RECONSTITUTION AFTER HSCT Severe immune suppression occurs during the time it takes for graft-derived cells to reconstitute the host haematopoeitic system with innate and adaptive immune mediators. Delayed immune reconstitution accounts for a large proportion of mortality and morbidity after HSCT due to opportunistic fungal, viral or bacterial infections such as aspergillus, streptococcus pneumonia, haemophilus influenza, adenovirus (ADV), cytomegalovirus (CMV) and Epstein Bar Virus (EBV), of which the latter two commonly become re-activated after HSCT [34, 35]. T-cells are also required to mediate GVT/GVL effects required to induce or sustain remission. GVHD causes delayed and impaired immune reconstitution due to the damage it causes on the bone marrow and thymus, and the high level of cytokines that are produced by GVHD effector T-cells which result in skewed lymphocyte differentiation and induce apoptosis through death receptor signaling (e.g., TNF/TNFR, FAS/FASLG dyads) [20]. This is made evident by the fact that GVHD is an independent risk factor for CMV, Varicella zoster virus (VZV) and ADV infection/re-activation, and that most fungal infections occur during 2-3 months post-HSCT when acute GVHD occurs [36].

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The kinetics of immune cell reconstitution vary with HSC source, donor age, conditioning regimen and acute GVHD status [37]. The first cells to engraft are the neutrophils and platelets that appear in circulation after 2-4 weeks of alloHSCT. Although slightly delayed after UCB HSCT, these kinetics are fairly consistent between HSC sources and are therefore used to monitor engraftment [10-12, 20]. During the aplastic, neutropenic stage prior to neutrophil engraftment patients are highly susceptible to bacterial infections and require prophylactic antibiotics [20]. Reconstituted natural killer (NK) cells are seen in circulation within the first 60 days of HSCT [14, 37-39], however, their numbers decline over the following year [20]. Rapid NK reconstitution correlated with reduced mortality in patients that received UCB or BM HSCT [10]. T-cell responses at early stages post-HSCT are primarily mediated by donor Tcells present in the graft. T-cell-replete grafts mediate an early wave of graftderived mature T-cell reconstitution that can mediate short term immunity at the cost of an increased risk of acute GVHD, especially in the case of haploidentical HSCT (haplo-HSCT) [40]. Homeostatic proliferation of mature graft-derived donor T-cells accounts for their peripheral expansion and reconstitution. Extensive proliferation of T-cells also results in their differentiation of into late memory T-cells that can be observed at early stages post-transplant [13]. Cytokine-driven proliferation of naïve T-cells acts to increase the circulating Tcell pool only by expanding existing T-cell receptor (TCR)-specific T-cell clones and therefore does not contribute to broadening the TCR repertoire. Homeostatic proliferation of T-cells supported by cytokines produced during GVHD pathophysiology, and immunomodulatory drugs used to control GVHD, cause naïve-to-memory T-cell differentiation [20]. Late memory T-cell skewing and thymic dysfunction are common after UCB HSCT [13]. High proportions of memory cells have also been described in recipients of matched sibling and unrelated BM HSCTs [10]. Late memory T-cell skewing can increase the risk of CMV re-activation if homeostatic proliferation of a narrow range of TCR-specific clones, without high affinity for CMV peptides, is the primary driver of peripheral T-cell reconstitution. To reduce the risk of acute GVHD after haplo-HSCT, T-cell-depletive agents are often administered as part of the pre-transplant conditioning regimen (e.g., anti-

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thymocyte globulin, CD34-selection). Detrimentally, T-cell depletion (TCD) delays T-cell reconstitution compared to non-TCD HSCT [41]. Thymusdependent de novo generation of naïve T-cells is the avenue to T-cell reconstitution after TCD haplo-HSCT. Stem cell-generated T-cells contribute to immune responses at later stages post-transplant. These cells can take 6-24 months to appear in circulation at normal levels leaving patients at high risk of viral infections [37]. B-cell reconstitution occurs more rapidly in recipients of UCB HSCT compared to PB HSCT recipients with their levels remaining higher over 2 years, after which time levels can be observed above normal [14]. Stem cell homing to the BM, stem cell lymphoid differentiation, thymus seeding by lymphoid precursors that migrate from the BM, and thymopoeisis are all required for efficient T-cell reconstitution post-HSCT. The major factor determining de novo T-cell development is the status of thymus. Chemotherapy and radiotherapy applied as transplant conditioning causes damage to thymic epithelial cells (TECs) that are required for normal thymocyte differentiation and expansion. Conditioning-induced thymic damage is less severe after reduced intensity conditioning (RIC). RIC is often applied as a means to permit older patients to receive allo-grafts. In these cases, the limited damage inflicted on the thymus by RIC is counterbalanced by age-related thymic involution that is caused by sex hormone activity starting at puberty [20]. Thymic function is required for the generation of naïve T-cells with a broad TCR repertoire for protection against a wide variety of infectious diseases and disease relapse. Thymic atrophy, involution and dysfunction reduce the diversity of the TCR repertoire by reducing the rate of naïve T-cell efflux from the thymus. Cytokine-dependent homeostatic expansion of de novo generated naïve T-cells in PB can compensate for a low efflux rate of naïve T-cells from the thymus, however, as in the case of graftderived T cells, de novo generated T-cells rapidly differentiate into late memory cells following expansion in PB and may therefore display attenuated immune responses [42]. STRATEGIES TO ACCELERATE T-CELL RECONSTITUTION AFTER HSCT Humanized mice are new tools to investigate human immune cell development post-HSCT. In the last decade, genetically engineered mice with combined

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immunodeficiencies were obtained to allow efficient engraftment of human HSCs and de novo generation of essentially all immune compartments [43-45]. Even though full human reconstitution is generally not achieved, these mice have already given valuable results for a better understanding of the development of human immune cells post-transplant and, more importantly, a model to test new strategies to accelerate immune reconstitution after HSCT [46, 47]. The choice of mouse model depends on availability of mice and stem cell sources as both syngeneic and xenogeneic mouse models are established. Syngeneic mouse models of HSCT have been around longer than xenogeneic humanized mouse models as graft rejection was an issue before severely immune-compromised mice were generated for human cell transplant modeling. Injection of C57BL/6 mice with mouse-derived TCD BM results in de novo T-cell reconstitution within 3 weeks [48]. Inoculation of 1x105 human UCB-derived CD34+ HSCs results in Tcell reconstitution in irradiated severely immune-compromised NSG (NOD/SCID/IL-2rγnull ) and BALB/c-Rag2nullIL-2rγnull (BALB/c-DKO) mice. The time to mature human T-cell reconstitution in the peripheral blood of graft recipients is approximately 10-20 weeks and gradually rises over this period. Increasing stem cell dose in the graft promotes T-cell reconstitution [49]. Functional CD4+ and CD8+ T-cell subsets, as well as B-cells, are reconstituted in mice receiving purified human CD34+ HSCs [50-52]. Human foetal thymus/liver implantation under the kidney capsule of mice was also shown to result in thymopoeisis and functional mature human T-cell reconstitution in NOD/SCID mice. Co-transplantation of human thymus/liver implant with purified CD34+ HSCs improved T-cell reconstitution in this model that was observed in the periphery at low levels after 6 weeks. A weekly increase in peripheral human Tcells was recorded until week +18. Functional B-cells reconstitution was also recorded in these mice [53]. In addition, TNF-α administration also promoted human T-cell regeneration in NOD/SCID mice [54]. Using immunocompromised NSG mice we and others have shown that human HSC rapidly home to the host BM, and early lymphoid differentiation can be registered by week 6 posttransplant [50, 55]. Transplanted human stem cells acquire lymphopoietic commitment in the BM [55] and then migrate to the thymus to undergo differentiation that results in the generation of CD4+ and CD8+ single positive (SP) thymocytes that survive negative selection and enter circulation to eliminate

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infection and transformed cells [50]. Once in circulation, naïve T-cells are quiescent prior to antigen stimulation. When thymic function is impaired as a result of chemotherapy or irradiation, naïve T-cells undergo homeostatic proliferation induced by cytokines, mostly IL-7, that results in peripheral selfrenewal in order to compensate for reduced thymic output [56]. Analysis of immune regeneration in NSG mice transplanted with human UCBderived CD34+ stem cells revealed that T-cell development was delayed as compared to B-cell and myeloid lineage apparently due to irradiation-impaired thymic function. Early pro-thymocyte development in the bone marrow of human stem cell reconstituted mice peaked 4 weeks after transplant, declined at 8 weeks and persisted at very low level thereafter with only few CD34+CD7low prothymocytes with limited T-cell potential detected 20 weeks after transplant [57]. Thus, both BM-lymphoid potential and thymopoiesis are largely exhausted by week 20, and homeostatic T-cell proliferation accounts for further T-cell expansion. Single positive CD4+ and CD8+ human T-cells expressing high levels of TCRαβ and TCRγδ were readily identifiable in the thymus at week 7 posttransplant and at week 20 in PB of reconstituted mice [58]. A large proportion of T-cells displayed a naïve T-cell phenotype, and phenotypic transition from naïve to central and effector memory was observed in PB and spleen of transplanted mice (submitted paper). The naïve T-cell/effector memory T-cell ratios was higher in the CD8+ than in the CD4+ T-cell compartment, consistent with the delayed CD8+ T-cell differentiation in this mouse model lacking human HLA class 1. Only a small proportion of PB CD4+ naïve T-cells expressed CD31, a recent thymic emigrant (RTE) marker [56]. Decline in CD31 expression in CD4+ naïve T-cells is associated with robust homeostatic expansion of T-cells in circulation in human patients suggesting that homeostatic T-cell proliferation contributed to T-cell expansion in PB of mice transplanted with human stem cells. No signs of Graft-versus-Host Disease (GVHD) morbidity as determined by an established grading system were observed in xeno-grafted mice [59]. Weak xenoreactivity is likely due to thymic deletion of xeno-reactive clones. Human CD3+ T-cells derived from graft recipient mice, however, exhibited robust proliferative response to mitogenic stimulation using anti-CD3/CD28 microbeads and expressed the human Th1 cytokine TNF-α indicating functional competence.

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Strategies to accelerate T-cell reconstitution aim to improve stem cell homing to the BM, thymic function, RTE efflux, and naïve T-cell expansion in PB. Thymic output can be achieved at multiple levels, including the promotion of BM lymphoid progenitor migration to the thymus, thymocyte expansion or differentiation in the thymus, protection of TECs from conditioning-induced or GVHD-mediated damage, or promotion of TEC repair after TEC-mediated damage (Fig. 1). Routes of T-cell reconstitution and factors that delay IR Allograft HSC (1) BM (3)

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Figure 1: Routes of T-cell reconstitution and factors that delay immune reconstitution (IR).

There are several ways in which T-cell reconstitution becomes delayed after HSCT: (1) Limited HSC numbers in the allograft delays IR. (2) TCD therapy reduces donor T-cell numbers in graft and delays resultant IR. (3) Impaired BM homing and damage to the BM niche from conditioning delays HSC engraftment and resultant IR. (4) Impaired homing of T-cell precursors to the thymus delays resultant IR. (5) Damage to the thymic microenvironment from conditioning and GVHD impairs thymic output, delays de novo T-cell generation and reduces TCR repertoire in periphery.

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(6) Immunomodulatory drugs suppress expansion of donor T-cells and induce differentiation causing memory T-cell skewing. These result in delayed IR and sub-optimal immunity. (7) Presence of residual host T-cells at time of transplant act to reject HSC engraftment, T-cell reconstitution and therefore delay overall IR. Several cytokines and growth factors promoting thymic function and expansion of circulating T-cells were tested using human immune system-engrafted mice and are now in early stage clinical trials. Administration of common γ chain cytokines IL-7, IL-2 and IL-15, keratinocyte growth factor, and sex steroid antagonists were shown to promote T-cell reconstitution in xeno-grafted mice [42]. IL-7, which is normally secreted by thymic epithelial cells, is a critical regulator of thymopoeisis and homeostatic proliferation of T-cells in the periphery [60]. IL-7 has been shown to improve T-cell reconstitution in mouse recipients of HSCT through improved thymopoeisis, and increased mature T-cell expansion and survival [61]. However, these effects are counterbalanced by worsened GVHD in recipients of non-TCD HSCT but not TCD HSCT [62]. IL-7 treatment promoted homeostatic T-cell proliferation in PB, but not thymopoeisis, in non-human primates [63]. Phase 1/2a clinical trials have demonstrated that recombinant human IL-7 treatment improved thymopoietic efflux of RTEs in immunodeficient patients without toxic effects. Perales et al., [64] reported on a Phase 1 clinical trial (NCT00684008) investigating immune reconstitution in recipients of allogeneic TCD HSCTs (N = 12) treated with recombinant human IL-7 (CYT107). IL-7 boosted functional (e.g., mediated CMV immunity) CD4+ and CD8+ T-cell numbers primarily through expansion of effector memory T-cells and importantly enhanced TCR diversity. No effect on Tregs, NKs or B-cells was seen, nor was toxicity, however, acute skin GVHD did occur in 1 patient. Thus, treatment of IL-7 should be considered as a boosting agent of immune reconstitution after TCD HSCT as de novo generated T-cells undergo thymopoietic selection resulting in tolerance to host antigens. Additionally, infusion of mesenchymal stem cells (MSCs) transduced with an IL-7 gene was able to significantly improve thymopoeisis and homeostatic proliferation of Tcells in mice after TCD BM transplantation whilst protecting from GVHD [65].

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IL-2 is a common-gamma chain cytokine that binds to T-cell bound IL-2R to regulate survival and proliferation of naïve and memory T-cells [66]. The role of IL-2 in reconstitution of different haematopoeitic subsets after HSCT has been well studied and appears to be dose-dependent. Administration of low dose IL-2 promoted NK reconstitution but had no effect on T-cell numbers in recipients of auto-HSCT and TCD allo-HSCT [67]. Additionally, low-dose IL-2 promoted regulatory T-cell reconstitution that acts to mitigate GVHD in allo-HSCT recipients [68]. Improved NK reconstitution has the potential to enhance the GVL effect post-transplant, however, these effects may be counterbalanced by an immunosuppressive effect mediated by regulatory T-cells. IL-2 therapy has been shown to boost naïve and memory CD4+ T-cell counts in HIV patients treated with anti-retroviral therapy [69]. More importantly, IL-2/donor lymphocyte infusion (DLI) co-administration resulted in complete or partial response in a small group of leukaemia patients that relapsed after allo-HSCT and were refractory to DLI therapy without IL-2 [70]. There is a role for IL-2 in the promotion of immune reconstitution however the dose-dependent effects on different subsets must be determined. Clinical trials have been designed to determine the potential of IL-2 as an immunity/GVL boosting therapeutic after HSCT (NCT00003962, NCT00539695, NCT01517347). IL-15 is another gamma-common chain cytokine that regulates lymphocyte development and survival. IL-15 has been shown to preferentially expand memory T-cells and promote their migration to non-lymphoid tissues [71]. Like IL-2 and IL-7, binding of IL-15 to its receptor leads to activation of the JAK/STAT signaling pathway, STAT3 in this case. Injection of IL-15 after alloHSCT in mice led to an improved reconstitution of CD8+ T-cells and NK cells that was associated with improved T-cell function [72]. IL-15 enhanced GVL but also exacerbated GVHD in recipients of T-cell replete grafts indicative of stronger donor T-cell activity [72]. IL-15 treatment also promoted CD8+ and NK reconstitution in mouse recipients of TCD allo-HSCT however survival after cancer cell inoculation was not improved unless T-cells were inoculated with the graft [73]. There is evidence that improvements in T-cell immunity after IL-15 are transient and return to baseline or below after treatment termination. SIV-infected macaques were treated with anti-retroviral therapy with or without IL-15. The IL-

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15 cohort showed improvements in memory cell immunity however when IL-15 treatment was interrupted, CD4+ T-cells were lost more rapidly in the IL-15 than the ART therapy alone group [71]. Thus, to reap the benefits of IL-15 therapy, patients may need to receive inoculations of the cytokine for as long as the period between lymphopaenia and maximal de novo T-cell reconstitution. Moreover, IL15 may be used as a GVL boosting agent at the risk of GVHD exacerbation after non-TCD HSCT. The potential of combination IL-2, IL-15 and IL-7 treatment is being examined to improve immune reconstitution however like so many therapies that boost T-cell immunity, GVHD remains a serious side effect. Keratinocyte growth factor (KGF, FGF7) acts through the FGF receptor 2 IIIb (FGFR2IIIB) that is expressed on gut epithelial cells, hepatocytes and skin keratinocytes. KGF is produced by thymic stromal cells and thymocytes, however, its action is limited to TECs that express FGFR2IIIb. Mouse models of syngeneic and allogeneic BMT have shown that KGF is required for normal thymopoeisis. In mice, KGF administration promoted RTE efflux in recipients of TCD HSCT, and protected gut and thymic epithelium from GVHD-induced damage [42]. KGF improves post-transplant thymic architecture by exerting a protective effect on the cytoablative effects of conditioning on TECs [74, 75], induces transient expansion of TECs, and promotes immature TEC differentiation in mice. KGF signaling activates p53 and NF-κB signaling in mouse TECs and induces the expression of BMP2, BMP4, Wnt5b and Wnt10b, factors that are critically important for thymocyte development [76]. Pre-clinical testing of KGF has yielded controversial data. KGF was shown to improve haematologic recovery, thymopoeisis and T-cell reconstitution in rhesus macaques that received CD34+ selected autologous BM HSCTs. However, protection from CMV reactivation and antibody response to the tetanus toxoid vaccination was not associated with improved T-cell recovery [77]. Several other studies have demonstrated that KGF modestly improves, or has no effect, on functional immune responses [77]. When KGF and an inhibitor of P53 (PFT-β) were administered to mice before BMT, improved T-cell reconstitution was associated with protection from Listeria monocytogenes infection [78]. KGF was tested in a phase 1/2 clinical trial as a novel therapeutic for GVHD for its ability to protect epithelial tissue from damage and facilitate repair (NCT00031148). This study,

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and others like it, may yield insight into the potential of KGF to promote T-cell reconstitution in human recipients of HSCT. Sex steroid inhibition is a treatment strategy used to reverse and inhibit agerelated thymic atrophy. Gradual loss of thymic function with age is mainly due to the action of sex steroids (e.g., androgens, progesterone, oestrogen) starting from puberty. Sex steroid inhibition, through surgical castration or pharmacological inhibition, reversed the signs of age-related thymic involution and increased thymocyte and TEC cellularity. Later it was demonstrated that sex steroid inhibition improved immune reconstitution after HSCT [79-81]. Carstration in mouse recipients of allo-HSCT increased donor-derived BM and thymic cellularity early after transplantation (2-4 weeks) without exacerbation of GVHD as RTEs are tolerant to host antigens following thymopoeisis [79]. Importantly, in mouse models of infectious disease involving aged mice, sex steroid ablation improved T-cell-mediated immunity towards viral infections indicating that RTEs in these models retain functional competence [82]. In humans, administration of chemical analogues of leutinizing-hormone-releasing hormone (LHRH) that are used to treat prostate cancer, breast cancer and endometriosis have been shown to improve thymic function and immune reconstitution in recipients of autologous and allogeneic HSCT. Guinan et al., presented data from adult recipients of autologous PB HSCT (N = 25) treated with Leuprolide (LHRH analog) at the 2010 American Society of Hematology Conference. They reported that Leuprolide increased IgM and IgG1 production after keyhole limpet haemocyanin vaccination, and increased the number of naïve CD4+ TREC+ cells 6 months posttransplant. Leuprolide did not delay engraftment [83]. A pilot study is currently recruiting patients to test the potential of Leuprolide to improve immune reconstition in patients (aged 15-40) being treated with an allogeneic BM HSCT for AML, ALL or high risk myelodysplastic syndrome (NCT01338987). Additionally, a phase 2 clinical trial testing the ability of Leuprolide to preserve ovarian function in recipients of HSCT may provide data demonstrating whether this treatment modality improves immune reconstitution (NCT01343368). Sex steroid inhibition causes common side effects that include shortening of the penis (when administered in conjunction with radiation), testicle shrinking, depression, and vaginal irritation. Patient concerns regarding the long-term effects of sex

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steroid inhibition on reproductive potential should be discussed as this therapy is believed to be reversible. Growth hormone (GH or somatotropin), which is primarily secreted by the anterior pituitary gland, plays a key role in the development and activity of many cells including T-cells. GH is also produced by lymphocytes [84]. The potential association of GH in T-cell development was first noticed in the 1930s when rats lacking a pituitary gland exhibited thymic involution. The activity of GH is largely mediated by insulin like growth factor 1 (IGF1) that is produced after binding of GH to its receptor. In vitro studies have demonstrated that GH can improve the function of B-cells, T-cells, NK cells, and macrophages. In vivo, thymic involution correlates with age-related reductions in GH production and GH treatment was shown to reverse thymic involution and improve T-cell reconstitution in GH-deficient mice [85]. GH acts on a wide variety of cells making it difficult to determine if its effects on T-cell reconstitution are caused by a direct effect on T-cell, progenitor cell or supporting cell signaling (e.g., TECs) biology. Developing and mature T-cell express GH and IGF1 receptors suggesting that GH, at least in part, regulates T-cell development through interactions with Tcells [86, 87]. The potential for GH to improve immune reconstitution after alloHSCT is currently being investigated in a phase 1 trial (NCT00737113). Improving Immune Reconstitution Through Pharmacological Activation of Wnt Signalling Wnt (Wingless) signaling plays an important role in T-cell development. Early activation of β-catenin/TCF-1 was demonstrated during thymocyte development [88-91]. Activation of β-catenin signaling plays a key role in thymocyte expansion and differentiation [92, 93]. In addition, inhibition of β-catenin signaling in mouse thymocytes blocks proliferation of thymocytes resulting in reduced thymic cellularity [94-97]. TCF1-deficient and TCF7 (gene encoding LEF1)-deficient mice showed a block in thymocyte differentiation suggesting a redundancy between these genes in developing thymocytes [98]. TCF1-deficient mice showed a block in thymocyte differentiation at DN1, DN2 and immature SP

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(ISP) stages whilst TCF7-deficient mice showed a block in ISP thymocyte differentiation [98]. Wnt3A-deficient mice, which die pre-natally around embryonic day 12.5, exhibit reduced numbers of immature myeloid cells, multipotent progenitor cells and thymocytes [99]. Thymocytes from Wnt3Adeificient mice undergo differentiation in in vitro culture however exhibit significant abberations in T-cell development at the ISP stage. The role of Wnt3A in thymocyte development has been described as TCF1-dependent [99]. The activity of another Wnt ligand, Wnt4, was shown to support thymopoeisis through regulation of TEC expansion [100]. Down-regulation of Wnt4 receptors (Frizzled 4 and 6) occur in the thymus of aging mice and thus correlate with age-related thymic involution [101]. Wnt has been shown to regulate T-cell differentiation in a dose-dependent manner. Luis et al., created a gradient of Wnt activation in the haematopoeitic system of mice through hypomorphic mutations of the APC gene using the Flox/Cre system [102]. Intermediate levels of Wnt activation (approximately 22fold above normal) promoted T-cell development in a HSC/OP9-DL1 co-culture system established to generate T-cell precursors from HSCs, whilst mild Wnt activation (approximately 2-fold above normal) had no effect on T-cell development in this culture system. Very high levels of Wnt activation (approximately 72-fold above normal) caused a partial block in thymocyte development at the DN3 stage leading to a reduced number of cells in culture. Similar data were registered in mice where again intermediate levels of Wnt activation enhanced T-cell development, mild levels had no effect on T-cell development, and high levels caused an accumulation of DN3 cells. Intermediate levels of Wnt activation did not affect TCRβ rearrangement in the majority of mice. As very high levels of Wnt activation impaired HSC engraftment in mice, and TCRβ rearrangement in thymocytes, administration of agents that activate Wnt should be applied at a time after HSC engraftment at doses that cause intermediate Wnt activation. All levels of Wnt activation caused a reduction in total DP cell numbers in thymii that were more pronounced in the higher levels of Wnt activation. This study confirmed that different subsets of developing thymocytes require different levels of Wnt activation [103, 104].

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WNT OFF Wnt

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Figure 2: Activation of Wnt signaling.

WNT OFF: Under normal conditions or during treatment of Wnt signaling inhibitors (e.g., DKK1, WIF), β-catenin is targeted for proteosomal degradation after phosphorylation by a member of the destruction complex, GSK3β. WNT ON: In the presence of Wnt ligands, and absence of Wnt inhibitors, association of Fzd and LRP5/6 triggers a cascade of cellular events that results in disassembly of the β-catenin destruction complex, and subsequent accumulation and nuclear translocation of β-catenin where it acts as transcriptional co-activator of TCF/LEF-mediated gene transcription. Alternatively, cytoplasmic accumulation of β-catenin can be induced by pharmacological inhibition of GSK3β (e.g., BIO, lithium, Chir99021, SB216763). We have recently examined the possibility of pharmacological activation of Wnt using the small molecule inhibitor of GSK3β, 6-bromoindirubin 3’-oxime (BIO) to modulate T-cell regeneration in mice transplanted with human stem cells. GSK3β-mediated phosphorylation marks β-catenin for proteosomal-mediated

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degradation [105, 106]. Inhibition of GSK3β results in cytoplasmic accumulation of β-catenin and subsequent nuclear translocation where it regulates gene transcription with the co-transcription factors TCF and LEF [106-108] (Fig. 2). When BIO was administered at early stages post-transplant, stem cell activity in the BM and T-cell reconstitution were inhibited suggestive of high levels of Wnt activation. When BIO was administered at week 6 post-transplant, when BM but not thymopoietic activity declines, inhibited thymopoiesis was observed including suppression of all T-cell subsets. Late application of the inhibitor when BM lymphoid potential declined and thymic output only marginally contributed to T-cell expansion, T-cell numbers in PB were not affected [52]. It is relevant that approximately 4-fold increase in β-catenin expression was observed in human T-cells when 3mg/kg BIO was applied daily, and increasing the dose of BIO up to 30mg/kg further up-regulated β-catenin and suppressed T-cell expansion in mice. Thus, pharmacological Wnt activation using GSK3β inhibitors did not produce any advantage in terms of T-cell numbers when given either at BM and thymusdependent or independent stages of T-cell regeneration. Remarkably, BIO given at post-thymic stage of T-cell expansion inhibited memory T-cell differentiation and preserved naïve T-cell pool which can potentially improve immune responses of de novo generated T-cells [58]. This effect was seen in both CD4+ and CD8+ T-cell compartments. Administration of BIO also delayed naïve T-cell differentiation into memory cells in mice transplanted with mature human cord blood or peripheral blood-derived T-cells. Collectively, our results are suggesting that low doses of inhibitor can be efficiently used to prevent memory T-cell skewing in expense of naïve T-cell differentiation in recipients of DLI or HSCT. Wnt signaling modulates mature T-cell differentiation. Our finding is consistent with the role of Wnt in the inhibition of T-cell differentiation demonstrated in βcatenin and TCF1 transgenic mouse models. The important components of Wnt signaling, β-catenin, TCF7 and LEF1, are all highly expressed in naïve CD8+ T-cells but their expression rapidly drops following interaction with antigen and differentiation into effector cells [109, 110]. CD8+ T-cells that persist long-term as central memory cells retain high expression of TCF7 and LEF1, and are able to selfrenew and expand following antigen recall [110]. The differential expression of TCF7 and LEF1 in CD8+ T-cell subsets is suggestive that Wnt sigalling is required

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for the function of memory T-cells. Activation of Wnt signaling through Wnt3A or GSK3β inhibitor treatment suppressed eomesoderin (EOMES) gene expression, a key regulator of effector CTL differentiation, in murine and human T-cells. An increased proportion of central memory T-cells has been identified in CD3/CD28stimulated CD3+ T-cells treated with a GSK3β inhibitor (BIO), however, when the same GSK3β inhibitor was used in PHA-activated T-cells we observed an increased proportion of naïve T-cells as differentiation was blocked [58]. Thus, it appears that Wnt signaling promotes central memory differentiation after TCR stimulation, and preserves naïve T-cell differentiation after mitogenic stimulation. In vivo, Wnt/βcatenin/TCF1 signaling inhibited effector T-cell differentiation by promoting memory T-cell differentiation in TCF1-transgenic mice [111-114]. β-catenin/TCF1 signaling was critical for induction of EOMES expression which promoted CD8+ memory T-cell persistence and differentiation of CD8+ memory T-cells from naïve CD8+ T-cells [115]. Wnt signaling was shown to promote the generation of a newly identified subset of CD8+ T-cells named stem cell-like memory T-cells (Tscm). These cells express a combination of biomarkers characteristic of naïve and memory T-cells, and display a functional competence more similar to memory T-cells than naïve T-cells. Compared to other memory subsets, Tscm were long-lived presumably through high expression of BCL2. More importantly, Tscm were able to selfrenew and differentiate into all memory T-cell subsets [116]. Tscm exhibited an improved proliferative potential, compared to other subsets, and mediated superior anti-tumour responses. All these features prove the ‘stemness’ of Tscm, however, it cannot be totally excluded that these cells represent the transitional phenotype between naïve T-cells and central memory T-cells. In any case, Wnt activation appears to promote generation of Tscm. Wnt activation was previously shown to arrest T-cell differentiation, however, it was not clear whether suppression of differentiation is a direct effect or a consequence of suppressed proliferation that acts to induce memory/effector differentiation [100]. By modulating Wnt activity using different doses of the GSK3β inhibitor BIO, we have shown that Wnt acts independently on T-cell expansion and differentiation. BIO used at low doses promoted IL-2-treated

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CD62L+ naïve T-cell divisions without inducing differentiation. When used at higher doses, naïve cell proliferation and differentiation was not affected in IL-2treated T-cells, however, PHA-induced proliferation and differentiation was inhibited. BIO activated Wnt signaling, preserved naive T-cell gene expression, and down-regulated effector T-cell cell gene expression in human T-cells [117]. Taken collectively, Wnt activation has the potential to increase the naïve T-cell pool by promoting naïve T-cell proliferation or inhibiting naïve to memory T-cell differentiation. Importantly, T-cells pre-treated with BIO in vitro showed similar or improved proliferative capacities compared to control cells. Additionally, Tcells harvested from BIO-treated mice mediated similar CTL effects towards cancer cells ex vivo compared to T-cells harvested from control mice [117]. This may be clinically applicable as a large naïve T-cell pool with broad TCR repertoire provides HSCT recipients with a more robust adaptive immune system needed to fight off opportunistic infections, and in the case of leukaemia patients, cancer cells that survive conditioning and GVL. The dose of GSK3β inhibitors should be optimized to allow for maximal T-cell benefits with minimal HSC detriment. Wnt activators such as BIO should be applied at a time point after robust HSC engraftment in the BM when they can promote post-thymic T-cell development. Cell Therapy To Improve Immune Reconstitution Infusion of ex vivo expanded autologous T-cells has been used to improve immune reconstitution. Porter and June described an in vitro method of expanding CD4+ T-cells by several thousand fold using anti-CD3 and anti-CD28 antibody coated beads that serve as artificial antigen presenting cells. This methodology was introduced as it was difficult to isolate a sufficient quantity of healthy T-cells from heavily pre-treated patients. In a phase 1 study, auto-HSCT in combination with ex vivo expanded T-cells was performed in patients being treated for nonHodgkin lymphoma. Infusion of 1x1010 T-cells was safe and feasible, and this therapy resulted in improved day +30 lymphocyte counts, Th1 function characterized by IFN-γ secretion, and delayed leukocytosis [118]. CD3/CD28 expansion results in T-cell activation and differentiation into a memory phenotype, and therefore, potential detrimental effects on T-cell exhaustion and memory skewing must be examined.

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Donor lymphocyte infusion (DLI) has been used to promote T-cell reconstitution and boost the GVL effect after haploidentical T-cell depleted alloHSCT [119]. However, this therapy is associated with a risk of GVHD. Optimization of the DLI dose is required for beneficial GVT/immunity/reconstitution effects without GVHD, however, this is difficult to ascertain as every patient will respond differently as will each disease type and status [120, 121]. Several graft engineering strategies have been developed that permit accelerated T-cell reconstitution and enhanced GVL/immunity without drastically increasing the risk of GVHD. Infusion of virus-specific T-cells after HSCT improved immunity towards specific pathogens and reduced tumour relapse. Several early clinical trials have been conducted to examine the safety and efficacy of virus-specific T-cells in patients at high risk of recurrent viral infection. CMV, EBV and ADV-specific T-cells were generated in vitro by cultures of donor lymphocytes and donor monocyte-derived dendritic cells pulsed with virus lysates [122]. No immediate toxicity or excess GVHD whilst virusspecific T-cell expansion was observed. Tumour specific T-cells were also used in pre-clinical and clinical studies to prevent cancer relapse following HSCT. Tumour specific T-cells can be isolated from tumours and expanded ex vivo. Alternatively, patients can be treated with antigen presenting cells pulsed with autologous tumour antigen [123]. Tumour specific T-cells require MHC/TCR interaction, however, MHC down-regulation on cancer cells helps them escape immune-surveillance thereby limiting the utility of tumour specific T-cells. In addition, autologous T-cells can be genetically modified to express a chimeric antigen receptor (CAR) that recognizes tumourassociated antigens such as CD19, GD2, HER2, and PSMA [124]. CAR T-cells express tumour-targeted monoclonal antibody-derived single fragment length antibodies (scFvs) fused to T-cell-derived cytoplasmic signaling domains that may be CD28, 41-BB or OX40. When expressed by a T-cell, CAR redirects T-cell specificity to an antigen expressed on the malignant cells. Genetically engineered with a CAR, T-cells are amplified ex vivo to numbers suitable for adoptive cell therapy and administered to the patient after pre-conditioning. CAR-transfected Tcell therapy is being tested in clinical trials against several malignancies including prostate cancer [125], B-cell cancer [126], neuroblastoma and melanoma [127]. A

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major benefit of CAR therapy is that it bypasses the requirement of MHC/TCR interactions. Preclinical and clinical data provide strong evidence that a patient’s own T-cells can be redirected to initiate an effective anti-tumour response [124]. Autologous T-cells transduced to express the CAR receptor lyse malignant B-cells and effectively traffic to distant tumour sites, penetrate even bulky disease and eradicate disseminated tumours. CAR based T-cell immunotherapy is currently under study in several clinical trials, and encouraging early response data is beginning to emerge. Ongoing clinical trials indicate that CAR T-cells targeting CD19, a B-cell-associated antigen expressed on normal and leukaemia B-cells, can be safely infused into patients and induce complete remission in a proportion of patients [128]. CAR T-cell therapy, however, is associated with a variety of problems, including limited availability of autologous T-cells, particularly at advanced stages of the disease. Another factor reducing the efficacy of CAR T-cell therapy is associated with functional T-cell exhaustion or anergy characteristic of cancer patients [129]. CAR T-cells generated from exhausted T-cells do not efficiently expand in vivo that is necessary to eradicate tumour. Using donor T-cells is an alternative approach to generate CAR T-cells, however, CAR T-cells generated from donor T-cells may induce graft-versus host disease (GVHD). We proposed that CARmodified donor HSCs or early T-cell precursors may prove to be more therapeutically relevant over CAR T-cells generated from mature donor T-cells by avoiding issues such as GVHD, since we and others have shown that T-cells generated from HSC or T-cell precursors develop tolerance to host antigens through normal thymopoiesis. We speculate that ex vivo generated CAR-modified stem cells may eventually serve as a viable option for cell-based therapies as these cells can generate large numbers of mature functional and tolerant T-cells targeting cancer. Another limiting factor to the potency of CAR T-cell therapy is the large degree of effector memory differentiation that occurs during the expansion phase, as these cells are relatively short-lived compared to naïve and central memory Tcells. We hypothesized that genetically modified HSC or T-cell precursors adoptively transplanted to the patient will provide long-lasting supply of naïve CAR T-cells and mediate the sustained anti-tumour activity. The feasibility of this

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novel approach to generate mature CAR T-cells from donor stem cells is currently being developed in our laboratory. We adopted a method of generating CD19_CAR gene-modified HSCs using a transposon-transposase based gene transfer system [130]. Genetically-modified HSC cultured in the presence of Notch ligand gene Delta-like 4 (DL4) differentiate into T-cell precursors expressing CARs. Furthermore, we and others have shown that in vitro generated human T-cell precursors effectively engrafted immunodeficient mice and developed mature T-cells [131]. In vivo generated mature T-cells exhibited a naïve T-cell phenotype suggesting that their infusion can produce better immune responses compared to ex vivo expanded mature T-cells that acquire late memory phenotype. Another method of developing T-cell precursors in vitro involves co-culturing HSCs with OP9-DL1 cells, a murine bone marrow stromal cell line (OP9) that secrete high levels of the Notch-1 ligand DL1 (Delta-like-1), to yield T-cell precursors. The use of recombinant human DL4 protein, however, eliminates the need of feeder stromal cells thereby allowing a cleaner culture system to generate T-cell precursors [131]. Mice that received an allogeneic TCD BM HSCT with the addition of T-cell precursors generated from HSC/OP9-DL1 cultures showed improved thymyic cellularity and donor T-cell chimerism, compared to recipients of BM or HSCs only. T-cell precursors generated functional CD4+ and CD8+ Tcells with a broad TCR repertoire that could provide resistance to Listeria monocytogenes infection, and mediate GVT effects without exacerbating GVHD. Adoptive transfer of T-cell precursors also promoted thymic-independent T-cell development in thymectomized mice [132]. Inoculation of precursor T-cells generated from DL4-treated UCB-derived HSCs resulted in improved thymicdependent generation of functional mature CD3+ T-cells, CD4+ and CD8+ T-cells, and CD19+ B-cells with absence of GVHD that has been observed in mice inoculated with mature human PB-derived T-cells [131]. There is potential to develop allogeneic T-cell precursors for addition to allografts in order to improve T-cell reconstitution through acceleration of thymic seeding with T-lymphoid progenitors without exacerbation of GVHD. Natural Killer (NK) cells have been shown to mediate GVT effects in leukaemia, and neuroblastoma patients that received an allogeneic transplant [21, 22, 133]. A

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major benefit to NK cell transplantation is that unlike donor T-cells that promote GVHD, NK cells have been shown to inhibit GVHD [21, 22, 134]. Therefore, it is rational to pursue donor NK cell transplantation as a means to elicit GVT effects in patients with high-risk cancer without causing harmful GVHD. A major challenge facing the implementation of NK cell transplantation is the low NK cell content available per blood donation. Currently, pre-clinical studies are seeking to determine an optimal method of increasing NK cell numbers in the laboratory using methods such as exposure to genetically engineered K562 cells expressing cell bound cytokines and co-stimulatory molecules, or exposure to EBV-transformed lymphoblastoid cells [135, 136]. Clinical studies are investigating whether ex vivo NK cell expansion yields functional NK cells that confer a beneficial GVT effect after TCD allogeneic or autologous HSCT [23, 137, 138]. Additionally, researchers are determining which NK cell bound stimulatory/inhibitory receptors should be matched or mismatched with donor MHC ligands to maximize the GVT effect [139]. SUMMARY Delayed immune reconstitution remains one of the major causes of morbidity and mortality after haematopoeitic stem cell transplantation. Although it is appreciated that a variety of circulating peripheral blood cell subpopulations contribute to immune integrity, including B-cells, NK cells, peripheral blood monocytes, dendritic cells, and more importantly, it is understood that T-cells are our most important immune mediators. Pre-transplant myeloablative conditioning depletes the patient of mature T-cells rendering them severely immune-compromised. T-cell reconstitution after HSCT depends on the survival and expansion of adoptively transferred T-cells from the graft and/or the de novo generation of T-cells in the recipient thymus. Inadequate thymic regeneration after HSCT is associated with lymphopenia, delayed functional immune recovery, and skewing of the T-cell compartment away from naive and toward memory T-cells. Therefore, improving immunological reconstitution, in particular T-cell reconstitution, after haematopoeitic stem cell transplantation is critically important. Attempts to improve immunological reconstitution depend on the success of novel experimental approaches in improving T-cell immune reconstitution. It is important to assess the effects of these various approaches on thymopoietic recovery. Several strategies already demonstrated to have efficacy in murine models and in human clinical trials,

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including the use of thymoprotective agents (e.g., keratinocyte growth factor or related family members) and the use of thymopoietic factors (e.g., androgen ablation using leuprolide, IL-7 administration, and growth hormone administration). The adoptive transfer of ex vivo generated CAR T-cells, virus-specific and tumourspecific T-cells, and NK cells are potential strategies proved to be successful in early stage clinical trials. Table 1 below summarises the several strategies adopted to improve immune reconstitution. Table 1: A summary of potential strategies to improve immune reconstitution after HSCT Strategy

Potential Benefits

Potential Side Effect

Tested in Clinical Trial

IL-2

Promotion of homeostatic T cell expansion. Promotion of NK reconstitution. Promotion of Treg development.

GVL mitigation through increased Treg development. Alloreactive T-cell expansion.

NCT00003962 NCT00539695 NCT01517347

IL-7

Promotion of homeostatic Tcell proliferation. Promotion of thymopoeisis.

Alloreactive T-cell expansion.

NCT00684008

IL-15

Promotion of GVL and immunity through enhanced alloreactive T-cell proliferation.

Promote GVHD through enhanced alloreactive T-cell proliferation.

NA

KGF

Promotion of thymopoeisis Protection of TEC from GVHD

No effect: Conflicting results in pre-clinical animal models have been reported.

NCT00031148

Sex Steroid Inhibition

Promotion of thymopoeisis. Prevention of thymic involution & loss TECs.

Inhibited sexual function.

NCT01338987

Growth Hormone

Promotion of lymphocyte development. Prevention of thymic involution.

Nerve, muscle, joint pain, oedema.

NCT00737113

Wnt Activation

Promotion of homeostatic Tcell proliferation. Promotion of thymopoeisis.

Alterations in glucose metabolism.

NA

DLI

Promotion of GVL and immunity.

Promotion of GVHD.

See reference 118

VirusSpecific TCells

Promotion of immunity without GVHD.

Off-target effects of T-cells (e.g., cytokine release syndrome).

NCT01945619

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Table 1: contd…

CAR T-cells

Promotion of GVL without GVHD.

Off-target effects of T-cells (e.g., cytokine release syndrome)

See reference 127

NK Cells

Promotion of GVL without GVHD. Mitigation of GVHD.

Off-target effects of NK cells (e.g., cytokine release syndrome).

See reference 137

T-Cell PreCursors

Promotion of GVL and immunity without GVHD.

Unknown.

NA

ACKNOWLEDGEMENTS Declared None. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. ABBREVIATIONS ALL

= Acute Lymphoblastic Leukaemia

AML

= Acute Myeloid Leukaemia

BIO

= 6-Bromoindirubin 3’-Oxime

BM

= Bone Marrow

BMT

= Bone Marrow Transplant

CAR

= Chimeric Antigen Receptor

CLL

= Chronic Lymphocytic Leukaemia

GVHD

= Graft-versus Host Disease

GVL

= Graft-versus-Leukaemia

GVT

= Graft-versus-Tumour

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HSCT

= Haematopoeitic Stem Cell Transplantation

IL

= Inteleukin

MA

= Myeloablative

MHC

= Major Histocompatibility Complex

NK

= Natural Killer

NMA

= Non-Myelablative

NRM

= Non-Relapse Mortality

PB

= Peripheral Blood

RIC

= Reduced Intensity Conditioning

TCD

= T-cell Depletion

TCR

= T-cell Receptor

TEC

= Thymic Epithelial Cell

TNF

= Tumour Necrosis Factor

TRM

= Transplant Related Mortality

UCB

= Umbilical Cord Blood

Klamer et al.

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[107] Holmes T, O'Brien TA, Knight R, et al. Glycogen synthase kinase-3beta inhibition preserves hematopoietic stem cell activity and inhibits leukemic cell growth. Stem Cells 2008; 26(5): 1288-97. [108] Holmes T, O'Brien TA, Knight R, et al. The role of glycogen synthase kinase-3beta in normal haematopoiesis, angiogenesis and leukaemia. Current medicinal chemistry 2008; 15(15): 14939. [109] Willinger T, Freeman T, Herbert M, et al. Human naive CD8 T cells down-regulate expression of the WNT pathway transcription factors lymphoid enhancer binding factor 1 and transcription factor 7 (T cell factor-1) following antigen encounter in vitro and in vivo. J Immunol 2006; 176(3): 1439-46. [110] Gattinoni L, Ji Y, Restifo NP. Wnt/{beta}-catenin signaling in T-cell immunity and cancer immunotherapy. Clin Cancer Res 2010; 16(19): 4695-701. [111] Muralidharan S, Hanley PJ, Liu E, et al. Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes. J Immunol 2011; 187(10): 5221-32. [112] Jeannet G, Boudousquie C, Gardiol N, et al. Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory. Proc Natl Acad Sci U S A 2010; 107(21): 9777-82. [113] Zhou X, Yu S, Zhao DM, et al. Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity 2010; 33(2): 229-40. [114] Gattinoni L, Zhong XS, Palmer DC, et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med 2009; 15(7): 808-13. [115] Zhao DM, Yu S, Zhou X, et al. Constitutive activation of Wnt signaling favors generation of memory CD8 T cells. J Immunol 2010; 184(3): 1191-9. [116] Gattinoni L, Lugli E, Ji Y, et al. A human memory T cell subset with stem cell-like properties. Nat Med 2011; 17(10): 1290-7. [117] Klamer G, Shen S, Song E, et al. GSK3 inhibition prevents lethal GVHD in mice. Exp Hematol 2013; 41(1): 39-55 e10. [118] Porter DL, June CH. T-cell reconstitution and expansion after hematopoietic stem cell transplantation: 'T' it up! Bone Marrow Transplant 2005; 35(10): 935-42. [119] Bellucci R, Alyea EP, Weller E, et al. Immunologic effects of prophylactic donor lymphocyte infusion after allogeneic marrow transplantation for multiple myeloma. Blood 2002; 99(12): 4610-7. [120] Frey NV, Porter DL. Graft-versus-host disease after donor leukocyte infusions: presentation and management. Best Pract Res Clin Haematol 2008; 21(2): 205-22. [121] Chalandon Y, Passweg JR, Schmid C, et al. Outcome of patients developing GVHD after DLI given to treat CML relapse: a study by the Chronic Leukemia Working Party of the EBMT. Bone Marrow Transplant 2010; 45(3): 558-64. [122] Peggs KS, Verfuerth S, Pizzey A, et al. Cytomegalovirus-specific T cell immunotherapy promotes restoration of durable functional antiviral immunity following allogeneic stem cell transplantation. Clin Infect Dis 2009; 49(12): 1851-60. [123] Brimnes MK, Gang AO, Donia M, et al. Generation of autologous tumor-specific T cells for adoptive transfer based on vaccination, in vitro restimulation and CD3/CD28 dynabeadinduced T cell expansion. Cancer immunology, immunotherapy : CII 2012; 61(8): 1221-31. [124] Curran KJ, Pegram HJ, Brentjens RJ. Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions. The journal of gene medicine 2012; 14(6): 405-15.

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[125] Hillerdal V, Nilsson B, Carlsson B, Eriksson F, Essand M. T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells. Proc Natl Acad Sci U S A 2012; 109(39): 15877-81. [126] Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013; 368(16): 1509-18. [127] Shen CJ, Yang YX, Han EQ, et al. Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma. J Hematol Oncol 2013; 6: 33. [128] Jena B, Maiti S, Huls H, et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PloS one 2013; 8(3): e57838. [129] Crespo J, Sun H, Welling TH, Tian Z, Zou W. T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment. Curr Opin Immunol 2013; 25(2): 214-21. [130] Manuri PV, Wilson MH, Maiti SN, et al. piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum Gene Ther 2010; 21(4): 427-37. [131] Reimann C, Six E, Dal-Cortivo L, et al. Human T-lymphoid progenitors generated in a feedercell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/SCID/gammac(-/-) mice. Stem Cells 2012; 30(8): 1771-80. [132] Zakrzewski JL, Kochman AA, Lu SX, et al. Adoptive transfer of T-cell precursors enhances Tcell reconstitution after allogeneic hematopoietic stem cell transplantation. Nat Med 2006; 12(9): 1039-47. [133] Kloess S, Huenecke S, Piechulek D, et al. IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA. Eur J Immunol 2010; 40(11): 3255-67. [134] Olson JA, Leveson-Gower DB, Gill S, et al. NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects. Blood 2010; 115(21): 4293-301. [135] Berg M, Lundqvist A, McCoy P, Jr., et al. Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells. Cytotherapy 2009; 11(3): 341-55. [136] Fujisaki H, Kakuda H, Shimasaki N, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res 2009; 69(9): 4010-7. [137] Foley B, Cooley S, Verneris MR, et al. NK cell education after allogeneic transplantation: dissociation between recovery of cytokine-producing and cytotoxic functions. Blood 2011; 118(10): 2784-92. [138] Klingemann H, Grodman C, Cutler E, et al. Autologous stem cell transplant recipients tolerate haploidentical related-donor natural killer cell-enriched infusions. Transfusion 2013; 53(2): 412-8; quiz 1. [139] Curti A, Ruggeri L, D'Addio A, et al. Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients. Blood 2011; 118(12): 3273-9.

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CHAPTER 3 Alternative Biomaterial Substrates for Human Embryonic Stem Cell Culture Deepak Kumar, Ying Yang and Nicholas R. Forsyth* Institute of Science and Technology in Medicine, University of Keele, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, UK Abstract: The additive effect of stem cell therapy and biomaterial substrates provides exciting opportunities for tissue engineering and regenerative medicine applications. Nanofibrous substrates can be fabricated to mimic the nano-architectural structure of specific, native tissue extracellular matrix. This provides topographical structure and contact guidance, which can impact stem cell biology as well as direct their differentiation towards specific lineages. This chapter highlights nanofibrous substrates as an alternative tool for the expansion and differentiation of embryonic stem cells. Future applications of such technology could promote the use of hESC-derived cells for clinical applications.

Keywords: Biomimetic, clonogenicity, embryonic stem cells, electrospinning, nanotechology, feeder layers, MatrigelTM, nanofiber substrates, synthetic polymers. INTRODUCTION Human embryonic stem cells (hESCs) are pluripotent with the potential to differentiate into all mature cell types found within the body. Therefore, ESCs have great potential for a wide range of therapeutic applications as they provide an unlimited source of several different cell types for tissue replacement and regeneration [1]. Popular methodology for the expansion of hESCs is largely reliant on either the mitotically-inactivated feeder cell method (using direct coculture with embryonic or adult fibroblasts), or the feeder-free method, which utilises feeder cell pre-conditioned media and a biological substrate, such as MatrigelTM [1, 2]. The inherent limitation of the MatrigelTM-based feeder-free *Corresponding author Nicholas R. Forsyth: Institute of Science and Technology in Medicine, University of Keele, Thornburrow Drive, Hartshill, Stoke-on-Trent, Staffordshire, ST4 7QB, UK; Tel: 01782 555 261; Fax: 01782 747 319; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds) All rights reserved-© 2015 Bentham Science Publishers

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method is that it is unsuitable for incorporation into hESC-based clinical trials due to the risk of xenocontamination alongside the batch to batch variability of MEFs used to condition hESC media. Furthermore, MatrigelTM limits hESC expansion to a two dimensional (2D) environment with subsequent interventions required prior to transplantation. Hence, innovative and novel tissue engineering strategies are urgently required to provide the opportunity of incorporating hESCs with synthetic, biomimetic substrates (scaffolds), with the potential to act as three dimensional (3D) carriers to facilitate ready transplantation into in vivo target sites and eliminate xenogenic contamination. One possible route could be the use of nanofibrous substrates, which can be fabricated via electrospinning. The natural 3D stem cell niche and ECM at the nanoscale level is dynamic featuring a complex mixture of pores, pits and a network of intricate nanofibres composed from various structural proteins including collagen fibrils. With relevance to tissue engineering applications, this architecture provides an important model for the design of artificial synthetic scaffolds, which can support, instruct and guide the behaviour of cells [3, 4]. Nanotechnology enables the provision of artificial templates able to mimic the architecture and topographical structure of the native ECM as closely and accurately as possible. This can enhance cell adhesion and biomimetic properties, which can in turn, attract stem cells, support stem cell proliferation and differentiation and also provide appropriate tissue functioning [3, 5]. Electrospun nanofibrous substrates have been investigated for application with many different types of stem cells including bone-marrow derived mesenchymal stem cells, neural stem cells and umbilical cord blood stem cells. However, only recently have researchers explored the use of electrospun nanofibrous substrates for hESC expansion and differentiation. This chapter highlights the current trends in hESC culture, emerging substrates (natural and synthetic) identified to hold potential for supporting hESC expansion in the future and, the progress made so far regarding hESCs and nanofibre technology. Embryonic Stem Cells Embryonic Stem Cell Characteristics Embryonic stem cells (ESCs) display unlimited self-renewal and are immortal in vitro. The immortality is attributed to ESCs having high telomerase activity (a

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ribonucleoprotein enzyme which maintains telomere length by adding repeats to chromosome ends), providing long-term proliferative potential [2] and preventing senescence, which usually occurs during tissue culture after a population doubling between 50-80 [6]. During expansion in vitro, ESCs have a high nucleus to cytoplasm ratio with prominent nucleoli [2]. Furthermore, as pluripotent hESCs proliferate (population doubling time of 36 hours) [7], they form flat, compact colonies that are tightly adherent (more so in hESCs, relative to mouse embryonic stem cells; mESCs). In suspension culture ESC’s aggregate to form embryoid bodies which support spontaneous differentiation whereas in vivo transplanted ESCs form teratomas [2, 8]. However, some progress has been made to scale up pluripotent hESCs using microcarrier suspension systems where a study by Amit et al., 2010 for example observed the expression of typical hESC markers up to 20 passages [9, 10]. Due to the possibility of hESCs forming teratomas after implantation in vivo, this has triggered a massive interest in the generation and use of induced pluripotent stem cells (iPS). Here, using somatic cell nuclear transfer, human somatic cells are programmed allowing trans-acting factors present in the mammalian oocyte to reprogramme cell nuclei to a pluripotent state. This allows the generation of patient-specific iPS cell lines, which eliminate the concern of immune rejection [11, 12]. Plasticity of Embryonic Stem Cells ESCs are pluripotent with the potential to differentiate in vitro into all mature cell types of the 3 somatic germ layers; endoderm, ectoderm and mesoderm (Fig. 1), including the male and female germ cells [2], with the exception of placental cells. Therefore, ESCs have great potential across a wide range of therapeutic applications as they can provide an unlimited source of several different cell types for tissue replacement and regeneration [1]. Certain growth factors can direct the differentiation of hESCs into specific germ layers; for example endoderm lineages can be stimulated using hepatocyte growth factor, mesoderm lineages can be encouraged using BMP-4 and TGF-β and ectoderm lineages using nerve growth factor and retinoic acid [13]. Examples of in vitro differentiation of hESCs include; oligodendrocytes, (induced using bFGF, epidermal GF and retinoic acid, the haematopoietic lineage (using a

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co ocktail of haaematopoietiic cytokiness SCF, Flt3L L, IL-3, IL-66, G-CSF annd BMP-4 [1 14]), cardiom myocytes (w when co-cultu ured with m mouse visceraal endoderm m gave rise to o beating heaart muscle colonies that expressed ccardiomyocyyte markers, α-myosin heeavy chain and cardiac troponins) and hepatoccytes (whichh expressedd markers, allbumin, α-1-anti-trypsin n and cytokeeratin 8 andd 18) [15]. E Examples oof in vitro hE ESC differrentiation into i mesod dermal lineeages incluude: chonddrogenesis (cconfirmed by b expressio on of proteo oglycans, Cool2a1, Sox99 and Col100a1) [16], osteogenesis (confirmed d by exprression of osteoblasticc markers; alkaline ph hosphatase, osteocalcin and collageen type I) [117] and adippogenesis (cconfirmed by y expression n of Adiponeectin, Leptin n, Adipophiliin and Perilippin) [18].

Fiigure 1: In vittro differentiatiion capacity off ESCs into sppecialised cell ttypes of the thhree somatic geerm layers; Ecttoderm, Endod derm and Meso oderm.

In n Vitro Cultture Method ds Current C techn niques for cullturing plurip potent hESC Cs in vitro caan involve eitther direct orr indirect feeeder layer meethods. How wever culturinng hESCs using these meethods can po otentially reesult in variied initial atttachment an and possible loss of cellls during .

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passaging. This may be due to a lack of understanding of the underlying mechanisms, which support their proliferation in an undifferentiated state. Feeder Method The feeder method involves the co-culture of hESCs with a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs) [19]. Recent modifications to this include the use of alternative mitotically inactivated feeder layers such as; human embryonic fibroblasts, adult Fallopian tube epithelium [20], human foreskin fibroblasts (with the ability to expand up to 42 passages) [21], human foetal muscle or skin fibroblasts and postnatal human bone marrow stromal cells [22]. Many of these feeder layers have claimed to support pluripotent hESC expansion with retention of expression of typical pluripotent markers [22]. Feeder-Free Method The feeder-free method uses pre-conditioned hESC media from MEFs (where cytokines, other growth factors and ECM proteins such as collagen I, IV, laminin and fibronectin are secreted) and is further supplemented with basic fibroblastic growth factor (bFGF; known to enhance cloning efficiency during hESC expansion in vitro) [6], in combination with a biological substrate such as MatrigelTM [1, 2]. This method supports the expansion of undifferentiated hESCs in vitro for up to 130 population doublings [1] whilst retaining typical characteristics (karyotype and phenotype) as well as forming more compact colonies when compared to hESC colonies formed using the MEF feeder layer method. MatrigelTM is a commercially available, loosely defined gel, sourced from Engelbreth-Holm-Swarm tumours. MatrigelTM is comprised of extracellular matrix (ECM) proteins including; laminin-111, collagen IV, heparin sulphate proteoglycans, entactin/nidogen, fibronectin, growth factors, matrix-degrading enzymes and their inhibitors; and other proteins yet to be defined [1, 23]. Combining ECM proteins secreted in conditioned media together with the proteins found in MatrigelTM probably enhances the signalling cues for corresponding integrins to interact and encourage initial attachment. MatrigelTM functions to artificially mimic the ESC ECM niche environment and provides the required chemical cues during for the expansion of hESCs in a pluripotent state,

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whilst inhibiting differentiation. Recent improvements related to this method have included eliminating animal based serum and replacing it with Knock-out™ serum replacement; whose components are proprietary [24]. Further developments to completely eliminate any xenogenic contaminations have included designing a medium which is totally serum and xeno-free; TeSR contains bFGF, TGF β1, Human Insulin, Human Holo-Transferrin, Human Serum Albumin and Glutathione in a DMEM/F12 base and retain the undifferentiated state of hESCs when used in combination with Matrigel™ [25, 26]. Despite various recent modifications to enhance hESC scale-up in vitro whilst retaining their pluripotency, feeder layers (MEFs) and Matrigel™ still remains as the popular conventional methods for hESC expansion in a research lab setting [27]. Other limitations associated with the current techniques of culturing hESCs using MatrigelTM include; batch to batch variability of MEFs, xenogenic contamination and expression of foreign oligosaccharide residues, and issues associated with scale-up [23]. Feeder layers also carry the risk of retroviral infections [28]. Sialic acid (Neu5Gc) has been identified on the surface of hESCs; this molecule is not of human origin and therefore may potentially elicit an immune response during transplantation. Originally it had been speculated that the Neu5Gc molecule was derived from MEFs but now its origin appears to have been from serum replacement [29]. The underlying mechanisms associated with Matrigel™ which support the attachment and undifferentiated expansion of hESCs are yet to be fully defined. Investigating these pathways would be important to help in understanding the associated pathways and proteins and perhaps mimic this phenomenon by using novel substrates that would eliminate the use of xenogenic materials and increase the efficiency of hESC numbers during routine culture. Natural ECM Proteins - As an Alternative Partial elucidation of proteins in MatrigelTM has encouraged the identification of some of the important corresponding integrins expressed on the surface of hESCs (Fig. 2). These integrins permit successful connection and adherance to ECM proteins [30]. This results in “inside-out” integrin signalling where integrinmediated signalling determines the type of ECM protein secreted and control how

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th hey are orgaanised in thee ECM bind ding surface of cells to eencourage ppluripotent ex xpansion [31]. Therefo ore, understaanding the hhESC integgrin profile will help im mprove curreent technolo ogy by creating novel suubstrates witth biologicall moieties th hat would prromote hESC adhesion via those inntegrins resuulting in an increased yiield of inittial hESC adhesion a an nd allowing an efficiennt expansionn rate of un ndifferentiatted cells. Wiith sufficientt numbers thhis would perrmit the use of hESCdeerived cells for clinical trials t [27].

Fiigure 2: Sch hematic repressenting the co onnection betw ween ECM pproteins and iintracellular co omponents proteins through integrins. i

A summary of o the criticaal integrins and a interactiive ECM liggands, whichh promote th he adhesion and undifferrentiated exp pansion of hE ESCs are lissted in Tablee 1 below. Furthermore, the impact of ECM M protein availability, concentraation and orresponding g hESC surfface integrin n expression on hESC diifferentiationn is yet to co bee fully eluccidated. Thiis would heelp to deterrmine the eexact integrrin-protein in nteractions, which w induce hESC diffe ferentiation.

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Table 1: A summary of the critical hESC integrins and corresponding ECM proteins involved in the adhesion and expansion of pluripotent hESCs Integrin

Integrin Function

Corresponding ECM Protein (Ligand)

References

α6β1 

Maintain hESC stemness.

Laminin

[17, 25, 32]

α5β1 

Maintain hESC stemness.

Fibronectin 

[25, 26, 32, 33]

αVβ5 

Mediates hESC adhesion and maintenance of pluripotency.

Vitronectin 

[26, 33]

α2β1

Major role in hESC adhesion to Matrigel™.

Collagen IV

[17, 23]

The limitations associated with the in vitro expansion of hESCs using Matrigel™ has driven researchers to explore other alternatives. Partial elucidation of the composition of Matrigel™ has identified the presence of various ECM proteins including fibronectin, laminin and collagen. This has encouraged investigation into the ability of various ECM proteins to support the in vitro expansion of undifferentiated hESCs. ECM proteins investigated have included: vitronectin, fibronectin, laminin, collagen type I and IV and hyaluronic acid; a summary of various studies is listed in Table 2. These proteins have been coated onto typical tissue culture plastic surfaces and cultured with MEF-conditioned media or more defined ESC media that are commercially available. Xeno-free commercial products, which have utilised human derived ECM proteins include Synthemax (Corning), CellStart (Life Technologies) and LN-521 Stem Cell Matrix (BioLamina Integrins). Despite extensive research into the use of ECM proteins as growth substrates the in vitro expansion of hESCs is not as straightforward, as previously thought. Limitations associated with using recombinant proteins include; prohibitive costing associated with production and purification, batch-to-batch variability, aseptic maintenance, degradation and denaturation during dehydration, an undefined thickness thresholds, and inefficiency in protein coating. As a result of these limitations, MatrigelTM still remains as a popular method for culture and expansion of undifferentiated hESCs in vitro in research labs [34].

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Table 2: A summary of studies, which have investigated the potential of purified ECM proteins as emerging substrates for the attachment and expansion of human embryonic stem cells during in vitro culture ECM Protein Substrate

Substrate Details

hESC Line

Observations

Collagen Type I

Biomatrix (10 µg/cm2).

H1 and H9

hESCs expanded on Collagen I [34] substrate with conditioned media from human embryonic germ-cell-derived cells had a population doubling time similar to hESCs expanded on MatrigelTM with ES conditioned media. hESC expressed typical pluripotent markers (Oct-4, Nanog and Tra-1-60).

Substrate coating on flask.

HUES-1 and SHEF-1

hESCs were expanded with KO- [35] DMEM/SR and defined media (HESF8). Both media’s in combination with Type I Collagen substrate demonstrated stable expression of pluripotent markers (Oct-3/4, Nanog, SSEA-1 and 3) as well as maintaining their differentiation capacity.

Collagen Type IV

Substrate coating onto 96-well plate.

HUES-1, HES2, HESCNL3

Supported undifferentiated expansion of [28] hESCs with MEF conditioned media, however in defined media (mTESR) proliferation was not as effective.

Vitronectin

Human purified and Recombinant Vitronectin coating onto 96-well plate.

HUES-1, HES2, HESCNL3

hESCs were expanded on Vitronectin [28] with mTESR-1 media or MEFconditioned media; mTESR-1 supported better hESC growth with retained pluripotency; although in both media’s, vitronectin supported the greatest proliferation of hESCs, in comparison to laminin + entactin, collagen IV and fibronectin.

Substrate coating.

hES1

hESCs were cultured in combination [36] with StemPro media.

Substrate coating.

H9

Good proliferation pluripotency.

Various recombinant laminins (511, 411, 332, 211 and 111).

KhES-1, KhES-2 and KhES-3

hESCs were cultured with MEF [37] conditioned media and observed a inconsistent cell attachment with laminin 332 showing better results.

Laminin

Laminin 511 coating. HS420, HS207, HS401 Substrate coating.

H1 and H9

References

and

retained [33]

Good attachment and proliferation with [38] retention of pluripotency. hESCs expanded with MEF conditioned [1] media. hESCs maintained a normal.

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Table 2: contd….

karyotype, stable proliferation rate and high telomerase activity. Expressed Oct4, hTERT, ALP and surface markers such as SSEA-4, Tra 1-60 and Tra 1-81 Laminin + Entactin

Substrate coating onto 96-well plate.

HUES-1, HES2, HESCNL3

Supported the proliferation of [38] undifferentiated hESCs; however proliferation activity was the lowest on this substrate, relative to Collagen IV, Vitronectin, Fibronectin and MatrigelTM.

Fibronectin

Human plasma fibronectin (Fn) coating.

HUES-1

Supported hESC colony expansion with [38] retained pluripotency when expanded in MEF-conditioned media. Proliferation rate was lower in comparison to hESCs grown on collagen IV and vitronectin.

Three types of fibronectin investigated: bovine Fn (bovine, human plasma and human cellular),

I-3, I-6 and H9

hESCs cultured on all 3 types of [39] fibronectin coated substrates with media (plus 15% SR and a combination of TGF-β1, LiF and bFGF were suitable for the undifferentiated hESC proliferation for more than 50 passages.

Hyaluronic Acid (HA) and dextran hydrogels

3D Hydrogel, modified with photoinitiator groups and photoinitiator cross-linked by UV curing for 10 mins HA hydrogels (Ø 3 mm and 2 mm thickness).

H1, H9 and H13

hESCs were encapsulated into HA [40] hydrogel and cultured in MEF conditioned ES media. hESCs retained their metabolic activity, supported their proliferation whilst retaining their pluripotency and capacity to differentiate for up to 20 days in culture. Dextran hydrogels supported EB formation instead.

Hyaluronic Acid surface functionalised with ECM proteins/peptides

Layer-by-layer selfassembled surface of HA functionalised with various ECM proteins using a cross-linking agent.

MEL1 and MEL2

hESC attachment observations after 2 [41] hours showed no attachment of hESCs to HA without any biofunctionalisation of proteins to its surface and that functionalization of recombinant Fn to HA enhanced hESC attachment. hESCs were cultured on these substrates using defined culture media.

E-cadherin

Human E-cadherin coating.

H1 and H9

hESCs maintained a typical morphology [42] and retained a similar proliferation rate to hESCs grown on MatrigelTM.

Tissue Engineering and Regenerative Medicine Strategies Major limitations of MatrigelTM include batch-to-batch variability, reproducibility, scale-up issues, xenogenic contaminations (such as Neu5Gc and lactate dehydrogenase elevating virus; LDEV) [42], and cost cause great concern for their clinical therapeutic use [43]. In addition to the limitations of using ECM proteins as alternatives, this has

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driven researchers to attempt to overcome these limitations and increase their applicability for clinical therapeutics. Currently, there are three main therapeutic approaches using stem cells: direct administration of adult stem cells (currently in clinical use), implantation of differentiated stem cells and tissue engineering [44]. Improved scale up of hESCs would have many beneficial implications for in vivo clinical application with the elimination of any xenogenic contact and applications relating to tissue engineering and regenerative medicine applications. The ultimate goal of regenerative medicine and tissue engineering is “to replace or regenerate cells, tissues or organs, to restore, improve or create normal function” [45]. A tissue engineering strategy involves a combinatory use of cells, engineering materials and biochemical factors [3, 46, 47]. The native stem cell ECM is an environmental structure to anchor and support cells and provides a template for tissue growth in 3D [37]. It is made of several fibrous proteins such as collagen, fibronectin, glycoproteins, proteoglycans, growth factors, bioactive molecules that maintain cell adhesion and growth [48] including chemical ligands which are able to interact with surface receptors on cells with mechanical stability [49]. Biomaterial scaffolds can be temporary or permanent substrates made of biological or artificial material, which fundamentally aim to provide a 3D environment (physically, chemically and biologically) [50] to mimic this native ECM. Scaffolds can be designed to support several cellular functions including: cell proliferation and differentiation, to allow isolation and expansion of cells, to function as a drug delivery system, growth factor delivery systems and to maintain the spatial environment to encourage the regeneration of tissue [51]. The more similar the structure and the ability of the scaffold to mimic the extracellular matrix, the better the end result of tissue engineering and its function. Specific to hESCs, biomaterials endeavour to mimic the stem cell microenvironment and niche, which would help maintain their typical undifferentiated cell phenotype expression (if desired) but also be able to support their differentiation function by providing the appropriate biochemical, topographical and mechanical signals. Incorporating biomaterial scaffolds into stem cell therapy would potentially provide the ability to expand a sufficient number of undifferentiated hESCs in comparison to 2D tissue culture plastic conditions due to greater surface area. Differentiated cells derived from hESCs would perhaps be more suitable for clinical therapies. By

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improving the ability to terminally differentiate expanded hESCs (using biochemical, topographical and mechanical signals) could result in a greater yield of the desired cell type subsequently increasing the efficiency of the culture method as well as minimising risk of teratoma formation. More importantly, scaffolds are able to act as a portable carrier to permit in vivo transplantation for the use of hESC derived differentiated cells in in vivo clinical implantations [52]. Naturally-Derived Polymer Scaffolds Polymers possess functional properties and flexibility in design. Broadly, there are two types of polymers, natural and synthetic; naturally-derived polymers include examples such as collagen, gelatin, chitin, chitosan and cellulose and synthetic polymers include polyurethane, poly (glycolic acid) and poly (lactic acid) amongst many others. Advantages of using natural polymers include: precise mimicking of the native ECM structure, available recognition sites which provide multiple cell attachment opportunities giving the substrate good adhesion properties to support subsequent cell growth, and improved interaction between the substrate and cells due to the bioactive nature of the substrate. Natural polymer hydrogels are also biocompatible, allow cell dependant degradability, and eliminate the limitations of 2D flat culture using tissue culture plastic surfaces [52]. However, limitations associated with the use of natural polymers as scaffolds includes: the dangers of eliciting an immune response, batch to batch variability, limited range of mechanical properties, rapid degradation rate and weak mechanical properties (though these limitations can be overcome by crosslinking), lack of consistency and structural malleability [44]. Currently, a limited number of studies have investigated the use of natural polymer scaffolds to support the expansion of hESCs. A 3D porous chitosan scaffold (Ø 13 mm and 2 mm thickness, with 95% porosity and 65% average pore size) with mechanical properties (compressive modulus (E) of 8.1 MPa and tensile E of 0.8 MPa) has been reported to support the expansion of hESCs in vitro where hESCs retained pluripotency (confirmed by ALP and SSEA-4 expression) and typical morphology. Further investigations included in vivo transplantation into an immunodeficient mouse where observations included hESCs population of the scaffold. Pluripotency was not maintained for longer than 30 days [53].

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Investigations have included the fabrication of hydrogels. Hydrogels are 3D networks formed from hydrophilic homopolymers, copolymers or macromers crosslinked to form insoluble polymer matrices [50]. Due to the ability of hydrogels to have high water retention this allows diffusion of nutrients and dissolved gases as well as being able to mimic the 3D spatial environment of natural ECM by the presentation of ligands in a similar way [50]. Properties such as biocompatibility, flexible method of formation, anticipated physical characteristics, the provision of structural integrity to tissue constructs, the essential structural and compositional similarities to ECM and an extensive framework which provides cellular proliferation and survival [50] makes them candidates for hESCs. Hydrogels fabricated from natural polymers have been demonstrated to successfully culture stem cells [54, 55] although issues exist in terms of controlling mechanical and degradation properties, which could be eliminated using synthetic hydrogels due to superior control over chemical composition and architecture. Hydrogels have the potential to mimic the 3D ECM environment (biologically, chemically and physically) that hESCs are exposed to during embryogenesis, within the inner cell mass whilst embedded in a 3D matrix. This native ECM environment can control their self-renewal and differentiation. hESCs have been encapsulated within calcium alginate hydrogels and expanded in typical hESC media; it was apparent that after 260 days of culture, hESCs retained pluripotency (expression of Oct-4, Nanog and SSEA-4) with the capacity to differentiate into all three germ layers. Furthermore hESCs were arranged in typical closely packed colonies. This study showed promising results and eliminated any xenogenic contamination [56]. Hyaluronic acid (HA) hydrogels (Ø 3 mm and 2 mm thickness) fabricated by UV cross-linking supported the expansion of undifferentiated hESCs for up to 20 days in culture, while dextran hydrogels caused EB formation and subsequent differentiation [40]. Biofunctionality of HA with ECM proteins/peptides, especially fibronectin improved and enhanced initial hESC attachment rates [41]. Synthetic Polymer Scaffolds Synthetic polymers are man-made polymers that have great potential in tissue engineering due to their controllable biodegradation and mechanical property [57]. Several advantages of using synthetic polymers for scaffolds include biocompatibility, their chemistry being versatile, able to incorporate mimicking biological properties and the ability to tailor a scaffolds’ mechanical properties

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(including porosity, spatial arrangement, strength and degradation rate to suit various applications) [44]. Synthetic polymers are also cheaper, can be produced in large uniform numbers with a long shelf life, and can be designed to show similar physicochemical and mechanical properties to biological tissue with reproducible mechanical properties such as tensile strength and elastic modulus. With regards to hESCs, synthetic polymers can contribute to the development of feeder-free culture systems with the ability to offer reproducible culture conditions. Furthermore, they can minimise the cost of hESC expansion and eliminate exposure to xenogenic contaminants. These contributions would increase the potential clinical applications of differentiated hESCs but would only be possible due to constructive collaborations between chemists, engineers, and stem cell biologists [52]. A summary of various synthetic polymer substrates that have demonstrated the ability to expand undifferentiated hESCs are stated in Table 3. However, synthetic polymers lack biological function and require physical or chemical modification for cells to function appropriately. An example of a simple modification is treatment of typical polystyrene flasks by plasma etching and plasma-deposited gradients of octadiene to acrylic acid with demonstrable support of ESC expansion and retention of pluripotency. This technique has been applied to surface chemistry of scaffolds (via charged gas plasma polymerisation deposition), which enhanced the adherence properties of cells to a scaffold [58]. However, although synthetic polymers such as (polyethylene glycol) PEG/PLGA have positive features such as the ease of control and reproduction, the surfaces have to be modified to enhance cell adhesion as they lack biological motifs in comparison to natural polymers [47]. As a result, more complex modifications have included the biofunctionalisation of synthetic materials using ECM/natural proteins; for example, ECM molecules/peptides have been deposited onto various substrates including Hyaluronic acid and chitosan using methods such as layer-bylayer self-assembly and covalent bonding to enhance ESC adhesion properties [41, 59, 60]. However, limitations associated with many of these substrates include: batch to batch variability with limited mechanical properties, many of the polymers are not FDA approved and are flat 2D surfaces such as oxygen plasma treatment to typical tissue culture plastic surfaces, which do not fully mimic the typical 3D nanoarchitectural, ECM structure and environment [61, 62]. Therefore,

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a substrate, which closely mimics the native nanoarchitectural structure may provide a better environment for the culture of hESCs. Table 3: A summary of synthetic substrates used to culture and expand, undifferentiated hESCs Synthetic Substrates

Scaffold Properties

hESC Line

Observations

Oxygen plasma etched tissue culture polystyrene

2D synthetic culture surface stable at room temperature for atleast a year.

HUES7 and NOTT1

hESCs cultured with MEF conditioned media and retained typical hESC morphology for up to 10-14 passages. Expressed typical pluripotent markers (Oct-4, TRA160 and SSEA-4).

[63]

Poly(ethylene terephthalate) PET

Porous membranes. 1-4 x 106 pores/cm2 (0.291-0.345 GPa).

Information not available

Pore density of 4 x 106 supported the greatest number of hESC colonies, increased cell proliferation and maintained uniform and undifferentiated hESCs.

[64]

Poly(ethylene terephthalate) PET

Porous membranes with different pore sizes (1, 3 and 8 µm).

CHAhESC3

Feeder layers at the bottom of the trans-well inserts whilst hESCs were seeded on top of the porous membranes. 3 µm pore size demonstrated optimal results with greatest number of hESC colonies formed, prevented direct interaction with feeder cells and helped retain undifferentiated state for up to 25 passages.

[65]

Aminopropylmethacrylate hydrochloride (APMAAm)

Hydrogel photointiated using UV light.

H1 and H9

In combination with chemically defined media (mTESR1). hESCs demonstrated pluripotent expansion similar to hESCs expanded on MatrigelTM for over 20 passages.

[62]

Poly (methyl vinyl ether-Alt-maleic anhydride) PMVEAlt-MA

Various polymers including PMVEAlt-MA were deposited onto acrylamide gel coated slides in a spot (Ø150 - 200 µm).

HUES1 HUES9

1 x 106 cells seeded per array slide (10-20 cells/polymer spot) and cultured. Supported long-term proliferation and self-renewal of hESCs (Oct-4 and Sox-2) with differentiation capacity to form all 3 germ layers.

[61]

Poly [2(methacryloyloxy) ethyl dimethyl-(3sulfopropyl) ammonium hydroxide] PMEDSAH

PMEDSAH coated onto typical tissue culture plastic dishes.

BG01 and H9

hESCs cultured long-term up to passage 15 with defined media including Stem-Pro and mTeSR. hESCs stained positive for pluripotent markers and all 3 germ layers.

[66]

References

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Application of Nanotechnology in Culture of Human Embryonic Stem Cells Nanotechnology can create structures at the atomic and molecular levels with a size range of 10-500 nm [5]. The natural 3D stem cell niche and ECM at the nanoscale level is dynamic with a complex mixture of pores, pits and a network of intricate nanofibres composed from various structural proteins including collagen fibrils. All these features provide fundamental cues at the cellular level that support and regulate various cell functions and activity as a consequence of topographical features [5]. Cells are highly sensitive to nanoscale ECM patterns and topography and can probe these features using their filopodia which encourage the retention of cell shape or induce changes resulting in subsequent differentiation via cytoskeletal arrangement modification [4, 5, 58]. Typically, cells are tens of micrometers in diameter but have components such as cytoskeletal elements and transmembrane proteins that are nano-sized. Furthermore, stem cells have the ability to react with features as small as 5 nm and thus are highly sensitive to nanotopography [67]. Anisotropic topography is also considered important at the nanoscale level in ECM where cells in tissues such as nerve, cardiac and tendon require high levels of organisation which direct secreted ECM and tissues structure organisation from nanoscale through to macroscale levels [68]. With relevance to tissue engineering applications, this architecture provides an important model for the design of artificial synthetic scaffolds, which can support, instruct and guide the behaviour of cells [3, 4]. Nanotechnology enables the provision of artificial templates, which are able to mimic the architecture and topographical structure of the native ECM as closely and accurately as possible. This enables the expectation of a cell response and behaviour to be similar to as it would react or perform in vivo. Scaffolds fabricated with a nanotexture such as nanofibres, whose topography can also be controlled are able to mimic this natural ECM architecture and provide a high surface area to volume ratio with a microporous structure [69]. This can enhance cell adhesion and biomimetic properties, which in turn attract stem cells and support stem cell activities such as proliferation, differentiation and also provide appropriate functioning of tissues (Fig. 3) [3, 5]. However, nanofiber scaffolds

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caan also havee disadvantaages includin ng small poore size, lim mited porositty and the in nability to mimic m the EC CM/tissue hydrated structture on theirr own [48].

Fiigure 3: Nano-scale topograp phy and archittecture influencce on cell attacchment abilitiees. (Adapted frrom Stevens an nd George, 200 05 [4]).

Electrospinni E ing The T electrosp pinning proceess utilises a polymer soolution whicch is chargedd up using hiigh voltage electrodes which allow ws the polym mer to be ddrawn from a needle (n nozzle) and is accelerateed towards an a oppositelyy charged or grounded collecting su ubstrate [49]]. During fliight from the needle to the collectoor, the intenssity of the ellectric field pulls p the pollymer solutio on by forminng a Taylor cone from thhe needle. Once O the eleectric field over powerrs the surfaace tension of the polyymer, the so olution resullts in the forrmation of an a instable j et which duuring flight uundergoes

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whipping and solvent evaporation causing the deposition of finer fibres on the collecting substrate [49]. Applications of Electrospun Nanofibre Scaffolds with Embryonic Stem Cells Current limitations associated with stem cells include; efficiency of expansion and adequate numbers suitable for clinical therapeutic use. To overcome this limitation, many researchers have attempted to expand stem cells using nanofibrous substrates. Previous reports have detailed the biocompatibility of electrospun nanofibre scaffolds to support the attachment, proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (hMSC), cord blood-derived somatic stem cells, neural stem cells, and haematopoietic stem cells [70-75]. Many of these researchers used synthetic polymers such as PCL, poly-L-lactide acid (PLLA), poly(lactic-co-glycolic acid) (PLGA), as they are FDA approved and their bulk degradation properties are well characterised [48]. With regards to embryonic stem cells, a limited number of investigations have explored the ability of electrospun nanofibrous scaffolds to support their activity including attachment, proliferation and differentiation and hence is still a very much unexplored area. Perhaps, the reason for this is due to various complications associated with the complex conventional culture of expanding ESCs and the partial understanding of the exact mechanisms associated with controlling embryonic stem cell behaviour. For this reason, many studies have found it easier to differentiate embryonic stem cells towards countless lineages, as they have potential to transform into specialised cell types of all three germ layers. For example, PLLA nanofibres were able to successfully differentiate mESCs towards the osteogenic lineage and expressed bone specific markers such as calcium and osteocalcin [76]. Considerable attention has been given to the application of electrospun nanofibres for nerve regeneration, as electrospinning provides the ability to generate nanofibres with great degree of anisotropy and alignment, which provides topographical cues to induce and direct the differentiation of ESCs towards nerve cells [5]. Electrospun polyurethane nanofibrous scaffolds (150 µm thickness, 84% porosity and 360 nm fibre Ø) supported the initial adhesion and undifferentiated expansion of hESCs, which were then differentiated towards neurones by culturing in neurobasal A basal media supplemented with

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1% B27, 1% N2, epidermal growth factor (EGF) and bFGF for up to 47 days [77]. Similarly, neurospheres generated from hESCs were cultured onto PLDLA electrospun nanofibrous 3D scaffolds (thin structures of 2-3 fibre thickness; thick structures of >10 fibre thickness); observations here included enhanced neuron growth on the thicker scaffolds relative to thinner scaffolds. Cells had a typical morphology for neuronal cells and followed fibre orientation [71, 78]. Another study by Wang et al., also showed the ability of hESC-derived neural progenitor cells seeded onto Tussah silk fibroin nanofibous scaffolds to successfully differentiate towards neurons. In particular, aligned, 400 nm diameter fiber substrates significantly elevated neuronal differentiation and neural outgrowth in comparison to 800 nm fiber diameter and random conformation [79]. Due to the difficulty of hESCs adhering to synthetic substrates, alternative methods have included inducing embryoid body formation and then seeding these embryoid bodies onto electrospun nanofibrous substrates in combination with biochemical cues to induce their differentiation into specific lineages. Examples include electropsun nanofibre scaffolds from PLA which supported the differentiation of hESC-derived EB cells in the presence of osteogenic media towards osteogenesis and implanted subcutaneously to the back of immunodeficient mice for 5 weeks. Observations included discrete mineralisation expression of typical bone markers including osteocalcin [80]. PCL aligned and random electrospun nanofibres have successfully supported the differentiation of hESC-derived EBs into neural progenitors in the presence of neurobasal media (supplemented with B27); aligned nanofibres particularly enhanced the neurite outgrowth which was directed parallel to the orientation of the nanofibres [81]. However, there are a limited number of studies that have attempted to culture and expand undifferentiated embryonic stem cells (mouse and human origin) on electrospun synthetic nanofibrous substrates, specifically of human origin. Few attempts have been made using synthetic biodegradable polymers such as PCL, polyurethane and polyamide. Furthermore, composite polymers where natural polymers such as collagen and gelatin have been electrospun with synthetic polymers such as PCL have successfully enhanced the adhesion properties of the nanofibres resulting in attachment and expansion of undifferentiated ESCs. It has also been identified that the geometry and topography of the electrospun

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nanofibres alone was sufficient to support of significantly larger colonies of undifferentiated ESCs in comparison to controls such as glass coverlips and relative polymer films [53]. A summary of studies, which have attempted to culture ESCs on electrospun nanofibrous substrates is stated in Table 4. Table 4: A summary of electrospun nanorfibrous substrates fabricated from various synthetic polymers to culture, expand and differentiate embryonic stem cells Synthetic Nanofibrous Substrates Polyurethane

Substrate Properties

hESC Line

150 µm thickness Exhibited high porosity 84% Pore size 5-6 and 1 µm Fibre Ø 360 nm.

SA002

PCL

Random and aligned nanofibres on coverslips Fibre Ø 250 nm.

mESCs

PCL/collagen and PCL/gelatin

Random topography nanofibrous substrates. PCL/collagen (Ø 275 nm) and PCL/gelatin (Ø 283 nm) fibre diameters.

HES3

PCL and calcium deficient hydroxyapatite

Electrospun nanohybrid mats (non-woven architecture) 2 and 55 wt.% of calcium hydroxyapatite content Average fibre size

mESCs

Observations Undifferentiated hESCs were cultured and expanded on scaffolds (in hESC media for 57 days) and then induced to differentiate (using neurobasal media supplemented with 1% B27 and 1% N2) towards neurones for up to 47 days. Differentiation confirmed by positive immunostaining of dopaminergic tyrosine hydroxylase. ESCs induced to form embryoid bodies, which were seeded onto the nanofibrous substrates and induced to differentiate towards neurons using retinoic acid and neural basal media. After 14 days expression of neuron marker Tuj1 was visible. Direct seeding of ESCs without forming EBs displayed that using retinoic acid and culturing in neural basal media with B27 supplement ESCs formed aggregates on both aligned and random nanofibrous substrates. hESCs were grown in the presence of MEFs. Larger hESC colonies were supported on both substrates with increased cell growth by 47.58% and 40.18% for (PCL/collagen and PCL /gelatin, respectively) in comparison to their control (hESCs on MEFs only). Colonies generated on substrates retained stemness characteristics. On aligned substrates cell migrated away from the EB an along the axis of the aligned fibres. mESC response to nanohybrid PCL and calcium deficient hydroxyapatite substrates and neat PCL was evaluated. mESCs were able to adhere (although 45% efficiency relative to control), expand in an undifferentiated, pluripotent state (Nanog and β-Tubilin) in a typical way.

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[82]

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Table 4: contd…

1.5m, porosity 8090% and specific surface area 16m2g-

1.

PCL

Thickness of scaffold 200 µm. Porosity of ~ 88%, Pore size 30 µm and average fibre diameter 691 nm.

mESCs

Polyamide

A 3D Ultra-thin nanofibrous substrate.

mESCs

mESCs differentiated towards adipogenesis using a 3D culture system. mESCs seeded into PCL matrices. mESCs seeded into PCL matrices sealed into transwell inserts with membrane removed and expanded for 2 days before 4-day treatment with RA, insulin and T3 induction. Upon inducing differentiation morphology changed from fibroblastic to a spherical with evidence of lipid accumulation (Oil-red-O-staining) with confirmation using PPAR-γ marker. Migration and penetration of differentiated mESCs 40 µm deep into substrate. Ultra-web nanofibrous substrates fabricated from polyamide demonstrated to support the expansion of significantly larger colonies of undifferentiated mESCs compared to glass coverslips and relative polymer film controls.

[84]

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CONCLUDING REMARKS Recent developments in the interaction between embryonic stem cell biology and tissue engineering have resulted in progress in identifying alternative methods for culturing hESCs. These developments are crucial and would encourage the expansion of hESCs without any xeno exposure, direct their differentiation towards specific lineage and more importantly provide opportunities for the transportation and transplantation of hESC-derived cells for clinical therapies. Artificial nanofibrous substrates mimic the natural 3D, nanoarchitectural ECM structure of native tissue but also provide the opportunity to completely eliminate MatrigelTM from hESC culture systems. These substrates in the future could be used in combination with defined media’s for the complete removal of xenocontamination. Importantly, by using FDA approved, biodegradable polymers this permits transplantation of hESC-derived cells on nanofibrous substrates, which can be transplanted in vivo; benefits would mean no secondary intervention to remove the scaffold due to the degradation of the substrate in a controlled manner where polymer by-products would be secreted via the metabolic pathway and

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prevent accumulation of such by-products at the target site, but also allow the localisation of stem cells within the target site. It appears the exploring of new biomimetic substrates through nanotechnology and incorporating artificial ECM molecules may be the answer to expand hESCs in xeno-free environment allowing hESCs for future clinical cell therapies. ACKNOWLEDGEMENTS Declared None. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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CHAPTER 4 Frontiers in Regenerative Medicine for Cornea and Ocular Surface Maria P. De Miguel1,*, Ricardo P. Casaroli-Marano2,3, Nuria Nieto-Nicolau2, Eva M. Martínez-Conesa2, Jorge L. Alió del Barrio4, Jorge L. Alió5, Sherezade Fuentes1 and Francisco Arnalich-Montiel4 1

Cell Engineering Laboratory, IdiPAZ, La Paz Hospital Research Institute, Madrid, Spain; 2Transplant Services Foundation (TSF) at Hospital Clinic de Barcelona, Spain; 3Department of Surgery, School of Medicine at University of Barcelona, Barcelona, Spain; 4Ophthalmology Department, Ramon y Cajal Hospital, Madrid, Spain and 5Vissum Ophthalmological Institute and Miguel Hernandez University, Alicante, Spain Abstract: The cornea represents two thirds of the eye’s refractive power and, together with the sclera, is the protective shield of intraocular structures. The cornea is composed of three cellular layers (epithelium, stroma and endothelium) and three acellular layers (Bowman’s, Dua’s and Descemet’s). Corneal pathologies can affect one or all corneal layers, producing corneal opacities. Penetrating keratoplasty is currently being displaced by lamellar techniques that selectively replace the diseased layer, but neither solves the classical difficulties encountered in corneal transplantation, such as immune rejection and a shortage of organs. The development of bioengineered corneas composed of prosthetic or natural scaffolds and autologous stem cells that differentiate into corneal cells could overcome these difficulties. In recent years, much research has been carried out to find the optimal scaffold and the best source of stem cells to regenerate the corneal layers. Limbal stem cells (LSC) have arised as one of these sources, and the need to find a marker to distinguish them from more differentiated cells has also emerged. Both limbal and extraocular stem cells have been tested, and some techniques are already being used in clinical practice. These novel techniques for tissue engineering of functional corneal equivalents represent a new and fascinating way to treat corneal diseases. The new techniques allow us to treat patients with autologous grafts and can prevent the use of corneal stem cells, which are scarce and often unavailable.

Keywords: Cell culture, cell differentiation, cell-based therapy, ex vivo expansion, human adult mesenchymal stromal cells, limbal stem cells, ocular surface regeneration. *Corresponding author Maria P. De Miguel: Cell Engineering Laboratory, IdiPAZ, La Paz Hospital Research Institute, Madrid, Spain; Tel: +34 91 2071458; E-mail: [email protected] Atta-ur-Rahman & Shazia Anjum (Eds) All rights reserved-© 2015 Bentham Science Publishers

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1. INTRODUCTION The cornea is the primary refractive element on the anterior surface of the eye, and it is responsible for approximately two-thirds of its total optical power. It consists of an outermost stratified, squamous epithelial layer (corneal epithelium) that is limited posteriorly by the Bowman’s layer. The underlying stroma, which accounts for about 90% of the thickness of the cornea, is comprised of aligned arrays of collagen fibrils interspersed with cellular components (keratocytes), and it is this highly organized arrangement of lamellae that is responsible for corneal transparency. The stroma is separated from the endothelial layer (corneal endothelium) by the Dua´s layer and the Descemet’s membrane, which acts as a basement membrane for these endothelial cells. The corneal endothelium is a single layer of metabolically active cells that are in direct contact with the aqueous in the anterior chamber. These cells help to maintain corneal transparency by actively pumping water out of the stroma (Fig. 1) [1].

Figure 1: Cross-section of the human cornea and details of its main layers. The cornea is composed of an epithelial layer (epithelium, ep), the stroma (st) and an endothelial layer (endothelium). Corneal epithelium (ep) consists of the outermost layer, which presents five to seven stratified cell layers,

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limited posteriorly by the Bowman’s layer (b). The stroma (st), composed of compacted collagen lamellae and keratocytes (k), offers transparency and scaffolding to maintain the corneal shape in the middle portion of the cornea. The stroma (st) is separated from the endothelial monolayer by the Descemet’s membrane, which acts as a basement membrane for the endothelial cells. The corneal endothelium is metabolically active and also helps to maintain corneal transparency. Upper part: thin section stained with hematoxilin-eosin and semithin section stained with toluidine blue. Bottom part: ultrathin section viewed by electron transmission microscopy.

2. CORNEAL EPITHELIUM The corneal epithelium plays a key role in keeping the cornea transparent and free of blood vessels. Permanent repair is essential for the conservation of the cornea’s physiology. The stem cells responsible for the renewal of the corneal epithelium are located in the basal layers of the sclerocorneal limbus. The human limbus is a circumferential anatomic (approximately 1.5 mm wide) area that separates the clear cornea from the opaque white sclera, which is covered by conjunctiva epithelium. The limbus serves as a ‘reservoir’ for stem cells and provides a barrier to the overgrowth of conjunctival epithelial cells and their blood vessels onto the cornea [1-3] (Fig. 2). A disappearance or deficiency of this cell population (limbal stem cell deficiency, LSCD) gives rise to significant changes on the ocular surface, such as persistent corneal defects, epithelial keratinization, conjunctivalization phenomena with the development of newly formed vessels in the corneal tissue, and scarring. All of these conditions compromise corneal physiology, reducing transparency and decreasing vision [1-4]. The role of limbal stem cells (LSCs) in the maintenance of corneal epithelium integrity is widely accepted, due to their capacity for self-renewal and proliferation. These cells are characterized as an oligopotent progenitor cell population found in the basal layer of the limbal epithelium, presenting a high nucleus/cytoplasm ratio with a slow cell cycle, high proliferative potential, and a significant capacity for self-renewal by asymmetric division [4]. In addition to LSCs, in the limbic region there are a variety of cell types, such as subpopulations of various progenies (typically progenitors and transient amplifying cells), melanocytes, antigen presenting cells (Langerhans’ cells), mesenchymal cells, nerve endings, and vascular elements, which form a unique characteristic micro-environment, or niche, which is responsible for the events of cell proliferation and self-renewal [2, 5]. It is believed that the various progenies are intended for the production of differentiated cells, initially in the basal cell layer

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of the cornea. They give rise to a population of post-mitotic cells in the suprabasal layer, finally leading to terminally differentiated cells in the superficial layers (Fig. 2). This continuous flow of cell differentiation (of the centripetal type, from the deepest layers to the most superficial (the XYZ hypothesis)) ensures the renewal of the corneal epithelium and the maintenance of its integrity [1-6]. However, this XYZ theory has recently been challenged by evidence suggesting that at least in mice, cells from the central cornea have regenerative epithelial capabilities and could also be responsible for the maintenance of the corneal epithelium [7]. However, this has not yet been confirmed in the adult human cornea. Furthermore, the exact roles of mesenchymal stromal cells, corneal nerve endings, perilimbic vascularization and cellular signaling pathways involved in controlling cellular activity in the niche remain to be defined [5].

Figure 2: Diagram showing the compartments and zones corresponding to the XYZ hypothesis for corneal epithelium regeneration. The limbus (B) acts as the ‘reservoir’ for limbal stem cells (LSC) and also provides a barrier to the overgrowth of epithelial cells from conjunctiva (A) and its blood vessels onto the peripheral (C) and central (D) cornea. LSC are a relatively small population with progenitor cell characteristics with high self-renewal capacity, and they comprise the first compartment (C1). Transient amplifying cells (TAC) appear to be a larger cellular population with a moderate selfrenewal capacity and some differentiation state into intermediate compartment (C2). C1 and C2 correspond to the proliferative zone. The last compartment (C3) contains terminally differentiated cells (TDC), with terminal differentiation characteristics and no self-renewal capability, which

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corresponds to the differentiating zone. LSC repopulate the cornea by proliferating at the basal layer of epithelium, giving rise to TAC (XY movement; B to C), and then migrate centripetally, giving rise to TDC, which naturally migrate to the surface of central corneal (YZ movement; C to D). Upper left part: lateral view of ocular surface; Bottom left part: frontal view of ocular surface. Major putative cellular and molecular markers are shown in the right part of the diagram.

The clonogenic capacity of limbic epithelial cells was studied in vitro, and a progenitor cell system stratified into three levels (cellular compartments) similar to those found in some self-renewing tissues such as skin [8] was observed. At the first level (compartment 1), a relatively small population with progenitor cell features with high self-renewal capacity is described. At the intermediate level (compartment 2) a larger population of transient amplifying cells (TAC), characterized by moderate self-renewal capacity and some differentiation toward epithelial phenotypes, appears. Both the first and intermediate levels correspond to the cell populations with proliferative capacity. Lastly, the third level (compartment 3) contains a cell population with terminal differentiation characteristics associated with little or no self-renewal capability, similar to the results observed in studies of human epidermis [9]. Once they have completed their epithelial differentiation events, these cells lose their ability to self-renew and will be incorporated as corneal epithelial cells on the surface of the central cornea (Fig. 2). 2.1. Characteristics of LSCs 2.1.1. Localization and Proliferative Potential The limbus represents the niche of LSCs; it is a pigmented, highly innervated and vascularized zone, resulting a protective microenvironment for stem cells, as it confers proper nutrition and UV-light protection [10]. LSCD by congenital or acquired causes results in severe damage to corneal regeneration and transparency, suggesting that LSC are located in this region [11]. Some studies [8, 12] have concluded that epithelial cells of the ocular limbic region could form holoclones with higher clonogenic potential, in contrast to epithelial cells from central cornea. In addition, epithelial cells isolated from basal layers in limbus exhibit a high proliferative potential in vitro during expansion or after activation in response to corneal injury [13]. 2.1.2. Slow Cell Cycling and Self-renewal Epithelial cells of the basal layer of the limbus show an undifferentiated phenotype, lacking the expression of markers of corneal differentiated cells, such

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as cytokeratin (CK) 3 and 12 [14]. They are probed to retain labeled precursors of DNA over an extended time period [15, 16]. This slow cell cycling is characteristic of the quiescence state of stem cells. LSCs rarely undergo mitosis, and when they do (to maintain normal homeostasis or during corneal wound healing), they undergo an asymmetric cell division [17]. 2.1.3. Cell Size, Morphology and Plasticity During the differentiation process, the volume of the cytoplasm increases as new proteins are synthesized. Thus, LSCs in the basal layer are characterized by a small size and a higher nucleus to cytoplasm ratio [18-20]. The cytoplasm is enriched in tonofilaments that exhibit slight melanin granules. LSCs show basal cytoplasmatic invaginations from the underlying matrix [21-23] as well. Previous studies demonstrated that LSCs differentiate into several neuroectodermal lineages, such as hair follicle cells [24-26], neuronal and photoreceptors cells [2729] and in addition, they undergo epithelial-mesenchymal transition [30]. 2.1.4. Molecular and Cellular Markers In recent years, several molecular and cellular markers have been proposed to identify LSCs, but none have achieved widespread acceptance. These putative markers are composed of nuclear proteins such as p63 [31-35] although some findings revealed great variability in p63 expression depending on inter-individual variations, age, species analyzed, tissue processing or the methodology used to detect this protein [36, 37], Bmi-1 [38], and C/EBPδ [39]; transporter proteins such as ABCG2 [40, 41]; and cytoskeletal proteins such as CK5/14 [42-44], CK19 [23] and vimentin [22, 45]. Enzymes such as enolase [46, 47] and growth factor receptors such as TrkA [48] and p75NTR [49] are also positive in LSCs but are not considered specific markers, likewise some extracellular matrix union proteins [22, 50-53]. The α9 integrin was found in human corneas in a certain subset of cells of the limbal basal epithelium [23]. In addition, there are markers for differentiation, which are considered negative for LSCs. Among these markers, the most important are: CK12, CK3, connexin-43, p-cadherin [22] and involucrin. Thus, a combination of the presence and absence of putative markers can be used to identify LSCs. Here, we present a brief review of the most important positive markers suggested for LSCs (Table 1 and Fig. 2).

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Table 1: Major cellular and molecular markers found in human ocular surface epithelia Markers

Localization

Method References

ABCG2

Limbal epithelium (basal layer)

IHC, IF

[22, 23, 40, 41]

Alpha-enolase

Limbal and Conjunctival epithelium (basal layer)

IF

[23]

β-catenin (cytoplasmatic)

Limbal and Corneal epithelium (all layers)

IF

[22]

β-catenin (nuclear)

Limbal epithelium (basal layer)

IF

[60]

Bmi-1

Limbal epithelium (basal layer)

IF, qPCR

[39]

C/EBPδ

Limbal epithelium (basal layer)

IF, qPCR

[39]

CD34

Corneal stroma

IHC

[26]

CD133

Limbal and Corneal epithelium (all layers)

IHC

[26]

CD271 (p75NTR)

Limbal epithelium (suprabasal layer) and Corneal epithelium (all layers)

IHC

[208]

Connexin 43

Corneal epithelium

IF

[22]

Cytokeratin 19

Limbal (basal layer) and Conjunctival (suprabasal layer) epithelium

IF

[22, 23]

Cytokeratin 3/12

Limbal (suprabasal layer) and Corneal eptihelium

IF

[22, 23]

Cytokeratin 5/14

Limbal and Conjunctival epithelium (basal layer)

IF

[22]

E-cadherin

Corneal epithelium

IF

[22]

P-cadherin

Corneal epithelium (basal layer)

IF

[22]

EGFr

Limbal, Corneal and Conjunctival epithelium (basal layer)

IF

[22]

α2 integrin

Corneal epithelium

IF

[22]

α6 integrin

Corneal epithelium

IF

[22]

α9 integrin

Limbal epithelium (basal layer)

IF

[22]

β1 integrin

Limbal and Corneal epithelium (basal layer)

IF

[22]

Involucrin

Corneal epithelium

IF

[23]

KGFr bek

Llimbal epithelium (basal layer; LE)

IF

[22]

HGFr met

Limbal and Corneal epithelium (basal layer)

IF

[22]

OCT4

Limbal epithelium (basal layer)

IF, qPCR

[64, 65]

Nestin

Corneal epithelium

IF

[22, 23]

p63

Limbal epithelium (basal layer)

IF

[22, 23, 34]

TrkA

Limbal and Corneal epithelium (basal layer)

IF

[23]

Vimentin

Limbal epithelium (basal layer)

IF

[23]

Wnt2, Wnt6, Wnt11 and Wnt16b

Limbal epithelium

qPCR

[60]

Wnt3, Wnt7a, Wnt7b, Corneal epithelium qPCR Wnt10a IHC, immunohistochemistry; IF, immunofluorescence; qPCR, real time polymerase chain reaction

[60]

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2.1.5. The Limbal Niche There is evidence that the basement membrane of the limbal region is composed of α1-α2 chains, type IV collagen and laminin α2-β2 chains, while the basement membrane of central cornea epithelium is composed of α3-α4-α5 chains, type IV collagen and laminin α1-α3-β1-β3-γ1-γ2 chains [54, 55]. These differences play a crucial role maintaining stem cell behavior. Close communication between limbal epithelial cells and stromal fibroblasts into Vogt palisades [56] has been observed. It has been shown in vitro that LSC interactions with limbal stromal fibroblasts are mediated via interleukin 6 (IL-6). Blocking IL-6 or its downstream effector STAT3 resulted in reduced clonogenic potential, suggesting that the interaction between both regulates the LSCs proliferation [56]. LSCs attract stromal cells via SDF-1 and its receptor CXCR4, a pathway that controls the homing of hematopoietic stem cells in the bone marrow niche [57]. When CXCR4 was blocked in vitro, LSCs disrupted their interaction with limbal stromal cells and showed a more differentiated phenotype with loss of clonogenic potential. These results suggest that the fate of LSCs depends on the close physical association with limbal stromal cells via SDF-1/ CXCR4 signaling [58]. In addition, there are cytokines and growth factors that are mainly expressed by limbal epithelium and that are thought to impact limbic cellular stemness [59]. For example, keratinocyte growth factor (KGF) or hepatocyte growth factor (HGF), expressed by stromal cells, present their receptors on epithelial cells. However, transforming growth factor β (TGF-β), interleukin 1β (IL-1β) and platelet derived growth factor β (PDGF-β) are expressed by epithelial cells and their receptors are found in stromal cells. Lastly, there are growth factors whose receptors are found indistinctly on epithelial or stromal cell membrane such as insulin growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), or fibroblast growth factor (FGF) [59]. Recent studies [60, 61] have shown that proliferation signaling pathways such as Wnt/β-catenin are essential to direct LSCs in the niche. Mice lacking Dkk2, an inhibitor of the canonical Wnt pathway, increases the Wnt/β-catenin signaling pathway in the limbal stroma, leading to epithelial differentiation with a loss of PAX6 expression on the ocular surface [62]. Moreover, nuclear localization of β-

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catenin was found only in some basal epithelial cells of the human limbus [60]. Wnt2, Wnt6, Wnt11, Wnt16b and four Wnt inhibitors were found specifically in the limbal region, suggesting that there could be a balance between Wnt canonical signaling and their inhibitors controlling LSCs proliferation [60]. In this way, the activation of Wnt canonical signaling increases the clonogenic efficiency of LSCs while maintaining its undifferentiated phenotype [61]. In addition OCT4, a marker for embryonic stem cells that can be promoted by β-catenin [63], was found mainly in the basal layer of the limbal epithelium and was present in small amounts in the basal layer of central corneal epithelium [64, 65]. However, the mechanism by which OCT4 expression impacts LSCs proliferation has not yet been determined. 2.2. Ocular Surface Reconstruction The concept of ocular surface reconstruction was first introduced with the use of autologous conjunctiva in cases of unilateral chemical alkali burns. Since then, various surgical approaches have been developed with the goal of restoring the viability of the corneal epithelium on the diseased ocular surface, but these approaches present technical difficulties and complications in both the short and long term. In recent decades, limbal transplantation techniques using auto- or allografts were introduced to improve and reconstruct the altered ocular surface, constituting a method of in vivo cell expansion applied to the bio-replacement of limbic tissues [66]. The epithelial cells of the ocular surface were obtained by cell culture techniques for ex vivo expansion. Subsequently, the ocular surface was successfully reconstructed by using LSCs in patients with severe unilateral ocular surface pathology, with good anatomic and functional results [67]. Since then, various translational approaches have been developed and optimized, with satisfactory long-term clinical results [68-72]. 2.3. Techniques for Corneal Epithelial Regeneration Penetrating keratoplasty is considered the conventional therapy used for regeneration of the corneal epithelium. However, this method is not a viable strategy for patients suffering from LSCD because it does not replace the limbal stem cell population [73, 74]. For this reason, the ex vivo expansion of LSCs is the most common approach for ocular surface bio-replacement. LSCD using limbal

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tissue from the same patient’s healthy eye (unilateral diseases) is the preferred method (Fig. 3), although this source of stem cells is not always available [2]. Ex vivo expansion of LSCs, using the enzymatic technique in one or two steps [75] is the most innovative bio-replacement approach, although the explant techniques are largely used with good results [72, 76-79] (Fig. 3). When the disease is bilateral, the transplant is only possible from a living related family member or cadaveric donor. For this reason, there are other sources of cells or stem cells (with autologous or heterologous origin) that are being tested for the regeneration of the ocular surface, which could be useful in situations in which both eyes are affected (Table 2). The use of stem cells for the regenerative purposes of organ and tissue repair is a subject of great current scientific interest. An excellent alternative to LSCs is the use of adult or somatic stem cells, which present significant advantages due to their immediate clinical applicability [80, 81].

Figure 3: LSC cultures. LSC could be expanded ex vivo from a minimally invasive biopsy of the healthy limbic region of the same or the contralateral eye. The limbic epithelial layer is separated from the fragment obtained from the limbus by an enzymatic method in one (trypsin) or two steps (dispase and trypsin). The acquired cell population (cell suspension) needs to be co-cultured in vitro using cell culture techniques on feeder-layers with 3T3 fibroblasts, previously arrested by irradiation or treated by mitomycin-C. The explant technique, using amniotic membrane (AM) as substrate, is also commonly used with good results. LSC: limbal stem cells.

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Table 2: Alternative adult progenitor cell sources for limbal stem cell deficiency Alternative Cell Source

Species

References

Cultured conjunctival epithelial cells

Human

[86, 87]

Ex vivo cultivated oral mucosa

Human

[88, 93, 95-100]

Cultured epidermal adult stem cells

Goat

[103-105]

Cultured embryonic stem cell

Mouse

[106, 107]

Cultured bone marrow-derived mesenchymal stem cells

Rat, Rabbit

[94, 110, 111]

Cultured immature dental pulp stem cells

Rabbit

[114, 115]

Cultured hair follicle bulge-derived stem cells

Mice

[116, 117]

Cultured umbilical cord-lining stem cells

Rabbit

[119]

Cultured orbital fat-derived stem cells

Mice

[121]

Cultured adipose derived mesenchymal stem cells

In vitro

[122-124]

Cultured induced pluripotent stem cells

In vitro

[125]

2.3.1. CLET: Cultured Limbal Epithelial Transplantation Ex vivo expansion of LSCs (Figs. 3 and 4) is the most innovative approach for ocular surface bio-replacement. From a minimally invasive biopsy (1-2 mm2) of the healthy limbic region (the same or contralateral eye), an explant culture technique can be applied to a suitable substrate (such as amniotic membrane) or by separating the epithelial layer from the fragment obtained by an enzymatic treatment in one or two stages [72, 76-79]. In the latter approach, the cells obtained need to be co-cultured using feeder-layers (3T3 murine fibroblasts arrested by irradiation or mitomycin-C) (Fig. 3). Once cell growth is achieved (Fig. 4), the cell suspensions can be transferred to suitable substrates such as fibrin, collagen or biocompatible polymers. The bio-replacement is carried out after removal of most of the diseased tissue from the ocular surface [67-70]. This methodology requires a substantially smaller limbic biopsy, which reduces the risk of induction of limbal deficiency in healthy donor tissue. Other advantages include a final cell population that is more homogeneous and theoretically, more enriched with progenitor cells [76, 78]. However, enzymatic techniques are characterized by a more complex approach, with additional manipulation of the tissue and the need for xenoproducts at various stages of the primary cell culture production. The explant technique has certain advantages, such as technical simplicity, the lack of xenoproducts and its cost-effectiveness, despite the

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heterogeneity of the cell population cultured (sclera fibroblasts, antigen presenting cells, melanocytes, conjunctival epithelium cells and others) [72, 76-79]. It is always desirable to use autologous cells for ex vivo expansion to avoid the risk of an immune response. However, in the presence of severe bilateral ocular pathology, the only current therapeutic options include COMET or limbal allografts. The use of heterologous epithelial cells is acceptable [68, 69, 72]. This approach is associated with potential adverse effects, including the risks of immunosuppressive therapy after heterologous graft application. Nowadays, these approaches are used as consolidated cell-based therapy for the ocular surface regeneration.

Figure 4: LSC expanded ex vivo. LSC suspension was co-cultured in vitro by a feeder-layer technique with 3T3 fibroblasts, which were previously arrested by irradiation (a). After five or seven days, clusters of progenitor cells (”embryonic bodies”) with high clonogenic capability (b) were observed in culture. A monolayer of cells (c), characterized by a high nucleus-cytoplasm ratio, could be seen after 10 to 15 days of culture. Once cell growth is achieved, the cell culture is transferred to suitable substrates and then, bio-replaced to a diseased ocular surface.

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2.3.2. Cultured Conjunctival Epithelial Cells In 2002, Meller and colleagues started to study the ex vivo expansion of conjunctival epithelial progenitor cells for the treatment of severe ocular surface disease [82-84]. These studies showed that microscopically, human conjunctival epithelial cells have features similar to human corneal epithelial cells, with clinically equivalent outcomes in rabbits [85]. Recently, Ricardo and coworkers have demonstrated an 83.3% of improvement in the clinical parameters for the transplantation of autologous conjunctival epithelial cells cultivated ex vivo in 12 eyes with total LSCD [86]. Moreover, the coculture of autologous limbal and conjunctival epithelial cells is a novel alternative approach to CLET with additional conjunctival transplantation for patients with severe ocular surface disorders. The technique offers a simple one-step surgical approach for extended ocular surface reconstruction, demonstrating the feasibility of cultivation and transplantation of two phenotypically different but contiguous epithelia [87]. 2.3.3. COMET: Cultured Oral Mucosal Epithelial Transplantation Sheets of oral mucosal epithelial cells expanded ex vivo have also been used successfully as an alternative source of autologous epithelial cells [88]. In 2003, Nakamura and Kinoshita investigated the use of the epithelial cells of the oral mucosa for ocular surface reconstruction [89]. Since then, this cell type has been successfully used to treat LSCD in animal models [89-92] and humans [88, 93-99]. Despite their strong expression of CK3, oral mucosal cells have greater angiogenic potential compared to limbal cells, which may explain some peripheral corneal neovascularization following transplantation [100-102]. These data suggest that COMET could be considered soon as an established cell therapy for ocular surface diseases, being an excellent choice for cases of bilateral involvement. 2.3.4. Cultured Epidermal Adult Stem Cells In 2007, the transdifferentiation of rhesus putative epidermal stem cells into corneal epithelium-like cells in vitro was demonstrated [103]. Thereafter, Yang and colleagues reported the isolation and characterization of pluripotent stem cells from adult goat skin (called epidermal adult stem cells) that were able to repair the damaged cornea of goats with total LSCD [104, 105]. Despite the encouraging preliminary results, this is still an experimental technique that requires further investigation before clinical use.

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2.3.5. Cultured Embryonic Stem Cells Epithelial progenitors were successfully induced in vitro from mouse embryonic stem cells and were applicable as grafts for treating corneal epithelial injury [106]. Moreover, the introduction of the transcription factor Pax6 into embryonic stem cells formed a monolayer of epithelium-like cells in vitro and also were applicable for treating injured corneas in mice [107]. Ahmad and colleagues showed the differentiation of human embryonic stem cells into epithelial like cells through limbal fibroblast conditioned medium [108]. Recently, it was also reported that cells originating from human embryonic stem cells were successfully transplanted onto a partially wounded human cornea and differentiated into corneal epithelial-like cells in vitro [109]. To date, the use of human embryonic stem cells has a number of limitations, among which ethical issues and their ability to induce tumors should be highlighted. 2.3.6. Cultured Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) The earliest evidence that BM-MSCs could be used for reconstruction of damaged corneas was discovered in 2006 [94]. Epithelial-like cells accompanied by the expression of CK12 and the presence of tight junctions connecting cells in a patchlike pattern were also observed in cocultures of rat BM-MSCs with corneal stromal cells [110]. It has been demonstrated that BM-MSCs could differentiate into corneal epithelial cells in vivo and ex vivo in a rabbit model [111]. BM-MSCs may also be used as alternative feeder layer cells for LSC cultures [112] or as conditioned medium [113]. 2.3.7. Cultured Immature Dental Pulp Stem Cells Human immature dental pulp stem cells are another type of MSC that share similar characteristics with LSCs. They express markers such as ABCG2, integrin beta1, vimentin, p63, connexin 43 and CK3/12. They were also capable of reconstructing the eye surface after the induction of unilateral total LSCD in rabbits [114, 115]. 2.3.8. Cultured Hair Follicle Bulge-derived Stem Cells (HFSCs) In 2009, Meyer-Blazejewska et al., showed that transdifferentiation of HFSCs into corneal epithelial phenotype cells was possible when the cells were cultured with corneal or limbus specific microenvironmental factors [126]. The positive

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expression of two corneal epithelial markers, Krt12 and Pax6, showed that the cultured hair follicle bulge-derived stem cells transplant in mice was also capable of differentiating into cells with a corneal epithelial phenotype [116, 117]. 2.3.9. Cultured Umbilical Cord-lining Stem Cells Umbilical cord tissue is gaining attention as a novel source of multipotent stem cells because it is easily obtainable, ethically acceptable and the cells are immunologically naïve [118]. Transplantation of a bioengineered umbilical cord lining epithelial cell sheet (expressing CD227) in rabbit eyes with LSCD resulted in regeneration of a smooth, clear corneal surface with phenotypic expression of the normal corneal specific epithelial markers CK3, CK12 but not CK4 or CK1/10 [119]. 2.3.10. Cultured Orbital Fat-derived Stem Cells (OFSCs) OFSCs possess multilineage differentiation potential to become osteoblasts, chondrocytes, and adipocytes. Moreover, corneal epithelial differentiation was demonstrated through expression of CK19 and CK3 after a mixed culture with corneal epithelial cells [120]. Topical administration of OFSCs promotes corneal tissue regeneration and is a simple and non-invasive method of delivering stem cells for corneal tissue regeneration [121]. 2.3.11. Cultured Adipose Derived Mesenchymal Stem Cells (ADSC) ADS cells can also be used as a source for the regeneration of the ocular surface [122]. The expression of CK12 marker in ADS cell cultures indicates the capacity to acquire epithelial characteristics in appropriate conditions [123, 124]. No in vivo studies have yet been published. 2.3.12. Cultured Induced Pluripotent Stem Cells (iPSC) One study [125] has shown the generation of corneal epithelial cells from iPSs derived from human dermal fibroblast and corneal limbal epithelium, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells. As discussed above the use of cell-based therapies are rational and effective methods for ocular surface recovery. All of them have a high translational

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potential in regenerative medicine of the ocular tissue. However, from the point of view of clinical applicability only the use of CLET and COMET techniques, and to a lesser extent, conjunctival epithelial cells expanded ex vivo, are well documented so far. Other alternative sources of progenitor cells for clinical use have some limitations despite their theoretical advantages. It should be noted that the application of progenitor cells from adult tissue have shown evidence of better adaptation to clinical use in other settings. 3. CORNEAL STROMA The stroma constitutes more than 90% of the corneal thickness. Many features of the cornea, including its strength, morphology and transparency, are attributable to the anatomy and biomechanical properties of the corneal stroma. The extracellular matrix of the corneal stroma is composed of: A) Collagen, which represents more than 70% of the weight of the dehydrated cornea, and the most abundant is type 1 (75%), followed by type VI (17%) and V (2%); and B) Proteoglycans, including keratan sulphate, chondroitin sulphate and dermatan sulphate. Keratan sulphate is the most abundant (65%) and its proteic core is composed of lumican, keratocan and mimecan. Corneal stroma is the only tissue of the complete organism where keratocan is expressed, thus, human keratocan is considered a specific marker of human keratocytic differentiation [126]. The uniform distribution, and the slow but steady replacement of the stromal extracellular matrix are essential for the maintenance of corneal transparency. The cellular component occupies only 2-3% of the stromal volume, where keratocytes are predominant. Keratocytes are cells derived from the neural crest, with a flattened look and stellate morphology, and they are distributed between the lamellae of stromal collagen. These cells project long processes that are connected by gap junctions with the processes of the neighboring cells, thereby forming a three-dimensional lattice that contains the extracellular matrix they secrete. Keratocytes are quiescent in a normal cornea, but in response to various types of aggression they activate their metabolism and transdifferentiate into fibroblasts and myofibroblasts, which are involved in the cicatrization of the tissue. In addition, keratocytes are essential not only for remodeling the corneal stroma (as they produce factors such as collagen, proteoglycans, glycosaminglycans and metalloproteinases necessary for the long-term maintenance of corneal

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transparency), but also stromal-epithelial interactions are key determinants of corneal function, as paracrine mediators are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions of epithelial cells [127] The renewal capacity of stromal keratocytes is not very well defined and until a few years ago, the real presence of stem cells in this population was debated. Recently, a keratocyte precursor located in the anterior limbal corneal stroma (near to stem cells of the corneal epithelium) has been reported, which expresses markers for adult stem cells such as ABCG2 (ATP binding cassette G2), and PAX6 (Paired box 6) [128-130]. 3.1. Stem Cells used for Stroma Regeneration 3.1.1. Keratocytes When keratocytes are expanded in vitro in a serum-containing medium, they lose their in vivo quiescent phenotype and acquire a fibroblastic phenotype with abnormal physiological properties [131]. However, in serum-free cultures they maintain their dendritic morphology and the production of keratan sulphate proteoglycans [132, 133]. Yoshida et al., described a new method for subculturing mouse keratocytes in large quantities in a serum free medium, aimed at maintaining their secretion of the cornea-specific proteoglycan keratocan and the aldehyde dehydrogenase enzyme (ALDH) [134]. To our knowledge, no in vivo studies have yet been reported, so their use in clinical settings is still far away. 3.1.2. Corneal Stromal Stem Cells (CSSC) Unlike keratocytes, human CSSCs (h-CSSCs) undergo extensive expansion in vitro without losing their ability to adopt a keratocyte phenotype [135]. These corneal mesenchymal stem cells have a demonstrated potential for differentiation into corneal epithelium and adult keratocytes in vitro [135, 136]. When cultured on a substratum of parallel aligned polymeric nanofibers, h-CSSCs produce layers of highly parallel collagen fibers with packing and fibril diameter indistinguishable from that of the human stromal lamellae [137]. The ability of hCSSCs to adopt a keratocyte function has been even more striking in vivo. When injected into mouse corneal stroma, h-CSSC express keratocyte mRNA and protein, replacing the mouse extracellular matrix with human matrix components. These injected cells remain viable for many months, apparently becoming

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quiescent keratocytes [138]. However, their use in the clinical setting is obviously restricted due to the need of a disease-free contralateral eye. 3.1.3. Bone Marrow Mesenchymal Stem Cells (BM-MSCs) Recently Park et al., [139], reported that human BM-MSCs differentiate in vitro into keratocyte-like cells when they are grown in specific keratocytedifferentiation conditions. They demonstrated a strong expression of keratocyte markers lumican and ALDH together with the loss of expression of stem cell markers such as α-smooth muscle actin, but they did not achieve an evident expression of keratocan on these differentiated cells [139]. The survival and differentiation of human BM-MSCs into keratocytes has also been demonstrated in vivo when these cells are transplanted inside the corneal stroma of an animal model. Keratocan expression was observed without any sign of immune or inflammatory responses [140]. These data suggest that in the short term these cells could be valable for their clinical use.

Figure 5: Cellular morphology and collagen production of h-ADASCs in culture. Observe the normal fusiform morphology of mesenchymal stem cells. (A) Phase contrast of living cells. (B) Cells stained with CM-DiI. C: Collagen I production in vitro. (D) Collagen VI production in vitro. (A) Magnification 200x. (B);(C);(D): magnification 400x.

3.1.4. Adipose Derived Adult Mesenchymal Stem Cells (ADASCs) Human ADASCs (h-ADASCs) cultured in vitro (Fig. 5) under keratocytedifferentiation conditions express collagens and other corneal-specific matrix

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components. This expression is quantitatively similar to that achieved by the differentiated h-CSSCs [141]. The differentiation of h-ADASCs in functional human keratocytes has also been demonstrated in vivo in a previous study by our group using a rabbit animal model [142]. These cells, once implanted intrastromally, express not only collagens type I and VI (the main components of corneal extracellular matrix) (Fig. 6) but also, keratocyte specific markers such as keratocan or ALDH, without inducing an immune or inflammatory response. The relatively easier extraction and isolation techniques of these cells respect to bone marrow sources, make these cells so far the best candidates for clinical use for stromal regeneration.

Figure 6: Extracellular matrix production profile by transdifferentiated h-ADASCs in vivo in a rabbit model. (A, B): Photomicrographs showing sections showing surviving h-ADASCs labeled with CM-DiI after 3 month follow-up. (C, D): Same sections showing collagen I and VI expression in host rabbit corneal stroma and transplanted h-ADASCs cells. Magnification : 200x. Abbreviations: Epi, epithelium; Str, stroma.

3.1.5. Umbilical Cord Mesenchymal Stem Cells (UCMSCs) Human mesenchymal stem cells isolated from neonatal umbilical cords have shown similar behavior to other types of mesenchymal stem cells when transplanted inside the corneal stroma in vivo, expressing keratocyte-specific markers such as keratocan without inducing rejection. Liu et al., reported that the injection of these cells inside the corneal stroma of lumican null mice improved corneal transparency and increased the stromal thickness with a re-organized collagen lamellae, also

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improving the host keratocyte function through enhanced expression of keratocan and ALDH in these mice [143]. These data are encouraging, but due to the nature of these cells, the autologous use is not possible. 3.1.6. Embryonic Stem Cells (ESCs) Experience with these human pluripotent stem cells is much more limited. Chan et al., reported recently that differentiation of these cells into the keratocyte lineage can be induced in vitro, demonstrating an upregulation of keratocyte markers including keratocan [144]. To the best of our knowledge no studies in vivo with these cells have been performed in the field of regenerative medicine of the corneal stroma. The use of these cells raises ethical issues, and together with the lack of in vivo data so far, discourages for their use in the clinical setting at this time. Summarizing (Table 3), it seems that all types of mesenchymal stem cells have similar behavior in vivo, and thus are able to achieve a keratocyte differentiation and modulate the corneal stroma with immunomodulatory properties. CSSCs may have enhanced functions due to the fact that they are already corneal cells with more directed differentiation potential. However, the number of corneal stromal stem cells that can be obtained from human corneas is quite limited and technically demanding, with inefficient cell subcultures and the inability to obtain them without damaging the donor cornea. These major drawbacks will significantly limit their use in clinical practice and precludes their autologous application, so an extraocular source of cells that could replace CSSCs is necessary to solve all these limitations. Human adult adipose tissue has shown to be an ideal source of autologous stem cells, as it satisfies all the requirements: easy accessibility to the tissue, high cell retrieval efficiency and the ability of its stem cells (h-ADASCs), to differentiate into multiple cell types (keratocytes, osteoblasts, chondroblasts, myoblasts, hepatocytes, neurons, etc.) [142]. This cellular differentiation occurs due to the effects of very specific stimulating factors or environments for each cell type, and avoiding the mix of multiple kinds of cells in different niches. BM-MSCs have the same profile as ADASCs, but their extraction by bone marrow puncture is a more complicated and painful procedure that requires general anesthesia. UMSCs present an attractive alternative, but their autologous use would be expensive and is currently

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impossible. Embryonic stem cells also present important ethical issues. The use of iPS technology [145] could solve such problems, but no studies have yet been performed. Table 3: Stem cells used for the regeneration of the corneal stroma. Evidence of keratocyte differentiation and their possible autologous applications CSSC

BM-MSC

ADASC

UMSC

ESC

Keratocyte differentiation in vitro demonstrated

Yes

Yes

Yes

Yes

Yes

Keratocyte differentiation in vivo demonstrated

Yes

Yes

Yes

Yes

No

Possible autologous use

No

Yes

Yes

Yes/No

No

3.2. Techniques used for Stromal Regeneration All these types of stem cells have been used in various ways in several research projects in order to find the optimal procedure to regenerate the human corneal stroma. These approaches can be classified into four techniques: 3.2.1. Intrastromal Injection of Stem Cells Alone Direct injection of stem cells inside the corneal stroma has been assayed in vivo in some studies, demonstrating the differentiation of the stem cells into adult keratocytes without signs of immune rejection. In our study we also demonstrated by immunohistochemistry the production of human extracellular matrix when hADASCs were transplanted inside the rabbit cornea [142]. As expected, collagens type I and type VI were founded to be expressed in the rabbits’ corneal stroma as well as the transplanted h-ADASCs (Fig. 6), and collagens III and IV, not expressed normally in corneal stroma, were detected neither in the host corneal stroma nor in the transplanted h-ADASCs. Du et al., [138] reported a restoration of the corneal transparency and thickness in lumican null mice (thin corneas, haze and disruption of normal stromal organization) three months after the intrastromal transplant of human CSSCs. They also confirmed that human keratan sulphate was deposited in the mouse stroma and the host collagen lamellae were re-organized, concluding that delivery of h-CSSCs to scarred human stroma may alleviate corneal scars without requiring surgery [138]. Very similar findings were reported by Liu et al., using human UMSCs in the same animal model [143]. Recently, Thomas et al., found that,

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in a mice model for mucopolysaccharidosis, transplanted human UMSC participate both in extracellular glycosaminoglycans (GAG) turnover and enable host keratocytes to catabolize accumulated GAG products [146]. In our experience, the production of human extracellular matrix by implanted mesenchymal stem cells occurs, but not quantitatively enough to be able to restore the thickness of a diseased human cornea. However, the direct injection of stem cells may provide a promising treatment for corneal dystrophies, corneal stroma progressive opacification in the context of systemic metabolic disorders, and for the modulation of corneal scarring. 3.2.2. Intrastromal Implantation of Stem Cells Together with a Biodegradable Scaffold To enhance the growth and development of the stem cells injected into the corneal stroma, transplantation together with biodegradable synthetic extracellular matrixes has been performed. Espandar et al., injected h-ADASCs with a semisolid hyaluronic acid hydrogel into rabbit corneal stroma, reporting better survival and keratocyte differentiation of the h-ADASCs when compared to their injection alone [147]. Ma et al., used rabbit ADSCs with a polylactic-co-glycolic (PLGA) biodegradable scaffold in a rabbit model of stromal injury, observing newly formed tissue with successful collagen remodeling and less stromal scarring [148]. Initial data show that these scaffolds could enhance stem cell effects over corneal stroma, although more research is required. 3.2.3. Intrastromal Implantation of Stem Cells with a Non-Biodegradable Scaffold At the present time, no clinically viable human corneal equivalents have been produced by tissue engineering methods. The major obstacle to the production of a successfully engineered cornea is the difficulty with reproducing (or at least simulating) the stromal architecture. The majority of stromal analogs for tissue engineered corneas have been created by seeding human corneal stromal cells into collagen-based scaffoldings, which are apparently designed to be remodeled (see [149] for a general review of corneal tissue engineering). In addition, new and improved biomaterials compatible with human corneas have been developed

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leading to advanced scaffolds that can be used to engineer an artificial cornea (keratoprostheses), such as poly-hydroxyethyl methacrylate hydrogels, collagenchondroitin sulphate hydrogels, and polyurethanes [150]. The combination of these scaffolds with cells can generate promising corneal equivalents, and some studies have already been published that use mainly corneal cell lines, including stromal keratocytes, providing positive results regarding adhesion and cellular survival in vitro [151]. However, limited research has been done in vivo and using stem cells in combination with this kind of synthetic scaffold. Mimura et al., used corneal fibroblast precursors together with porous gelatin hydrogels in vivo in a rabbit model, detecting an increased expression of type I collagen, but the authors later stated that these scaffolds were weak and unstable [152]. In a recent study by our group, we analyzed the survival and biointegration of scaffolds composed by macroporous membranes of poly-ethyl-acrylate (PEA) colonized by h-ADASCs, inside the rabbit corneal stroma in vivo (Alió del Barrio et al., in press). Despite demonstrating stem cell survival in vivo after 12 weeks, h-ADASCs did not improve the extrusion rate of the biomaterial when compared with PEA membranes implanted without stem cells. Our opinion is that stem cells do not differentiate properly into keratocytes in the presence of these synthetic biomaterials. They lose the potential benefits demonstrated with biodegradable scaffolds or by isolated injection, and fail to encroach on the biomaterial despite a macroporous structure. However, these types of materials could be used to anchor rings of future keratoprosthesis developments. 3.2.4. Intrastromal Implantation of Stem Cells with a Decellularized Corneal Stromal Scaffold The complex structure of the corneal stroma has not been yet replicated, and there are well-known drawbacks to the use of synthetic scaffold-based designs: strong inflammatory responses induced upon their biodegradation, and nearly all polymer materials cause a nonspecific inflammatory response. Recently, several corneal decellularization techniques have been described, which provide an acellular corneal extracellular matrix (ECM) [153]. These scaffolds have gained attention in the last few years as they provide a more natural environment for the growth and differentiation of cells when compared with synthetic scaffolds. In addition, components of the ECM are generally conserved among species and are

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tolerated well even by xenogeneic recipients. As previously noted, keratocytes are essential for remodeling the corneal stroma and for normal epithelial physiology [127]. This highlights the importance of transplanting a cellular substitute together with the structural support (acellular ECM) to undertake these critical functions in corneal homeostasis. To the best of our knowledge, all attempts to repopulate decellularized corneal scaffolds have used corneal cells [154-156] but as already discussed, these cells have major drawbacks that preclude their autologous use in clinical practice (damage of the donor tissue, lack of cells and inefficient cell subcultures), thus the efforts to find an extraocular source of autologous cells. In a recent study by our group, we showed the perfect biointegration of human decellularized corneal stromal sheets (100 µm thickness) with and without hADASCs colonization inside the rabbit cornea in vivo (Fig. 7A, B), without observing any rejection response despite the graft being xenogeneic (Alió del Barrio et al., sin press. We also demonstrated the differentiation of h-ADASCs into functional keratocytes inside these implants in vivo, which then achieved their proper biofunctionalization (Fig. 7C, D). In our opinion the transplant of stem cells together with decellularized corneal ECM would be the best technique to effectively restore the thickness of a diseased human cornea. Through this technique, and using extraocular mesenchymal stem cells from a patient, it is possible to transform allergenic grafts into functional autologous grafts, theoretically avoiding the risk of rejection. 4. CORNEAL ENDOTHELIUM The human corneal endothelium plays a crucial role in corneal transparency because it controls the movement of ions and water into the hydrophilic stroma [157] by actively moving fluid from the stroma back into the anterior chamber [158, 159]. It covers the entire posterior corneal surface, and at this periphery it reaches the trabecular meshwork (TM) in the angle between the cornea and iris root. Corneal endothelial cells (CECs) are derived for the cranial neural crest [160], and they form a monolayer of hexagonal cells organized in a tessellated mosaic on their basal membrane, the Descemet´s membrane (DM). Central endothelial cell density (ECD, expressed in cells per mm2) decreases at an average rate of about a 0.6% per year in normal corneas throughout adult life, with gradual increases in polymegathism and pleomorphism [161]. In a normal individual, this decline in endothelial cells (EC)

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does not impair corneal transparency, even in centenarians. However if the density falls below the threshold of 300-500 cells per mm2, irreversible corneal edema can lead to blindness [162]. This decline can occur following intraocular surgeries, traumas, or in dystrophies. In fact, blindness due to corneal edema is the indication for corneal grafting in one of every three recipients.

Figure 7: Reconstruction of corneal stroma. (A) Hematoxylin-eosin staining of a rabbit cornea with an implanted graft of decellularized human corneal stroma with h-ADASC colonization. (magnification 200X); (B) The graft remains totally transparent after 12 weeks of follow-up (magnification 2X) (arrows point to the slightly visible edge of the graft). (C) Human cells labeled with CM-DiI around and inside the implant confirming the presence of living human cells inside the corneal stroma (D) Same section showing human keratocan and thus their differentiation into human keratocytes (arrows) (magnification 400x). Abbreviations: Epi: epithelium; Str: stroma.

As the human corneal endothelium is held in a nonreplicative state within the eye [163], currently, the only effective and proven way to restore endothelial function is to perform an allogenic graft. Recently, corneal grafting techniques have evolved into endothelial keratoplasty, which has replaced the more invasive penetrating keratoplasty for the treatment of endothelial dysfunction [164]. These techniques include partial-thickness posterior lamellar grafts such as Descemet stripping automated endothelial keratoplasty (DSAEK) or even isolated transplantation of Descemet membrane such as in Descemet membrane endothelial keratoplasty (DMEK).

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Although human corneal endothelial cells (HCECs) are not able to actively divide within the eye, ex vivo mechanical wounding studies and treatment of HCECs using EDTA have shown that they retain the capacity to proliferate [165]. The possibility of using one donor cornea for the treatment of multiple patients by expansion of primary HCECs in vitro and transplantation of functional tissueengineered HCECs could alleviate the projected growing demand for transplant tissue and the global shortage of donor corneas. Immune rejection may also be reduced with the use of isolated Descemet membrane transplantation instead of other corneal graft techniques [166], because a monolayer of EC transplantation is less antigenic than full corneas or posterior lamellar grafts as in DSAEK. 4.1. Homeostasis of Corneal Endothelium It has been a common belief that in vivo, corneal endothelium has limited woundhealing capacity, mainly through residual EC which, by enlargement and migration, cover the space left by the lost cells without division [167]. Joyce et al., demonstrated that HCECs are arrested in the G1-phase of the cell cycle in vivo [168]. Mitotic inhibition has been suggested to be due to contact dependent inhibition and the transforming growth factor beta (TGF-ß) found within the aqueous humor [163]. However, a series of clinical observations suggests the possibility of endothelial regeneration in vivo from the human corneal periphery. Higher ECD in the endothelial periphery was described a few decades ago [169]. There is a longer survival of corneal grafts in patients with high ECD in the endothelium periphery such as in keratoconus, suggesting that the central endothelium is continuously sustained by the peripheral reserve [162]. Similarly, there is a rapid decrease in central ECD after corneal grafts when no ECs persist in the recipient’s corneal periphery, which typically occurs in diseases with corneal edema including the periphery. Recently, corneal clearance was observed after re-endothelization of the recipient posterior stroma after transplanting a free floating donor DM graft in the recipient anterior chamber after removing the recipient endothelium and DM. This corneal clearance was reported in corneas with endothelial dystrophy (primarily a DM disorder not affecting the periphery) but not in bullous

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keratopathy (primarily an endothelial depletion in all the cornea). This difference suggests that the remaining rim of recipient endothelium (after descemetorhexis) is involved in the re-endothelization of the recipient posterior stroma rather than the free floating cells from the donor [170]. In vitro, HCECs have shown residual proliferative capacity, even in very old donors [165], and this mitotic activity is higher in the peripheral ECs than in central ECs [171]. Whikehart et al., in 2005 discovered endothelial precursors in the human corneal endothelium for the first time [172]. They reported the presence of these cells adjacent to the endothelial periphery at the limbal zone, and found stem cell markers (nestin, alkaline phosphate, telomerase, Oct-3/4 and Wnt-1) [173] in these cells, sequestered in niches located deep in the TM or in the so-called transition zone (TZ) between the endothelial edge and the TM. Recently He et al., identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches [162]. They propose that there is a renewal zone in the “non-visual” periphery of the cornea in which ECs divide very slowly and migrate towards the center but probably desquamate, while the central ECs in the optical axis remains stable. Cell progenitors would emerge from specific niches located in the periphery of the cornea and lose their dividing capacity, but not their migrating potential, once they make contact with the aqueous humor and form a monolayer. These cells may then be continuously pushed toward the center and retain some proliferative capacity in experimental conditions. 4.2. Cells used for Endothelial Regeneration 4.2.1. Isolation and Culture of HCECs Isolation and cultivation of primary HCECs used to be performed by using an explant culture method and allowing endothelial cells to migrate and grow outside the explant [174]. Currently, isolation of HCECs involves a two-step, peel-anddigest method [175]. The DM-endothelial layer is carefully peeled off from the donor cornea and then subjected to enzymatic digestion to free the ECs from the DM, using collagenase [176], dispase [177] or trypsin/EDTA [178] (Fig. 8).

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Figure 8: Culture of human endothelium. Magnification 200x.

The use of an extracellular matrix (ECM) coated surface has also been used to promote endothelial cell attachment, including collagen, laminin, chondroitin sulfate, fibronectin or a mixture of several of these products [175]. A wide range of complex serum-supplemented culture media developed from various basal media with the addition of mitogenic factors have been shown to induce HCEC proliferation (Table 4). Table 4: Culture conditions for corneal endothelial cells Researcher Group

Cells

Difestion

Basal Medium Serum Supplement

Coating

References

Amano S and Yamagami

HCECs

Explants no digestion

DMEM

15% 2 ng/ml b-FGF

Bovine CE EM

[197]

HCECs

Collagenase A 2mg/ml and then 0,05% trypsinEDTA

DMEM

15% 2 ng/mL bFGF Asc-2P (0.3 mM)

Aterocollagen [209]

Joyce NC

HCECs

0.2 mg/mL EDTA 1h

OPTIMEM-1

8% 5 ng/ml EGF FNC coating mix 20 ng/ml NGF 100 µg/ml pituitary extract 20 µg/ml ascorbic acid, 200 mg/l calcium chloride 0.08% chondroitin sulfate

[181]

HCECs

0.2 mg/mL EDTA 1h

OPTIMEM-1

8% 40 ng/ml of FGF 5 ng/ml of EGF 20 ng/ml of NGF 20 µg/ml of ascorbic acid 0.005% human lipids 200 mg/l of calcium

[178]

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Table 4: contd… chloride 0.08% chondroitin sulfate 1% RPMI-1640 multiple vitamin solution Chen KH and Hsiue GH

HCECs

1.2 U/ml of Dispase OPTIMEM-1 II 1h

15% 40 ng/ml FGF PNIPAAm 5 ng/ml EGF 20 ng/ml NGF 20 µg/ml ascorbic acid, 0.005% human lipids 0.2 mg/ml of calcium chloride 0.08% chondroitin sulfate 1%1640 vitamin solution

[191

Kinoshita S

HCECs

1.2 U/mL Dispase 1h

DMEM

10% 2 ng/ml b-FGF

[177]

monkey CECs

0.6 U/mL of Dispase II 1h

DMEM

10% 2 ng/ml b-FGF ( and Y-27632 at 10 µM)

monkey CECs

0.6 U/mL of Dispase II 1h

DMEM

10% 2 ng/ml b-FGF ( and Y-27632 at 10 µM)

FNC Coating Mix

[207]

monkey CECs

1.2 U/mL dispase 20'

DMEM

10 % 2 ng/ml b-FGF

FNC Coating Mix

[193]

Mehta JS

HCECs

Collagenase 2 mg/mL 2h + TrypLE express 5'

Human Endothelial SFM unsuppleme ted O/N and then F99

5% 21 μg/ml ascorbic acid FNC Coating Mix insulin-transferrinselenium 10 ng/ml bFGF

[185]

Engelmann

HCECs

F99

5% 20 μg/ml ascorbic acid 20 μg/ml insulin 2,5 μg/ml transferrin 0,6 ng/ml selenium 10 ng/ml bFGF

[210]

Blake

HCECs

Trypsin over endothelial surface

MEM

10% horse

0,25 µg/ml amphotericin B 5 µg/ml insulina 5 µg/ml transferrina 5 ng/ml sodium selenite (hCE basal medium) 150 µg/ml ECGS. HGF and EGF (0.1-1-10-100 µg/ml)

Collagen IV

[183]

Bovine CE EM

[111]

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Table 4: contd… Soker S

HCECs

0.2% Collagenase II EGM-2 1h

10% EGF, VEGF, FGF, IGF, hydrocortisone,

Fibronectin or [154] Collagen type IV

Tseng SCG

HCECs agregates

2mg/ml Collagenase A 1,516h

SHEM

5% 0,5% DMSO 2 ng/ml EGF 5 µg/ml insulin+5µg/ml transferrin+5ng/ml selenium 0,5µg/ml hidrocortisone 1nm cholera toxin 40 ng/mLbFGF 0.1 mg/mL BPE 20 ng/mL NGF

[176]

Wang Z

HCECs

Explants no digestion

OPTIMEM1+25% ESCCM

8% 40 ng/ml FGF 5 ng/ml EGF 20 ng/ml NGF 20 μg/ml ascorbic acid 0.005% human lipids 200 mg/l calcium chloride 0.08% chondroitin sulfate 1% RPMI-1640 multiple vitamin solution

[212]

HCEC proliferation and consistent long-term culture are still very challenging. The correlation between donor age [179] (older donor HCEC cultures proliferate less and slower) and endothelial topography [180] (HCEC cultures from the peripheral ring have more proliferative cells) needs to be taken into consideration, as well as the length of time between death, enucleation, and preservation of donor corneas to the isolation and culture of CECs [181]. Cellular replicative senescence [165] and endothelial mesenchymal transition [182] are common obstacles to establishing consistent long-term cultures of HCECs for ex-vivo corneas or for corneal bioengineering. A recent study reported that selective ROCK inhibitor, Y-27632, inhibited dissociation induced apoptosis and promoted adhesion and proliferation of CECs isolated from cynomologous monkeys [183]. Rho-kinase (ROCK) is a serine/threonine kinase, which serves as a target protein for Rho, and the

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Rho/ROCK pathway is involved in regulating cytoskeleton, cell migration, cell proliferation, and apoptosis [184]. Other authors have improved the culture of HCEC by applying a system consisting in a first proliferation medium and a second expansion medium that prevents the endothelial to mesenchymal transition described in sequential passages of these cells [185]. 4.2.2. Use of Endothelial Cell Precursors Some authors have derived CECs from corneal endothelial precursors using a sphere forming assay, these cells retain their pump functionality [186]. Moreover, corneal endothelial precursors reached successful outcome when injected into the anterior chamber of a pathological cornea model [187]. An sphere system was also used to differentiate CECs from corneal stroma precursors [188]. These cells also had an appropriate pump function and reached successful results when cultured over collagen sheets and adhered to corneal buttons in a rabbit model of penetrating keratoplasty [188]. 4.2.3. Differentiation from Neural Crest Cells Likewise, EC-like cells have been derived from neural crest cells and transplanted into host corneal endothelium [189] following Mimura´s system [187]. These cells were able to cover the full edthelial surface, and decrease corneal thickness and edema [189]. 4.3. New Techniques used for Corneal Endothelial Regeneration 4.3.1. Transplantation of Tissue Engineered Corneal Endothelial from HCEC There is growing interest in developing clinically suitable alternatives for donor graft material to alleviate the increasing demand for transplantable corneal tissue worldwide. Efforts have been directed towards expansion of primary HCECs in vitro and the development of a system that enables delivery and transplantation of functional tissue-engineered endothelium, so one donor can provide treatment for multiple recipients. For transplantation experiments several cell carriers have been tested, such as: amniotic membrane [177], collagen derived carriers [190-194], full cornea or corneal derived carriers [154, 188, 195-202] and other biomaterials [203].

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Cultivated monkey corneal endothelial sheets using collagen type I as a carrier have been successfully implanted into monkey eyes, and acquire adherence to the posterior surface of the cornea in a similar manner as with other endothelial keratoplasty procedures [204]. Interestingly, in the postoperative period the cell sheets became detached from the posterior surface of the cornea, but the corneas still achieved full clarity. This finding lead to speculation that once cultivated in vitro, monkey corneal endothelial cells might recover their proliferative ability and migrate onto the host Descemet’s membrane and proliferate in vivo. Human corneal endothelial cells have also successfully been delivered into animal models using carrier donor posterior stroma tissue as in DSAEK [195, 204]. Other carriers have been suggested for this purpose, such as plastic compressed collagen [205], or nanocomposite gel sheet [206]. 4.3.2. Direct Injection-Cell Therapy The observations previously mentioned [170], as well as the finding that monkey CECs were able to migrate out of the transplanted bio-engineered cell sheet into the posterior corneal stroma and proliferate until reaching cell confluence [204], have created high expectations for direct cell therapy with no need for a sophisticated bio-engineered carrier. Transplantation of cultivated corneal endothelia by cell-injection into the anterior chamber of the eye, combined with Rock inhibitors and strict eye positioning, has allowed deposition of the cells onto the posterior surface of the cornea. Further attachment and proliferation lead to the recovery of host cornea transparency in a monkey model [204]. 4.3.3. Eye Drop Therapy for In Vivo Endothelial Regeneration The success of ROCK inhibitor with in vitro and in vivo transplanted HCEC proliferation and adhesion lead to the idea that they may also promote proliferation of healthy CECs in the periphery of corneas with central disease. In an animal model of corneal endothelial injury, topical administration of selective ROCK inhibitor, Y-27632, enhanced corneal endothelial wound healing [207]. Similar results were obtained in a partial corneal endothelial dysfunction model in cynomolgus monkeys [204].

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Annex Figure: Most frequent corneal pathologies that can be addressed using cell-based therapies approaches. (a) Limbic deficiency secondary to ocular cicatricial pemphigoid with "conjuntivalization" of corneal epithelium and the presence of newly formed vessels which lead to a loss of corneal transparency. Limbal stem cell deficiency (LSCD) is the only corneal pathology which can be treated using current cell therapy techniques, such as CLET or COMET. (b) Central corneal leucoma due secondary scarring from Acanthamoeba keratitis. (c) Fuchs endothelial dystrophy with loss of central corneal transparency due to the failure of the corneal endothelium function.

CONCLUSION To date, the search for innovative strategies and approaches in the field of ocular surface reconstruction has produced some encouraging results. An emerging alternative source is adult stem cells for therapeutic purposes, which provide high proliferative potential, differentiation capability, less immunogenicity, are nontumorigenic, and can be obtained by minimally invasive methods. They represent a more physiological, more rational, and less invasive treatment. However, the selection of the best cell source and techniques still need to be investigated. One of the key elements to consider is the role of the cellular microenvironment or niche. In the future, greater understanding of the behavioral characteristics of the ocular stem cell niche as well as proliferation and differentiation pathway events will allow us to generate corneal replacements of one or several of its layers.

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Annex Table: Etiologic classification of corneal pathology that can be treated by cell-based therapy Epithelial diseases

Primary LSCD

Hereditary

Aniridia Ectodermic dysplasia Polyglandular autoimmune syndromes

Secundary LSCD

Neoplasic

Intraepithelial neoplasia

Degenerative

Recurrent pterygium

Trophic

Neurotrophic keratitis

Mechanical

Alkali, acids, thermal burns

Anoxic

Contact lenses misuse or prolonged use

Infeccitions

Severe infeccious keratitis

Chronic inflammations

Collagen diseases related ulcers Mooren ulcer Stevens-Johnson syndrome Ocular pemphigoid Atopic keratoconjunctivitis Superior limbic keratoconjunctivitis Ocular rosacea Vitamin A deficiency

Stromal diseases

Melting

Infectious

Herpes, Bacterial, Parasitic diseases

Autoimmune

Rheumatoid arthritis Sjogren´s syndrome

Thinning

Keratoconus Pellucid marginal degeneration

Endothelial diseases

Primary Endothelial Failure

Fuchs Dystrophy

Secondary Endothelial Failure

Surgical trauma

Glaucoma, retinal or cataract surgery

Infectious

Keratouveitis (Herpes)

LSCD: limbal stem cell deficiency

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ACKNOWLEDGEMENTS Part of this work was funded by Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III (FIS09-PI040654), MICINN SAF2010-19230, Dirección General de Terapias Avanzadas y Trasplantes del Ministerio de Sanidad y Politica Social (TRA-53, TRA-072 and EC-11-129), and Fundació Marató TV3 (120630). The authors acknowledge Juliette Siegfried (ServingMed.com) for the English editing of this manuscript. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. REFERENCES [1] [2]

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139

Subject Index A Abnormalties 10, 14, 18 Acute GVHD 34, 35 Acute myelogenous leukaemia (AML) 31, 43 Adhesion 71, 72, 114, 123 cell 66, 75, 78, 80 Adipose stromal cell (ASC) 10, 11, 13, 15, 18, 19 ALDH 108-111 Anti-retroviral therapy 41 APC 46 Aqueous humor 117, 118 Areas, hypercellular 21, 22 Ascorbic acid 119-121 Auto-HSCT 31, 32, 49 Autologous applications 111, 112 Autologous cells 103, 115 Autologous epithelial cells 104 Autologous stem cells 92, 111 Autologous T-cells 50, 51

B Basal layer 94, 96-98, 100 Basal media, neural 84 B-cells 37, 38, 40, 44, 52 including 53 Biocompatibility 77, 82 Biology, stem cell 7, 65 Biomaterials 114, 122 Biomimetic 65, 66, 80 Blindness 116 Blood vessels 94, 95 Bone engineering 3 Bone marrow stromal cell (BMSC) 4, 7, 9, 11, 13, 15, 18, 19 Bone marrow transplant (BMT) 31, 42 Bowman’s layer 93, 94

C Calcium chloride 119-121 Calvarial 10, 11 Cancer cells 49, 50 residual 32 Cancer patients 32, 51 Capacity 68, 74, 77, 94, 106, 117, 118 high self-renewal 95, 96 moderate self-renewal 95, 96 Carcinoma growth, sites of 5 CAR redirects T-cell specificity 50 CAR T-cells 50, 51

mature 52 naïve 51 CAR T-cell therapy 51 Cartilage engineering 3 Catenin signaling 44 Cell-based therapy 92, 103 Cell culture 65, 92, 100-103, 106 Cell growth 102, 103 Cell population 5, 94, 96, 103 Cell proliferation 3, 8, 12, 21, 75, 94, 122 Cells 4, 6, 10, 18, 22, 30, 35, 36, 43-45, 48, 51, 52, 66, 68, 71, 75, 76, 78-80, 83, 94-97, 101-107, 109-111, 114-116, 118, 119, 122, 123 active 93 conjunctival epithelium 103 corneal epithelial phenotype 105 effector 47 engineered K562 53 feeder 65, 79 feeder layer 105 female germ 67 free floating 118 germ-cell-derived 73 graft-derived 34 hair follicle 97 haploidentical Natural Killer 32 hESC-derived 65 hESC-derived EB 83 hexagonal 115 human somatic 67 immature myeloid 45 included multinucleated giant 9 injected 108 malignant 50 mesenchymal 94 mononuclear 22 murine 4 neighboring 107 nerve 82 neuronal 83 oral mucosal 104 photoreceptors 97 placental 67 plasma 22 post-mitotic 95 presenting 49, 50, 94, 103 proliferative 121 regenerate 75 rived 71 rounded typical chondrocytic 13

Atta-ur-Rahman & Shazia Anjum (Eds) All rights reserved-© 2015 Bentham Science Publishers

140 Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 1 support 75 transformed 38 transient amplifying 94-96 transplanted 7, 9, 13 transplanted h-ADASCs 110 Cells used for endothelial regeneration 118 Cell suspensions 101, 102 Cellular proliferation 7, 8, 12, 16, 17, 21, 22, 77 Chemotherapy 30, 32, 33, 36, 38 Chimeric antigen receptor (CAR) 50-52 Chondrogenesis 3, 9, 12, 17 Chondroitin sulfate 119-121 Chronic lymphocytic leukaemia (CLL) 31 Collagen IV 69, 72-74 Collagens type 110, 112 Collagen type 72, 73, 121 Contralateral eye 101, 102 Cornea damaged 104, 105 engineered 113 Corneal cells 92, 111 Corneal clearance 117 Corneal edema 116, 117 Corneal endothelial cells (CECs) 115, 117, 119, 121-123 Corneal endothelial precursors 122 Corneal endothelium 93, 94, 115, 117 human 115, 116, 118 Corneal epithelial cells 96, 105, 106 Corneal epithelial differentiation 106 Corneal epithelium 93-95, 98, 100, 108 Corneal grafts 117 Corneal layers 92 Corneal stroma 98, 107, 109-116 Corneal stromal cells 105 Corneal tissue 94, 113 Corneal tissue regeneration 106 Corneal transparency 93, 94, 107, 112, 115, 116 Cultured conjunctival epithelial cells 102, 104 Cultured embryonic stem cell 102,105 Cultured epidermal adult stem cells 104 Cultured immature dental pulp stem cells 102, 105 Cultured induced pluripotent stem cells 102, 106 Cultured Orbital Fat-derived Stem Cells 106 Cultured umbilical cord-lining stem cells 102, 106 Culture stem cells 77 Cytokeratin 97, 98 Cytokines 5, 34, 35, 38, 40, 42, 68, 69, 99 Cytologic atypia 9, 13, 17, 21 Cytology 8, 17, 21, 22 Cytoplasm 97

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D Deficiency, limbal stem cell 94, 102 Dendritic cells 53 monocyte-derived 50 Descemet´s membrane (DM) 115, 117, 118 Descemet membrane endothelial keratoplasty (DMEK) 116 Descemet’s membrane 93, 94 Descemet stripping automated endothelial keratoplasty (DSAEK) 116, 117, 123 Development, thymocyte 42, 44, 45 Differentiated cells 75, 92, 94-96, 109 derived 76 Differentiation keratocyte 111-113 subsequent 77, 80 Differentiation capacity 73, 79 Direct injection of stem cells 112 Disease type 33, 34, 50 DN3 cells 45 Donor corneas 111, 117, 118, 121 Donor lymphocyte infusion (DLI) 41, 47, 50 Donor T-cells 32, 35, 40, 51, 53 mature 51

E EBV-transformed lymphoblastoid cells 53 EC-like cells 122 ECM proteins 69, 70, 72 various 72, 74 Effects, mediate GVT 52 Electrospinning 65, 66, 82 Electrospun nanofibrous substrates 66, 83, 84 Embryonic stem cells 65-67, 82, 84, 100, 105, 111, 112 Embryonic stem cells (ESCs) 65-68, 71, 73, 80, 8284, 100, 105, 111, 112 Endothelial cells human corneal 117, 123 monkey corneal 123 Endothelial regeneration 117, 118 Endothelium, recipient 117, 118 Epidermal growth factor (EGF) 83, 119-121 Epithelial cells81, 93-96, 99, 100, 104, 108, 115, 118 autologous conjunctival 104 basal 100 conjunctival 94, 104, 107 gastrointestinal 33 gut 42 heterologous 103 human conjunctival 104 human corneal 104

Subject Index

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limbal 99 limbic 96 thymic 36, 40 Epithelium 92, 93, 96, 98, 110, 116 Epstein Bar Virus (EBV) 34, 50 ESCs Ethylene terephthalate 79 Event-free survival (EFS) 34 Explant techniques 101, 102 Extracellular matrix 69, 75, 107, 119 production of human 112, 113 Ex vivo expansion of LSCs 101, 102

F Feeder layers 65, 69, 70, 79 Fibroblast growth factor (FGF) 99, 119-121 Fibroblasts 101, 103, 107 Fibronectin 69, 72-75, 77, 119, 121 FNC coating Mix 119, 120 Formation, sarcoma 4, 5, 17 Functional B-cells reconstitution 37

G Gastrocnemius muscle (GM) 19 Germ layers 67, 77, 79, 82 Graft-versus-host disease (GVHD) 32, 34, 38-40, 42, 50-53 Graft-versus-Leukaemia (GVL) 32, 49 Graft-versus-Tumour (GVT) 32 Growth factors 40, 44, 67, 69, 75, 99 keratinocyte 30, 40, 42, 99 transforming 99 GSK3 30, 46-49 GVHD exacerbation of 42, 43, 52 GVHD effector T-cells 34 GVT effect 53

H H-ADASCs 109, 111-114 differentiation of 110, 115 transplanted 112 Haematopoeitic stem cell transplantation (HSCT) 30, 31, 33-37, 39-41, 43, 47, 50, 53 Haplo-HSCT 35 HCEC proliferation 119, 121 HCECs collagenase 119, 120 HCECs explants 119, 121 Hepatocyte-growth factor (HGF) 5, 99, 120 HESC colonies 69, 79

HESC expansion 65, 66, 69, 70, 76, 78 HESCs culturing 70 pluripotent 67, 72 surface of 70 HESC stemness 72 H&E staining 21, 22 Homeostatic proliferation of T-cells 35 Homeostatic T-cell proliferation 38 promoted 40 Homeostatic T-cell proliferation accounts 38 Homing, stem cell 32, 36, 39 HSCs 37, 39, 45, 51, 52 human 37 HSC sources 35 HSCT, non-TCD 36, 40, 42 Human corneal endothelial cells (HCECs) 117-123 Human corneas 93, 97, 111, 113 Human embryonic stem cell culture 65 Human embryonic stem cells 65, 73, 80, 105 Human extracellular matrix 112, 113 Human immature dental pulp stem cells 105 Human lipids 119-121 Human mesenchymal stem cells 110 Human MSC 4-6, 20 Hyaluronic acid 72, 74, 77 Hydrogels 74, 77, 79 dextran 74, 77

I IL-7 treatment 40 IL-15 treatment 41, 42 IL-2-treated T-cells 49 Immune reconstitution (IR) 30, 34, 37, 39-44, 49, 53 Immuno-suppression 10, 14, 18 Infarcted myocardium (IM) 18, 19 Inflammatory responses 109, 110 Inoculation of precursor T-cells 52 Intermediate levels of Wnt activation 45 Intrastromal implantation of stem cells 113, 114 Intrastromal Injection of Stem Cells 112 Invasive biopsy 101, 102

K Keratinocyte growth factor (KGF) 30, 40, 42, 43, 99 Keratocan 107, 109-111 Keratocyte-differentiation conditions 109 Keratocytes 93, 94, 107-111, 114, 115

142 Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 1

L Laminin 69, 72-74, 99, 119 Late memory T-cell skewing 35 Layers endothelial 93 suprabasal 98 Leukaemia B-cells 51 Leukaemia patients 31, 41, 49 Leuprolide 43 Leutinizing-hormone-releasing hormone (LHRH) 43 Limbal and Conjunctival epithelium 98 Limbal and Corneal epithelium 98 Limbal cells 104 Limbal epithelium 94, 98-100 Limbal stem cells (LSCs) 92, 94-97, 99-103, 105 Limbic region, healthy 101, 102 Limbus 94-96, 101, 105 Living cells 109 Long-term culture 4 LSCs proliferation 99, 100

M Malignant transformation 4, 5, 20 Markers 9, 13, 17, 38, 68, 82, 92, 96-98, 100, 105, 107, 108, 110 cellular 97 molecular 96, 98 putative 97 Marrow-derived mesenchymal stem cells Rat 102 MatrigelTM 65, 66, 69, 72-74, 79 Mature graft-derived donor T-cells accounts 35 Mature T-cells 44, 53 developed 52 expanded 52 generated 52 Mechanical properties 76-78 Mechanical signals 75, 76 MEF-conditioned media 72-74 Membrane basement 93, 94, 99 synovial 13, 14 Memory cells 35, 47 central 47 late 36 Memory T-cells 41, 48, 53 central 48, 51 effector 40 Mesenchymal stem cells 3, 4, 7, 9, 11, 13, 15, 19, 22, 40, 66, 82, 102, 105, 106, 108-111, 113, 115 Metastasis 5, 23 Method, feeder 69

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MHC matching 33, 34 MHC/TCR interactions 50, 51 Monolayer 103, 105, 115, 117, 118 Morbidity 33, 34, 38, 53 Mouse embryonic fibroblasts (MEFs) 69, 70, 73, 74, 79, 84 MSC implantation 3, 6, 13, 17, 23 MSC muscle formation 16, 18 Muscle engineering 3 Muscle tissue 3, 6 Myogenesis 3, 16, 17

N Naïve T-cell proliferation 49 Naïve T-cells 36, 38, 48 generated 36 Naïve T-cells acts 35 Nanofiber substrates 65 Nanofibres 80, 82-84 aligned 83, 84 Nanofibrous substrates 65, 66, 84 Natural killer (NK) 30, 35, 52 Natural polymers 76-78, 83 Neural crest cells 122 Neurons 83, 84, 111 NGF 119-121 Niches, stem cell 66, 80 NK cells 44, 53 functional 53 NK cell transplantation 53 NMA conditioning 33

O Ocular surface 92, 94, 96, 99-102, 106 Ocular surface bio-replacement 100, 102 Ocular surface reconstruction 100, 104 Ocular surface regeneration 92, 103 Oral mucosal epithelial cells 104 Osteogenesis 3, 7, 17, 83 Overall survival (OS) 33, 34

P P-cadherin 97, 98 Peripheral blood (PB) 31, 32, 36-40 Periphery, endothelial 117, 118 Perivascular stem cells (PSC) 5 PHA-activated T-cells 48 Phase contrast of living cells 109 Phenotype, naïve T-cell 38, 52 Plasticity of Embryonic Stem Cells 67 Pluripotency 70, 72, 74, 76 retained 73, 74, 76, 77

Subject Index

Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 1 143

Poly-ethyl-acrylate (PEA) 114 Poly-L-lactide acid (PLLA) 82 Polymers 76, 78, 81 Polymorphonuclear 22 Polyurethane 76, 83, 84, 114 Post-transplant 37, 38, 43, 47 human immune cells 37 Precursor T-cells 52 Primary HCECs 117, 118, 122 Progenies, various 94 Progenitor cells 44, 102, 103, 107 conjunctival epithelial 104 hESC-derived neural 83 multipotent 45 Progression free survival (PFS) 33 Proliferation, homeostatic 35, 38, 39 Proliferation rate 74 Proportion, increased 48 Proteoglycans 75, 107

R Recent thymic emigrant (RTEs) 38, 40, 43 Reduced intensity conditioning (RIC) 33, 36 Re-endothelization 117, 118 Residual host T-cells 39, 40 Retinoic acid 67 ROCK inhibitor, selective 121, 123 Routes of T-cell reconstitution 39

S Safety assessments 3, 7, 10, 14, 16, 18, 21 Safety assessments performed 10, 14, 18 Safety parameters 8, 12, 16, 20 evaluation of 6 Sarcomagenesis 3 Sarcomas 5, 21, 23 Scaffolds 66, 75-78, 80, 83, 84, 113, 114 biodegradable 113, 114 synthetic 114 Self-renewal capability 95, 96 Serum replacement 70 Severe combined immunodeficiency disease (SCID) 31 Sex steroid inhibition 43 Similarities, immunophenotypic 5 Skewing, memory T-cell 40, 47 Soleous Muscle 18 Stem cell engraftment 8, 12 Stem cell lymphoid differentiation 36 Stem cell markers 109, 118 Stem cells 3, 4, 6, 9, 31, 38, 66, 80, 82, 92, 94, 96, 97, 101, 106, 108, 111-115

adult 75, 102, 104, 108 blood-derived somatic 82 bone marrow mesenchymal 7, 9, 13, 109 bulge-derived 102 corneal 92 corneal mesenchymal 108 corneal stromal 108, 111 derived 13 derived mesenchymal 66, 102 differentiated 75 donor 52 extraocular 92 generated CAR-modified 51 haematopoietic 82 hematopoietic 99 human 38, 46 human pluripotent 111 implanted mesenchymal 113 induced pluripotent 4, 67 limbal 92, 94, 95, 101 marrow-derived mesenchymal 82 mobilized peripheral blood 31 mouse embryonic 67, 105 multipotent 106 murine bone marrow mesenchymal 4 neural 66, 82 perivascular 5 pluripotent 4, 104 putative epidermal 104 somatic 101 umbilical cord blood 66 undifferentiated embryonic 83 using extraocular mesenchymal 115 Stem cell therapy 65, 75 Stroma, recipient posterior 117, 118 Stromal cells 99 feeder 52 human adult mesenchymal 92 human bone marrow 69 human corneal 113 limbal 99 mesenchymal 95 thymic 42 Substrate coating 73, 74 Surfaces, typical tissue culture plastic 72, 78 Synovial membrane stem cells 19 Synthetic polymers 65, 76-78, 83

T T-cell compartments 38, 47, 53 T-cell development 30, 38, 44, 45 T-cell differentiation arrest 48

144 Frontiers in Stem Cell and Regenerative Medicine Research, Vol. 1 delayed naïve 47 inhibited effector 48 inhibited memory 47 mature 47 memory 48, 49 naïve 47, 48 naïve-to-memory 35 T-cell expansion 38, 47, 48, 50 T-cell expansion in PB of mice 38 T-cell immunity 41, 42 T-cell numbers 40, 41, 47 T-cell precursors 30, 39, 45, 51, 52 T-cell receptor (TCR) 35, 38, 45 T-cell reconstitution 30, 36, 37, 39, 40, 42-44, 50, 52, 53 improved 37, 42 T-cell reconstitution in GH-deficient mice 44 T-cells 30, 32, 34, 35, 38, 39, 41, 42, 44, 47-53 blood-derived 47 cell-generated 36 circulating 40 exhausted 51 expanded 49 generated 30, 36, 40, 47 haploidentical 50 healthy 49 homeostatic proliferation of 40 human 37, 38, 47-49 mature human PB-derived 52 regulatory 41 tolerant 51 transferred 53 T-cells and NK cells 41 T-cell subsets 37, 47 TCR repertoire 35, 36, 39, 49, 52 Terminally differentiated cells (TDC) 95, 96 TGF 67, 70, 74, 99 Thymic epithelial cells (TECs) 36, 39, 40, 42, 44 Thymic function 30, 36, 38-40, 43 Thymic output 39, 47 Thymocyte differentiation 44 Thymocytes 37, 42, 44, 45 developing 44, 45 Thymopoeisis 36, 37, 40, 42, 43 Thymus-dependent de novo generation of naïve Tcells 36

Atta-ur-Rahman and Anjum

Tibialis anterior muscle (TAM) 19 Tissue engineering 3, 6, 9, 20, 21, 23, 65, 66, 74, 75, 77, 80, 92, 113 Tissue replacement 65, 67 Tissue typing 31, 32 Trabecular meshwork (TM) 115, 118 Transferrin 120, 121 Transformation, spontaneous 4 Transient amplifying cells (TAC) 94-96 Transplant, bulge-derived stem cells 106 Transplantation 30, 32, 43, 66, 70, 104, 106, 113, 117, 122 dual cord blood 32, 33 haematopoeitic stem cell 30, 53 haematopoietic stem cell 30 Transplant conditioning 31, 32, 36 Transplanted human stem cells 37 Transplant related mortality (TRM) 34 Treatment modality 32, 33, 43 Trypsin 101 Tumorigenesis 3, 6

U Undifferentiated hESC proliferation 74 Undifferentiated hESCs 72, 74, 75, 78, 79, 84 Undifferentiated state 69, 70, 79 Using donor T-cells 51

V Varicella zoster virus (VZV) 34 Vitamin solution 120, 121 Vitronectin 72-74

W Wnt activation 45, 48, 49 high levels of 45, 47 Wnt canonical signaling 100 Wnt ligands 45, 46 Wnt signaling 30, 46-48

X Xenogenic contaminations 66, 70, 74, 77