A Roadmap to Nonhematopoietic Stem Cell-Based Therapeutics: From the Bench to the Clinic

By Xiao-Dong Chen

A Roadmap to Non-hematopoietic Stem Cell-Based Therapeutics: From the Bench to the Clinic is a resource that provides an overview of the principles of stem cell therapy, the promises and challenges of using stem cells for treating various clinical conditions, and future perspectives. The overall goal is to facilitate the translation of basic research on stem cells to clinical applications. The properties of stem cells from various sources are reviewed and the advantages and disadvantages of each for clinical use are discussed. Modifying stem cell properties through preconditioning strategies using physical, chemical, genetic, and molecular manipulation to improve cell survival, increase cell differentiation potential, enhance production of paracrine factors, and facilitate homing to the site of injury or disease upon transplantation are reviewed. Various routes of stem cell administration and dosing, and the duration of effects, are explored. Individual chapters are written by experts in the field and focus on the use of stem cells in treating various degenerative diseases, autoimmune diseases, wound healing, cardiovascular disease, spinal cord injury, oral and dental diseases, and skeletal disorders. Finally, experts in the regulatory arena discuss mechanisms used in different countries for approving the use of stem cells to treat diseases and many common issues that are typically encountered while seeking approval for this class of therapeutic agent.

• Offers advanced students, as well as new researchers, an overview of the principles of stem cell therapy
• Discusses a wide array of pressing clinical issues with stem cell-based therapies so that new ideas in the laboratory can be efficiently translated to the clinic through better designed clinical trials
• Helps clarify current regulatory mechanisms so that the safe use of stem cells for treating a variety of diseases can move forward
• Fosters cross-disciplinary dialogue between research scientists and physicians to accelerate the safe implementation of efficacious cell therapies

• Language: English
• Publisher: Academic Press
• Release date: Aug 31, 2018
• ISBN: 9780128119211

Book preview

A Roadmap to Nonhematopoietic Stem Cell-Based Therapeutics

From the Bench to the Clinic

Editor

Xiao-Dong Chen

Department of Comprehensive Dentistry and Chief, Regenerative Medicine Program, School of Dentistry, University of Texas Health Science Center, San Antonio, United States

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Part I. Principles & Concepts

Chapter 1. What Can We Learn From This Book?

1. Introduction

2. What Can We Learn From Hematopoietic Stem Cell Therapy?

3. Why Is the Term Mesenchymal Stem Cells (MSCs) So Confusing?

4. What Is the Origin of MSCs?

5. What Are the Characteristics of a Culture System That Maintains MSC Properties During Expansion?

6. What Are the Potential Challenges Facing the Development and Translation of MSC-Based Therapies?

7. What Are the Effects of Government Regulation?

8. Conclusions

Chapter 2. Features of Mesenchymal Stem Cells

1. Introduction

2. Prospective Isolation of Human BMSC

3. Perivascular Origin of BMSC

4. Plasticity of BMSC

5. Self-Renewal and Growth Potential of BMSC

6. Immunomodulatory Properties of BMSC

7. MSC-Like Populations From Other Tissues

8. iPSC-Derived MSC

9. Conclusions

List of Acronyms and Abbreviations

Chapter 3. Maintenance and Culture of MSCs

1. Introduction: Therapeutic Potential of MSCs and Obstacles to Clinical Translation

2. Why Are Current Culture Systems Unsuitable for MSC-Based Regenerative Medicine?

3. How Must Stem Cell Behavior Be Measured and Analyzed in Order to Develop a More Appropriate Culture System?

4. What Is Stem Cell Niche and How Does It Regulate Cell Fate?

5. Is It Possible to Reconstruct the Stem Cell Niche Ex Vivo?

6. Does ECM Produced by Stromal Cells Derived From Different Tissues Exhibit a Tissue-Specific Role in Controlling the Fate of MSCs?

7. Can Stromal Cell–Derived ECMs Be Used to Study the Impact of Aging and Disease-Related Pathologies on the Stem Cell Niche?

8. Conclusions

List of Abbreviations

Chapter 4. Manufacturing Mesenchymal Stromal Cell Banks

1. Regulatory Requirements

2. Cell Banks

3. Conclusions

Part II. Promises and Challenges of MSC-Based Therapies

Chapter 5. Mesenchymal Stem Cell-Based Therapy of Osteoarthritis: Current Clinical Developments and Future Therapeutic Strategies

1. Introduction

2. Functional Properties of MSCs

3. Evidence From Preclinical Models That Support a Therapeutic Role for MSCs in OA

4. Recent Clinical Results of MSC-Based Therapy of OA

5. Novel Perspectives for Enhancing the Efficacy of MSC Therapy of OA

6. Conclusions

List of Acronyms and Abbreviations

Chapter 6. Mesenchymal Stem Cell Therapy in Graft Versus Host Disease

1. Introduction

2. Hematopoietic Cell Transplantation

3. Graft Versus Host Disease

4. MSC Therapy in GVHD

5. Future Directions

Chapter 7. Systemic Lupus Erythematosus

1. SLE Is a Chronic Autoimmune Disease With Challenging Therapies

2. MSC Deficiency in SLE

3. Preclinical Study of Allogeneic MSC Therapy in Lupus Models

4. Clinical Trials of MSC Transplantation in SLE Patients

5. The Mechanism of Allogeneic MSC Transplantation in Lupus Mice and Humans

6. Clinical Challenges of Allogeneic MSCT for SLE

Chapter 8. Mesenchymal Stem Cell–Based Therapies for Repair and Regeneration of Skin Wounds

1. Introduction

2. Mesenchymal Stem Cells

3. Skin—A Reservoir of MSCs

4. Other Nonskin Reservoirs of MSCs

5. Clinical Evaluation of Stem Cell–Based Therapies

6. Manufacturing and FDA Requirements

7. Future Prospects

Glossary

List of Acronyms and Abbreviations

Chapter 9. Myocardial Infarction

1. Introduction: Mesenchymal Stem Cells

2. Mechanism of Action

3. Clinical Trials

4. New Perspectives

5. Conclusions

List of Abbreviations

Chapter 10. Bone Marrow Mesenchymal Stem Cells as a New Therapeutic Approach for Diabetes Mellitus

1. Introduction

2. BM-MSCs Support Human Islets and Show Promise in Diabetes Therapy

3. BM-MSCs Affect Islets via Multiple Mechanisms

4. BM-MSCs Are Inhibited by the Diabetic State

5. Clinical Effects of BM-MSC Transplantation in Patients With Diabetes Mellitus

6. Conclusions and Future Studies

List of Abbreviations

Chapter 11. Mesenchymal Stem Cell–Based Therapy for Chronic Kidney Disease

1. Introduction

2. Efficacy of MSCs in Genetic CKD (PKD)

3. MSCs for the Treatment of Diabetic Nephropathy

4. MSCs in Hypertensive Renal Disease

5. MSCs for Other Causes of CKD

6. MSC-Derived Extracellular Vesicles

7. Challenges for Clinical Translation

8. Summary and Conclusions

List of Acronyms and Abbreviations

Chapter 12. Promises and Challenges of MSC-Based Therapies: Parkinson Disease and Parkinsonism

1. Background: Parkinson Disease and Parkinsonism

2. The Current Treatment Landscape and Its Limitations

3. How Could MSCs Provide Advanced Therapeutic Options?

4. Clinical Trials of MSCs in PD and Parkinsonism

5. Clinical Trials in PD

6. Clinical Trials in Atypical Parkinsonian Disorders: Parkinson Plus Syndromes

7. Multiple System Atrophy

8. Progressive Supranuclear Palsy

9. Pipeline, Challenges, and Future Prospects

10. Conclusions

Acronyms and Abbreviations

Chapter 13. Spinal Cord Injury

1. Introduction

2. Pathophysiology of Spinal Cord Injury

3. Cellular Therapies and Their Targets

4. Preclinical and Clinical Trials

5. Future Directions in Cell Therapy for SCI

6. Conclusions

Glossary

List of Abbreviations and Acronyms

Chapter 14. Stem Cell–Based Restoration of Salivary Gland Function

1. Introduction

2. Salivary Gland Stem Cells

3. Stem Cell–Based Regeneration and Repair

4. Nonsalivary Gland Stem Cell–Based Therapies

5. Current Challenges With MSC-Based Therapies

6. Conclusions

Chapter 15. Tooth and Dental Pulp Regeneration

1. Introduction

2. Stem Cells of Dental Origin

3. Cell Therapy for Periodontal Tissue Engineering

4. Cell Therapy for Dental Pulp Regeneration

5. Challenges and Future Directions

6. Conclusions

Part III. Future Perspectives

Chapter 16. MSCs as Biological Drugs

1. Introduction

2. Developing Stem Cell Therapies: Current Challenges

3. Strategies for Increasing the Efficacy of Stem Cell–Based Therapies: Learning From Drug-Based Therapies

4. Therapeutic Road Maps for Autologous Versus Allogeneic Cell Products Are Different

5. Lessons From Drug-Based Therapies Which Can Be Translated to Stem Cell–Based Therapies (vs. Those Which Cannot)

6. Stem Cell Therapies: Future Perspective in Regenerative Medicine

7. Conclusions

Abbreviations

Chapter 17. Mesenchymal Stem Cell–Derived Products for Tissue Repair and Regeneration

1. Introduction to MSC-Derived Products and Their Applications

2. Cadaveric Donor Tissues as a Source for Mesenchymal Stem Cells

3. Mesenchymal Stem Cell–Derived Products in Research and Development Phase

4. Mesenchymal Stem Cell–Based Products on the Market

5. Mesenchymal Stem Cell–Based Products in Clinical Trials

6. Conclusions

Glossary

List of Acronyms and Abbreviations

Chapter 18. Use of MSCs in Antiaging Strategies

1. Introduction

2. The Principle of MSC Antiaging

3. Rescuing the Quantity and Quality of Elderly MSCs for Autologous Cell-Based Therapies

4. Conclusions

Chapter 19. Regulatory Developments for Nonhematopoietic Stem Cell Therapeutics: Perspectives From the EU, the USA, Japan, China, India, Argentina, and Brazil

1. Introduction:

2. The Regulatory Comparison

3. Three Dynamics of Regulatory Diversification

4. The Advantages and Disadvantages of Different Regulatory Approaches

5. Roadblocks to the Clinical Translation of Nonhematopoietic Stem Cell–Based Therapies

6. Conclusions

Index

Copyright

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List of Contributors

Ashley E. Aaroe,     Department of Neurology, Weill Cornell Medical College, New York, NY, United States

Travis J. Block

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States

Gabriela Bortz

Institute of Science and Technology Studies, National University of Quilmes, Buenos Aires, Argentina

National Council of Scientific and Technical Research (CONICET), Buenos Aires, Argentina

Rodney K. Chan

Q-SCARR™ (Quality Skin Collaborative for Advanced Reconstruction and Regeneration) Research Program, US Army Institute of Surgical Research, Joint Base San Antonio, TX, United States

Dental and Craniofacial Trauma Research and Tissue Regeneration, US Army Institute of Surgical Research, Joint Base San Antonio, Ft. Sam Houston, TX, United States

Xi Chen

State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, China

Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China

Xiao-Dong Chen

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States

Audie Murphy VA Medical Center, San Antonio, TX, United States

Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Research Service and Geriatric Research, Education, and Clinical Center, Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, United States

Robert J. Christy,     Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, Joint Base San Antonio, Ft. Sam Houston, TX, United States

C.S. Cox,     Department of Pediatric Surgery, Regenerative Medicine Division, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States

David D. Dean

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States

Alfonso Eirin,     Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, United States

Roberto Esquivel,     BioBridge Global, San Antonio, TX, United States

Michelle Lynn Fults,     GenCure, San Antonio, TX, United States

Stan Gronthos

Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia

South Australian Health and Medical Research Institute, Adelaide, SA, Australia

Patrick J. Hanley

Program for Cell Enhancement and Technologies for Immunotherapy, Center for Cancer and Immunology Research, Sheikh Zayed Institute for Pediatric Surgical Innovation, The George Washington University, Washington, DC, United States

Division of Blood and Marrow Transplantation, Children's National Health System, The George Washington University, Washington, DC, United States

Claire Henchcliffe,     Department of Neurology, Weill Cornell Medical College, New York, NY, United States

LaTonya J. Hickson,     Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, United States

Jamie Hoover,     Program for Cell Enhancement and Technologies for Immunotherapy, Center for Cancer and Immunology Research, Sheikh Zayed Institute for Pediatric Surgical Innovation, The George Washington University, Washington, DC, United States

Maria V. Irazabal,     Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, United States

Yan Jin,     State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, China

Christian Jorgensen

IRMB, INSERM, University of Montpellier, Montpellier, France

Clinical Immunology and Osteoarticular Diseases Therapeutic Unit, Hôpital Lapeyronie, Montpellier, France

Noor Hayaty Abu Kasim,     Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia

Joseph W. Kim,     Department of Medicine, School of Medicine, Boston University, Boston, MA, United States

Maciej Kurpisz,     Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland

Bei Li,     State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, China

John Z.Q. Luo,     Insure Health, Inc., Warwick, RI, United States

Luguang Luo

Department of Medicine, School of Medicine, Boston University, Boston, MA, United States

The Center for Natural Healing Rhode Island, Pawtucket, RI, United States

Shivaprasad Manjappa,     Department of Medicine, Washington University in St. Louis, St. Louis, MO, United States

Milos Marinkovic

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States

Marie Maumus,     IRMB, INSERM, University of Montpellier, Montpellier, France

Shanmugasundaram Natesan,     Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, Joint Base San Antonio, Ft. Sam Houston, TX, United States

Danièle Noël

IRMB, INSERM, University of Montpellier, Montpellier, France

Clinical Immunology and Osteoarticular Diseases Therapeutic Unit, Hôpital Lapeyronie, Montpellier, France

S.D. Olson,     Department of Pediatric Surgery, Regenerative Medicine Division, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States

Armin Rashidi,     Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MN, United States

Rizwan Romee,     Clinical Director of Haploidentical Transplant Program, Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, United States

Achim Rosemann

Department of Sociology, University of Exeter, Exeter, United Kingdom

Centre for Bionetworking, School of Global Studies, University of Sussex, Brighton, United Kingdom

Natalia Rozwadowska,     Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland

Maxime Ruiz,     IRMB, INSERM, University of Montpellier, Montpellier, France

K.A. Ruppert,     Department of Pediatric Surgery, Regenerative Medicine Division, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States

Thekkeparambil Chandrabose Srijaya,     Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia

Anand Srinivasan

GenCure, San Antonio, TX, United States

BioBridge Global, San Antonio, TX, United States

Sandhya Sriram,     Fat Metabolism and Stem Cell Group (FMSCG), Singapore Bioimaging Consortium (SBIC), A*STAR Research Entities, Biopolis Way, Helios, Singapore

Randolph Stone II ,     Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, Joint Base San Antonio, Ft. Sam Houston, TX, United States

Shigeki Sugii

Fat Metabolism and Stem Cell Group (FMSCG), Singapore Bioimaging Consortium (SBIC), A*STAR Research Entities, Biopolis Way, Helios, Singapore

Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, College Road, Singapore

Lingyun Sun,     Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, China

Peter Supronowicz,     GenCure, San Antonio, TX, United States

Olivia N. Tran

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States

Federico Vasen

National Council of Scientific and Technical Research (CONICET), Buenos Aires, Argentina

Instituto de Investigaciones en Ciencias de la Educación, Universidad de Buenos Aires, Buenos Aires, Argentina

Dandan Wang,     Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, China

Hanzhou Wang,     Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Chih-Ko Yeh

Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Geriatric Research, Education, and Clinical Center, Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, United States

Rogelio Zamilpa,     GenCure, San Antonio, TX, United States

Preface

Why the Book Is Needed

Since 1993, I have been studying nonhematopoietic stem cells (so called mesenchymal stem cells). As a postdoctoral fellow, I was actively involved in identifying a group of specific cell surface antigens that were associated with mesenchymal stem cell (MSC) maturation. During that time, the term stem cell was typically used to refer to hematopoietic stem cells (HSCs). Indeed, MSCs were far less characterized and understood than HSCs. Over the last 25  years, the field of MSC biology has developed into one of the most exciting and rapidly advancing areas of scientific endeavor. Although MSCs were originally identified in bone marrow, they have been discovered in almost every tissue of the body, including those that were previously thought to be non-regenerative, as well as other more unusual sources (e.g., umbilical cord blood, umbilical cord tissue [Wharton jelly], amniotic fluid, dental pulp, and periodontal ligament). Furthermore, the technology for preparing embryonic stem cells (ESCs) is now well-established and even artificial ESCs (i.e., inducible pluripotent stem cells [iPSCs]) can be created in the lab. The boom in stem cell research and associated industries has led to the establishment of regenerative medicine as a major treatment for repairing damaged tissues caused by trauma or, more frequently, age-related diseases. However, this rapid growth in information has also created much confusion and controversy, which has negatively impacted the translation of basic research on MSCs to clinical applications. Since my career has spanned much of the time during which this boom occurred, I have witnessed and contributed to many of these new developments. Thus, it has motivated me to assemble the topics covered in this text and express my thoughts, as well as share my personal experiences, with others in the field. My hope is that this book will help generate discussion aimed at addressing many of the critical questions in the field today and lead to the identification of new solutions for navigating the roadblocks that obstruct translation of stem cell research to new therapeutics.

Audience for This Book

In this book, many important clinically relevant concepts, as well as research philosophy, are reviewed as a background in MSC biology for college and graduate level biological science students and postdoctoral fellows. In addition, the principles of MSC therapeutics for a number of common age-related diseases are discussed for physicians/surgeons/dentists and research scientists/engineers in the regenerative medicine field. For a future perspective, regulation of MSC-based therapies, the potential of MSC-related products and drugs, and use of MSCs in antiaging are also discussed. These topics will be of interest to a broad audience, including nonresearchers or researchers in other fields (e.g., sociologists, politicians). Finally, another important goal that I hope this book will achieve is to gain the attention of policymakers and/or regulatory officials. At present, regulations often ignore the reality of MSC biology (e.g., MSCs are frequently obtained from tissues in small numbers and must be expanded before use in therapeutic applications which presents an impossible hurdle to overcome the requirement that cells be minimally manipulated), which dramatically slows the approval/translation of cell-based therapies to the clinic. In the future, regulations need to be structured so that translation can take place in both a safe and timely manner to provide valuable new therapies for large numbers of elderly patients with age-related diseases.

Structure of This Book

The book is divided into three sections. In the first section, we provide a rationale for the book and briefly introduce a number of issues/questions for the reader to consider while reading the text. The following chapters in this section then focus on the features of MSCs from various sources and describe the relative advantages and disadvantages of each of the cell-based therapies, current issues related to the preparation of stem cells, and the establishment of standardized high-quality stem cell banks. In Section 2, we focus on the use of MSCs for treating various types of diseases. The current status of each of the following diseases is reviewed by experts in the field: autoimmune diseases, osteoarthritis, neurological disorders, cardiovascular diseases, chronic kidney disease, diabetes, wound healing, spinal cord injury, and dental/oral diseases. In Section 3, we focus on future perspectives and review the development of stem cell–based products, as well as biological drugs, and discuss a new paradigm for the use of MSCs as an antiaging strategy. In addition, experts compare pathways leading to the approval of stem cell–based therapies in the US, UK, and other countries and address a number of critical issues/questions commonly encountered in developing this class of drugs/therapeutic agents.

Uniqueness of This Book

The content of this book is different from other regular texts. We only focus on addressing the most critical issues that may block the translation of MSCs from the laboratory to the clinic. In order to provide first-hand information, all of the contributing authors are worldwide experts from the frontline. They bring their own experience and expertise to make the book unique.

Acknowledgments

I would like to sincerely thank each of the authors for devoting their time and effort towards this book. It would not have been possible to accomplish this project without their enthusiastic participation. I am particularly grateful to my colleague, Dr. David D. Dean, who has worked closely with me throughout the preparation of this text, carefully edited each chapter, and provided wise advice about its organization and content.

I would also like to thank two very special people, my father and my lovely wife, who have both had major impacts on my life. My father always provided strong support while I was growing up so that I could obtain the best education possible during some very tough years in China. I really believe that no matter how much I achieve, it would never be enough to exceed his expectations. Even now, our weekly phone conversations always begin with him asking me What's the good news? This book will be a very special gift for him as he celebrates his 90th birthday this year. And to my wife, Lily, who has sacrificed her own career and dreams so I could have the freedom to pursue my own. I can never repay her for everything she has given up. Therefore, I dedicate this book to her and our upcoming 25th anniversary. I am deeply grateful for her continued companionship and I thank her for all the encouragement and moral support over the years!

Finally, I would like to thank all of the people and organizations that have supported me during my research career. I am especially grateful for the financial support provided by the National Institutes of Health, Department of Veterans Affairs (VA Merit Review), and the Owens Medical Research Foundation.

Part I

Principles & Concepts

Outline

Chapter 1. What Can We Learn From This Book?

Chapter 2. Features of Mesenchymal Stem Cells

Chapter 3. Maintenance and Culture of MSCs

Chapter 4. Manufacturing Mesenchymal Stromal Cell Banks

Chapter 1

What Can We Learn From This Book?

Travis J. Block¹,², David D. Dean¹,², and Xiao-Dong Chen¹,²,³,⁴     ¹Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States     ²Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, United States     ³Audie Murphy VA Medical Center, San Antonio, TX, United States     ⁴Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Abstract

As an introduction to this book on stem cell therapies, this chapter presents the rationale for preparing this volume and describes its key features. A brief historical overview is presented, describing some useful observations from the past to provide context and help us to overcome some of the current obstacles toward clinical translation. We attempt to clarify some of the confusion regarding terminology in the field (e.g., the term mesenchymal stem cells), as well as raise some specific questions/issues that we believe have hindered efforts in achieving effective stem cell–based therapies. The hope is that this volume will foster more critical thinking and generate robust discussion among basic and social scientists and clinicians, which may help us identify new solutions for navigating the roadblocks that continue to impede the translation of MSC-based therapies to the clinic.

Keywords

Critical issues and challenges; Guidance for readers; History of stem cell–based therapy; Rationale for the book; Stem cell therapies; Terminology

1. Introduction

During the 21st century, stem cell therapy will play an increasingly important role in treating many previously incurable diseases. More importantly, stem cells are likely to hold the key to delaying the aging process and significantly improve human longevity and healthspan. This is due to the unique ability of stem cells to serve as: (1) a cell reservoir that can continually replace aged and/or damaged cells over a lifetime and (2) a drug store that can immediately respond to pathological processes by rapidly secreting trophic factors to repair damaged tissues and restore homeostasis. Moreover, stem cell–based therapies are often able to simultaneously and precisely target multiple signaling pathways resulting in a systemic treatment for diseases with fewer side effects. This multipronged therapeutic approach stands in striking contrast to traditional pharmaceutical agents that usually target a single metabolic pathway by either blocking or catalyzing a specific biological process resulting in regional treatment with more side effects.

In the early 1970s, Friedenstein et al. were the first to identify bone marrow-derived nonhematopoietic stem cells (now called mesenchymal stem cells [MSCs]) that are capable of differentiating into osteoblasts, chondrocytes, and adipocytes [1–3]. These cells have been found in almost every tissue or organ, including tissues that were previously thought to be nonregenerative (e.g., central nervous system, myocardium, skeletal muscle, and fat), as well as other more unusual sources (e.g., umbilical cord blood, umbilical cord tissue [Wharton jelly], amniotic fluid, dental pulp, and periodontal ligament). Further, pluripotent stem cells, such as embryonic stem cells (ESCs), and artificial ESCs (i.e., inducible pluripotent stem cells ) have been found to generate virtually all types of tissue in the body. Although the overall phenotype of these cells appears to be very similar, each tissue source contains stem cells with a unique set of properties. Thus, it is essential that the type/source of stem cells be carefully investigated, so that the best cells (or combination of cells) for treating a specific disease are selected.

Although basic and preclinical research studies have provided results that are very encouraging and support the potential of stem cells for treating various diseases, the translation of these discoveries to the clinic has been slow. With the anticipated dramatic increase in the elderly population over the next decade(s) rapidly approaching, it is essential that the field moves more decisively to meet the expected clinical demand for treating large numbers of patients with age-related diseases [4].

In planning and organizing this text, critical issues have been highlighted, which must be addressed as a research community for progress in stem cell transplantation to accelerate. Cross-disciplinary dialogue among research and social scientists and physicians must be encouraged to resolve many of these issues so that stem cell therapies can provide benefits to patients in the near future.

The text is divided into three sections (Fig. 1.1). In the first section, the features of stem cells from various sources are reviewed to illuminate the relative advantages and disadvantages of each for cell-based therapies. This is followed by a discussion of current issues related to the preparation of stem cells, along with potential solutions, and the establishment of standardized high-quality stem cell banks. We believe that this introduction to stem cells and their preparation provides a foundation for understanding sections two and three and emphasizes the importance of ensuring both the efficacy and uniformity of stem cells for achieving consistent clinical outcomes. In the second section, the current status of stem cell–based therapies for common diseases, including autoimmune diseases, osteoarthritis, neurological disorders, cardiovascular diseases, chronic kidney disease, diabetes, wound healing, spinal cord injury, and dental/oral diseases, are each reviewed by experts from the frontline. The information contained in these chapters will guide the research efforts of scientists in the field and enable physicians to translate ideas from the laboratory to the clinic through better designed clinical trials. In the third section, future perspectives are considered. Issues related to the development of stem cell–based products and biological drugs are reviewed; it is anticipated that these new stem cell–based therapies will surpass the market for conventional synthetic drugs and also provide a new paradigm for antiaging therapies based on stem cells. In addition, current regulation of stem cell–based therapies is also reviewed; experts discuss pathways leading to the approval of stem cell–based therapies in the US, UK, and other countries and address a number of critical issues/questions commonly encountered in developing this class of drugs/therapeutic agents.

Figure 1.1  A roadmap to mesenchymal stem cell–based therapeutics: from the bench to the clinic.

The topics covered in this book have been presented by the authors with a broad audience, including college students, graduate students, postdoctoral fellows, physicians/surgeons/dentists, residents, and scientists/engineers, in mind. Aspects of both basic and clinical research, focused on developing stem cell/cell-based therapies, have been included. The topics covered may also be of interest to policy-makers and regulatory officials, including those in the FDA, and all others associated with policy and regulatory matters related to cell-based therapeutics. Since each chapter has been written by different experts in the field, readers may find that some of the information presented is occasionally redundant, especially with regard to the features of stem cells, and that opinions on the same subject may differ. We respect these diverse thoughts, and hope they spur students or young investigators to develop their critical thinking and problem-solving skills in this area and contribute to the discussion.

The remainder of this chapter will briefly review several common issues/questions that the reader is encouraged to consider while reading the text.

2. What Can We Learn From Hematopoietic Stem Cell Therapy?

In 1957, Thomas et al. were the first to attempt treating human patients by transplanting whole bone marrow cells from blood type-matched unrelated donors [5]. In a total of six patients, only two patients, who had received a large dose of total body irradiation, appeared to have successfully restored hematopoiesis. Since the transplants were administered by intravenous infusion, the authors also paid close attention to the formation of pulmonary emboli and failed to find any related signs and symptoms. The investigators concluded that patients receiving a high dose of total-body irradiation may benefit from this type of cell-based treatment. We now know that these high-dose irradiated patients had a completely destroyed immune system that prevented (or delayed) graft rejection. After more than 50  years of intense research and many major breakthroughs in the field, it is interesting to note that the unsuccessful cases from that pioneering study could have been due to graft versus host disease (GVHD) (due to contamination of T cells in the transplanted whole bone marrow from the donors) and/or transplanted bone marrow cells from human leukocyte antigen (HLA)–mismatched donors (even transplanted cells from blood-type matched donors are not good enough). Nevertheless, this pioneering work has been widely appreciated because it made an important step in advancing the field to treat human diseases.

Today, hematopoietic stem cell (HSC) transplantation is routinely used to treat a number of malignant and nonmalignant diseases, with approximately 45,000 cases reported worldwide annually [6]. Since the identification of CD34 as a specific HSC surface marker [7], the transplantation of CD34 positive cells, instead of whole bone marrow cells, significantly reduce the risk of GVHD. Because HSCs are able to be isolated, based on CD34 expression, peripheral blood has become a major source of HSCs for transplantation. An important advantage of peripheral blood relative to bone marrow is the decreased likelihood of transplanting tumor cells in the donor bone marrow to the recipient, which significantly increases the chance for cancer patients to use their own HSCs (autologous) to reconstitute their immune and hematopoietic systems after receiving high dose chemo- or radiotherapy.

Several important observations from HSC therapy may be helpful in developing MSC applications: (1) the established cluster of differentiation (CD) system allows precise identification of blood cells at different stages and lineages; (2) HSCs transplanted intravenously are able to specifically home to the bone marrow cavity and repopulate the full complement of blood cells; and (3) a large established pool of unrelated donors is available worldwide and increases the chances for patients to rapidly find HLA-matched donors; this bank of cells would also be instrumental for obtaining large numbers of high-quality of MSCs to achieve predictable therapeutic outcomes. All related details are discussed in Chapters 4 and 18.

3. Why Is the Term Mesenchymal Stem Cells (MSCs) So Confusing?

In the early 1970s, Friedenstein et al. identified a subpopulation of nonhematopoietic cells in bone marrow that were able to form colonies (i.e., colony forming unit-fibroblasts or CFU-Fs) in culture using very low cell seeding densities [1]; further, when cells from a single CFU-F were transplanted into an appropriate in vivo animal model bone, cartilage and fat formed [8]. Since these cells mainly give rise to mesoderm-derived tissues, they were named mesenchymal stem cells or MSCs [9]. More recently, it has been found that MSCs not only differentiate into mesoderm-derived tissues, but also endoderm- and/or ectoderm-derived tissues by a process called transdifferentiation. Logically, mesenchymal is not an appropriate term for describing these cells. However, the formation of three-germ layer tissues might also be due to heterogeneity within the MSC population, which contains cells at varying stages, in addition to the mature stromal cells, and each subpopulation has its own properties (e.g., capability for self-renewal and differentiation). This heterogeneity may also explain why inconsistent results in both in vitro and in vivo studies have been reported by different laboratories that have used different procedures for preparing their cells (i.e., they likely obtain subpopulations of cells in varying proportions). Since MSC-specific surface markers and reliable culture systems are lacking, it is very difficult to isolate and retain a pure or homogeneous population of MSCs. As a result, these mixed populations of cells are often safely referred to as marrow stromal cells (or MSCs) or stromal stem cells. Recently, Caplan has suggested that MSCs be renamed medicinal signaling cells (MSCs) [10]. His rationale for this revised nomenclature is based on the fact that therapeutic outcomes in vivo are mainly due to the production of trophic factors by MSCs, rather than their stem cell features (i.e., cell reservoir properties) such as the ability to regenerate new tissues.

Although it is true that MSCs are able to differentiate into many cell lineages in vitro, this often occurs under very strong inducing conditions that never happen in the body. This is the reason why it is always important to validate the retention of MSC self-renewal and differentiation capacity using in vivo animal studies. It is also true that only a few reports have been able to demonstrate that transplanted MSCs have been responsible for new tissue formation in vivo [10]. However, it is very likely that transplanted allogeneic cells are rejected after they differentiate in an immune-competent recipient. Based on what has been learned from HSCs, as well as other mounting evidence, it is illogical to continue believing that MSCs are immune privileged. These ideas are discussed further in Chapter 18. Indeed, the classical model for evaluating MSC quality (i.e., self-renewal and differentiation capacity) involves the implantation of human/mouse bone MSCs in immunodeficient mice. In this model, skeletal tissue is generated, which directly reflects the number and quality (e.g., young vs. old) of CFU-Fs within the implanted cells [11–15]. Recently, we provided strong evidence suggesting that nonhematopoietic stem cells from umbilical cord blood were able to form multiple types of tissues, including blood vessels, and regenerate muscle after cryoinjury using immunodeficient mice [16].

Mesenchymal stem cells (MSCs) appear to be present in almost every tissue. In Chapter 3, we discuss the importance of the microenvironment during cell culture and its role in the retention of MSC tissue-specific properties. Interestingly, when adipose-derived stem cells were loaded onto hydroxyapatite/tricalcium phosphate (HA/TCP) particles and implanted subcutaneously in immunodeficient mice, only adipose tissue was formed, even though these cells were able to differentiate into osteoblasts in vitro (unpublished data from the authors' lab). Based on these data, some investigators in the field have referred to these cells as tissue-specific stem/progenitor cells [17]. In each of the following chapters, authors have been given the latitude to choose nomenclature that is customary in their field of research. Predominantly, the authors have continued to use the term mesenchymal stem cells or MSCs in order to refer to the same cells discussed in this chapter.

4. What Is the Origin of MSCs?

Unlike HSCs, MSCs do not have a collection of cell surface markers (i.e., the CD system) to identify cells from different sources and their stage of differentiation. As a result, it is difficult to identify the origin of MSCs. In 2006, the International Society for Cellular Therapy (ISCT) promulgated minimal criteria for defining MSCs that included: (1) adherence to tissue culture plastic (polystyrene), (2) the presence of three stromal cell-related surface antigens (CD105, CD90, and CD73) and the absence of two blood cell–related antigens (CD34 and CD45), and (3) trilineage differentiation potential [18]. However, these criteria do not consider the widely accepted requirement that authentic MSCs demonstrate both self-renewal and differentiation capacity or the ability to distinguish MSCs in aging versus youth. In fact, this set of standard surface markers (i.e., CD105, CD90, and CD73) are just stromal cell adhesion–related antigens that are expressed by most fibroblasts. Other candidate markers that are being debated in the literature and discussed in Chapters 2, 3, and 18 include SSEA-4 (stage specific embryonic antigen-4), which may be a good marker for defining bone marrow–derived MSC proliferation/cell division, and STRO-1 for identifying early stage MSCs.

In 2003, Shi and Gronthos reported that MSCs were localized to regions of the microvasculature in human bone marrow and dental pulp [19]. These cells that expressed high levels of Stro-1 (bright) and CD146 were significantly enriched in CFU-Fs. Subcutaneous implantation of these double-positive cells from bone marrow or dental pulp in immunodeficient mice resulted in the formation of bone or dental tissue, respectively. This study not only provides evidence for the origin of MSCs, but also remarkably demonstrates the existence of tissue-specific MSCs by showing that MSCs from two different tissue sources, implanted into immunodeficient mice, faithfully generated the same tissues from where they were originally derived. This work has been further confirmed by several other groups suggesting that pericytes form the HSC niche which supports hematopoiesis and exhibits high levels of self-renewal and multipotentiality [20–22]. The perivascular origin for MSCs explains why MSCs are found in almost all the tissues and, in particular, why adipose-derived MSCs are found at such high concentrations in stromal vascular fraction (SVF).

However, MSCs are not exclusively localized to the perivascular region. For example, MSCs (or osteoblast precursors) have been found in cortical bone; these cells are highly proliferative and express CD90 [23]. Overall, it is likely that MSCs are a mixed population of cells, derived from various sources/origins, and express both tissue-specific and common stem-cell features. MSCs are very sensitive to their local microenvironment and their phenotype rapidly adapts to it, which makes it very challenging to expand MSCs that retain their original properties.

5. What Are the Characteristics of a Culture System That Maintains MSC Properties During Expansion?

Almost 20  years ago, as a postdoctoral fellow, I (Xiao-Dong Chen) tried isolating BM-MSCs using an approach that was very similar to the one used to isolate HSCs. Obviously, it was unsuccessful. Then, we used so-called negative selection to remove all bone marrow mononuclear cells with all known surface antigens and saved a very small number of cells that did not express any known surface markers. These cells, surviving from the negative selection, were then cultured with a very low cell seeding density. It was noticed that they proliferated very rapidly. However, after 2  weeks of culture, we surprisingly found that the phenotype of the cells that had been removed reappeared. This suggested that the phenotype of these cells had changed during expansion. The result made us realize that it would be impossible to obtain a homogeneous population of MSCs without having a culture system that was able to retain their original phenotypes. As mentioned previously with regard to the discovery of tissue-specific MSCs, it is now easy to understand the necessity of a unique culture system, which replicates the in vivo microenvironment, for expanding MSCs so that their MSC properties (i.e., self-renewal and differentiation capacity) are maintained.

Our laboratory was the first to describe a three-dimensional (3D) decellularized BM-derived extracellular matrix (BM-ECM) cell culture system for expanding BM-MSCs. This native culture surface provides many of the critical biochemical and physical cues for initiating and sustaining cell function [14,24] and is primarily composed of collagen types VI, I, and III, fibronectin, small leucine-rich proteoglycans (e.g., biglycan, decorin), and several major basement membrane components (e.g., perlecan, laminin), which have been shown to play a key role in regulating cell adhesion, migration, proliferation, differentiation, and survival [25–27]. Our published results, as well as studies by others, indicate that mouse and human BM-MSCs cultured on this native ECM display enhanced attachment and proliferation and retention of stem cell properties as compared to tissue culture plastic (TCP) or TCP coated with purified matrix proteins (e.g., fibronectin, collagens, Matrigel) or synthesized materials [14,24,28–31]. More about the preparation of MSCs is discussed in Chapter 3.

6. What Are the Potential Challenges Facing the Development and Translation of MSC-Based Therapies?

Currently, there are about 600 ongoing clinical trials in the US that are evaluating the use of MSCs for a variety of diseases (www.clinicaltrials.gov). In general, the clinical outcomes of completed and published trials have showed modest or inconsistent effects [32]. We believe that the main reasons for these results are: (1) there is no meaningful standard criterion for controlling MSC quality; (2) there is no standard procedure for preparing MSCs (isolation and expansion), resulting in large variations in the efficacy of the transplanted cells; (3) the dose, route, and timing of MSC administration may not be optimal due to the lack of an effective method for tracking cells in vivo; and (4) the medical condition of the recipients varies (even for individuals with the same disease) and this may impact the efficacy of the transplanted cells. All of these issues are extensively discussed in many of the following chapters. In addition, we strongly believe that the establishment of a high-quality MSC bank, providing standardized MSCs nationwide, is extremely important for improving the efficacy and reproducibility of MSC-based therapies.

7. What Are the Effects of Government Regulation?

The current regulations regarding the Minimal Manipulation of Human Cells are generally unsuitable for MSC-based therapies. Unlike HSCs, MSCs are extremely rare in their source tissues. Furthermore, many of these primary cells may be quiescent. Increasing the number of cells and reactivating them are necessary steps in order to enhance clinical efficacy. In this book, we pursue a global perspective and compare the regulatory climate for clinical stem cell research and market approval in different countries including Argentina, Brazil, China, the European Union, India, Japan, and the USA. What are the advantages and disadvantages of the regulatory climate in each of these countries? What can we learn from the different regulatory approaches/processes adopted in each? It is important that roadblocks, which prevent the translation of valuable and effective stem cell-based therapies to the clinic, be identified and strategies developed to safely remove those barriers. It is our hope that the ideas presented here will promote a better understanding of current government policies that regulate the use of stem cells in the clinic and also influence policy-makers in this challenging arena. In addition, these ideas may stimulate the development of new pathways for promoting safe application of stem cells for treating diseases of degeneration and aging.

8. Conclusions

With the benefit of hindsight and historical perspective, we understand the significance and meaning of some of the pioneering work on stem cells better than ever before. Hopefully, the questions raised and the challenges reviewed in this chapter will lead to more discussion and help identify new solutions for navigating the roadblocks that continue to obstruct the translation of stem cell–based therapies to the clinic. We are encouraged by the progress made so far. At this time, we are faced with the opportunity to redouble our efforts to remove these roadblocks and bring exciting new MSC-based therapies and their tremendous benefits to many patients.

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Chapter 2

Features of Mesenchymal Stem Cells

Stan Gronthos¹,²     ¹Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia     ²South Australian Health and Medical Research Institute, Adelaide, SA, Australia

Abstract

Human bone marrow contains self-renewing, multipotential stromal precursor cells, with the capacity to give rise to skeletal and myelosupportive stromal tissues. The progeny of culture expanded stromal precursor cells have been described as bone marrow stromal cells (BMSC) or mesenchymal stem cells (MSC). Over time, this terminology has been adopted as a generic label to describe similar populations with perivascular characteristics derived from different neonatal and adult tissues such as adipose, dental, umbilical cord, and placenta. However, given the diverse ontogeny and properties of MSC-like populations identified in different tissues, the question still remains whether tissue-specific stromal precursors are derived from resident smooth muscle cells or pericytes. The current chapter describes the identification and basic characteristics of BMSC, and highlights our current understanding of their identity, biological properties, and similarities with other MSC-like populations derived from nonskeletal tissues.

Keywords

Bone marrow stromal cells; Colony forming unit-fibroblast; Induced pluripotent stem cells; Mesenchymal precursor cells; Mesenchymal stem cells; Skeletal stem cells

1. Introduction

Skeletal tissue is capable of regeneration following disease or trauma, where bone repair and remodeling is attributed to the balance between bone resorbing osteoclasts, derived from hematopoietic myeloid progenitor cells, and bone forming osteoblasts, derived from bone marrow stromal cells (BMSC) [1]. However, the exact nature of the cellular elements and molecular pathways that mediate skeletal tissue maintenance/repair during normal aging or under pathological/trauma conditions requires further study.

The concept of resident bone marrow stromal cells (BMSC) endowed with stem cell-like qualities was first described by Friedenstein and colleagues, who identified clonogenic adherent colonies comprised of cells with a fibroblastic morphology in aspirates of rodent bone marrow [2]. Seminal experiments assessed the developmental capacity of the progeny of individual culture expanded colony-forming fibroblasts (CFU–F), following ectopic implantation into syngeneic animals [3]. Harvested ectopic implants showed that a minor proportion of the CFU-F formed a vascularized fibrous network, which supported local hematopoiesis adjacent to spicules of calcified bone tissue. A similar proportion of bone marrow CFU-F clones were only capable of producing calcified bone tissue, while the majority of clones synthesized a fibrous connective tissue [3]. Interestingly, the hematopoietic cells contained within the stromal organs were identified as being derived from the host animal, while the stromal tissues were determined to be of donor origin, based on immunological and chromosomal analyses. Other studies reported that a proportion of rabbit derived CFU-F clonal cell lines could be induced to differentiate into lipid-laden adipocytes under defined culture conditions [4]. Furthermore, less than half of the CFU-F clones comprising either fibroblastic or adipocytic cells could also be induced to form a calcified bone matrix when transplanted in vivo using diffusion chambers implanted into the peritoneal cavity of nude mice [4]. The minor population of CFU-F clones which formed bone marrow organs were hypothesized to be derived from multipotential BMSC, while those CFU-F clones, which gave rise to only bone or soft connective tissue, were proposed to be committed progenitors with restricted developmental potential [5].

Subsequent studies identified the presence of CFU-F in a number of other species, including humans, and confirmed their potential to develop into stromal multiple tissue types in vivo [6–10]. Experiments assessing the potential of ex vivo expanded adult human CFU-F reported the formation of bone, cartilage, and hematopoietic supportive stroma, when transplanted into immunocompromised animals, using a variety of carrier vehicles [11–15]. Studies investigating the properties of individually expanded human bone marrow CFU-F observed that more than half of the colonies were capable of forming bone in vivo, following cotransplantation with hydroxyapatite/tricalcium phosphate ceramic particles into immunocompromised mice [14,15] (Fig. 2.1). In analogy with the studies of Friedenstein and Owen described above, only about a third of CFU-F clones demonstrated the capacity to form stromal tissue able to support local hematopoiesis. Comparative in vitro experiments between different human-derived CFU-F clones have also reported a differential capacity for multidifferentiation into myelosupportive stroma, osteoblasts, chondrocytes, smooth muscle cells, and adipocytes, which subsequently became to be known as mesenchymal stem cells (MSC) [15–18]. Collectively, these studies provide a substantial body of data in support of the stromal stem cell hypothesis (Fig. 2.2) [5,19].

Figure 2.1  Developmental potential of BMSC in vivo. The image represents human BMSC seeded into hydroxyapatite-tricalcium phosphate bioscaffolds and transplanted subcutaneously into immune deficient Nude mice for 8  weeks. Harvested transplants were stained with hematoxylin and eosin. Histological examination of the transplants identified new bone comprised of bone lining osteoblasts and osteocytes encapsulated in the bone matrix. The transplants also showed the presence of blood vessels, hematopoietic marrow, and adipose tissue.

Three decades after their discovery, the International Society for Cellular Therapy proposed that the term MSC be reserved for cells that met specified stem cell criteria [20]. During this time, minimal criteria defining MSC were established, based on their capacity to adhere to plastic in standard culture conditions, and the positive expression of cell surface antigens, CD105, CD73, and CD90, and negative expression of CD45, CD34, CD14, or CD11b, CD79alpha, or CD19 and HLA-DR surface molecules [21]. However, the use of plastic adherence as an MSC selection protocol is grossly inadequate, since many other cell types exhibit the ability to adhere to plastic including mature bone cells, endothelial cells, smooth muscle cells, and monocytes/macrophages. Furthermore, it must also be stressed that the MSC-associated markers described above are also expressed on nonMSC fibroblast populations derived from different tissues. Therefore, the minimal criteria defining MSC falls well short in providing any meaningful standards for evaluating the findings between different research groups, because of the nonspecific nature of the markers used, and lack of rigorous assessment of multidifferentiation and self-renewal capacity in vivo. Therefore, it is not surprising that the reliance on traditional plastic adherence selection to generate primary bone marrow MSC cultures is fundamentally flawed, due to the presence of contaminating cell populations consisting of an assortment of progenitor and differentiated stromal cells, endothelial cells, and hematopoietic progenitors and macrophages that can persist from primary cultures through to early cell passages, which can severely influence the growth and functional properties of MSC [22].

2. Prospective Isolation of Human BMSC

Until recently, the identification and exact tissue locality of multipotential MSC have not been fully determined due to an inability to discriminate MSC from perivascular and other more differentiated stromal cell types residing in the bone marrow microenvironment. Electron microscopic examination and cytochemical staining of marrow tissue sections first identified reticular cells as the predominant stromal cell type present in the bone marrow spaces responsible for secreting the extracellular matrix fibers which support the hematopoietic tissue [23–25]. Early work in this field proposed that a subset of reticular cells in the vicinity of the endosteal bone surface or adventitial reticular cells lining the marrow sinuses could be a source of osteogenic, adipogenic, and myelosupportive stromal precursor cells [25].

Figure 2.2  Bone marrow stromal system. Bone marrow stromal cell differentiation hierarchy as first proposed by Owen and Friedenstein [5,19].

The ability to distinguish hematopoietic elements with clonogenic BMSCs based on their cell surface antigen expression profiles was a major step forward to designing strategies for the purification of MSC. Early studies of murine bone marrow identified a population of 5-fluoracil-resistant CFU-F exhibiting an immunophenotype lacking lymphoid and myeloid cell lineage markers (Lin−), but expressing stem cell antigen-1 (Sca-1+) and reacting strongly with wheat germ agglutinin (WGAbright) [26,27]. The Lin−/Sca-1+/WGAbright cell population was shown to possess the potential to support hematopoiesis and develop a calcified bone matrix in vitro when cultured under osteogenic inductive conditions. Multipotential murine MSCs have been further characterized by direct immunomagnetic and fluorescence activated cell-sorting techniques based on the cell surface expression of Lin−/c-fms+/c-kitlow/VCAM-1+ [28]. More recently, compact bone-derived MSCs were characterized as having an immunophenotype of Sca-1+/Lin−/CD31-/CD51− [29]. However, the exact relationship between compact bone and bone marrow–derived MSC requires more thorough interrogation.

Over the last decade the generation of transgenic reporter mice based on markers associated with mesodermal development or perivascular populations has given us a better insight into the identity and characteristics of different bone marrow–derived stromal populations. These include Leptin-receptor+, PDGF-receptor+, CXCL12+, α-smooth muscle+, Prx1+, Nestin+, Mx1+, multipotential perivascular cells, or reticular cells, with varying capacity to support hematopoiesis [30–35]. Other markers such as CD200 [36] and Gremlin1 [37] have been shown to identify a distinct stromal subpopulation within subchondral bone that represents a precursor population to osteoblasts and chondrocytes but not adipocytes. However, more studies are required to