Cord Blood Stem Cells Medicine

Cord Blood Stem Cells Medicine

Editors

Catherine Stavropoulos-Giokas

Dominique Charron

Cristina Navarrete

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Foreword

Section I. Introduction

Chapter 1. Introduction to Cord Blood Stem Cells

Section II. Cord Blood Cells Biology

Chapter 2. Cord Blood Content

1. Biological Background of Cord Blood Cells—Development of Hematopoietic and Nonhematopoietic Cells

2. Endothelial Cells in CB

3. Stromal Cells in CB and Cord Tissue as Compared to BM

4. Isolation, Expansion, and Characterization of CB-derived Adherent Cells from CB

5. Generation of Cell Clones and Clonal Populations

6. USSC and CB MSC

7. Generation of Adherent Cells from the Wharton’s Jelly (Cord)

8. Gene Expression Profiles

9. Correlation of HOX-gene Expression and Regenerative Potential

10. Bone and Cartilage Forming Potential of Cord Blood Stromal Cells

11. Why Do We Have These Progenitors or Elusive Cells in CB?

12. USSC and MSC from CB Support Hematopoietic Cells

13. Liver Regeneration and Potential of CB-derived Stem Cells to Undergo Hepatic Differentiation

14. Cardial Regeneration In vivo

15. In vitro Differentiation Potential toward Cardiomyocytes

16. CB Subpopulations for Neuronal Regeneration

17. Reprogrammed Subpopulations from CB

18. Conclusion

List of Acronyms and Abbreviations

Chapter 3. Cord Blood Hematopoiesis: The Road to Transplantation

1. Use of Placental Cord Blood as a Source for Hematopoietic Stem/Progenitor Cells Transplantation: A Historical Perspective

2. Characterization of PCB HSPCs

3. Strategies to Overcome Current Limitations in PCB Transplantation

4. Increasing the HSPCs of the Graft

5. Improving the Lodging Capacity of the PCB Graft

6. Improving the Stem Cell Receptivity or Immunological Status of the Host

7. Additional Clinical Uses of Cord Blood under Investigation

8. Concluding Remarks

Chapter 4. Immunobiology of Cord Blood Cells

1. Introduction

2. Immune Properties of Cord Blood Cells

3. Immune Reconstitution after CBT

4. Generation of Immune Competent Cells from Cord Blood

5. Conclusion

List of Abbreviations

Chapter 5. Cord and Cord Blood-derived Endothelial Cells

1. Introduction

2. The Human Endothelial Colony-Forming Cell Hierarchy

3. Differences Between Human Umbilical Cord Blood ECFCs and Those Sourced from Adult Peripheral Blood, Umbilical Cord, and Placenta

4. Obstetric Factors and ECFC Content in Umbilical Cord Blood at Term

5. ECFC Content and Function in Umbilical Cord Blood Based on Gestational Age

6. The Diabetic Environment Affects Umbilical Cord Blood ECFCs

7. Processing and Cryopreservation Affect ECFC Numbers in Umbilical Cord Blood

8. The Phenotypic Identity of ECFCs

9. The Use of Umbilical Cord Blood or Placental ECFCs as a Cellular Product in Regenerative Medicine

10. Conclusions

Glossary

List of Acronyms and Abbreviations

Chapter 6. HLA and Immunogenetics in Cord Blood Transplantation

1. Introduction

2. Structure, Function, and Polymorphism of HLA

3. HLA Typing Techniques

4. CBT Results

5. Other HLA Criteria for CBU Selection

6. Conclusion

List of Abbreviations

Section III. Cord Blood Cells for Clinical Use

Chapter 7. Clinical Use of Umbilical Cord Blood Cells

1. Introduction

2. Clinical CB Transplantation

3. Clinical Use of Related CB Cells for Allogeneic Transplantation

4. Clinical Use of Unrelated CB Cells for Allogeneic Transplantation

5. Selection of CB Units for Transplantation

6. New Strategies to Improve Outcomes after CB Transplantation

7. Conclusion

List of Acronyms and Abbreviations

Chapter 8. Immunodeficiencies and Metabolic Diseases

1. Immunodeficiencies

2. Metabolic Diseases

Chapter 9. Cord Blood Cells and Autoimmune Diseases

1. Introduction

2. New Insights in the Pathogenesis of ADs

3. Hematopoietic Stem Cell Transplantation for the Treatment of AD

4. Use of UCB-derived Cells and Cord Blood MSCs for Treating AD

5. Conclusion

Chapter 10. Umbilical Cord as a Source of Immunomodulatory Reagents

1. Regulatory T-cells

2. Mesenchymal Stromal Cells

3. Conclusions

Chapter 11. Cord Blood Cells for Clinical Use: Expansion and Manipulation

1. Introduction

2. CB Transplantation in Pediatric Patients

3. CB Transplantation in Adult Patients

4. Double CB Transplantation

5. CB Transplantation after Reduced Intensity Regimens

6. CB Graft Manipulation

7. CB Stem Cell and Progenitor Cell Expansion to Enhance Engraftment

8. Improving CB Homing to BM

9. Prostaglandin and Homing

10. CB Immune Cells to Improve Outcome

11. Expanding Multivirus-Specific Cytotoxic T Lymphocytes from CB

12. CB-Derived Natural Killer (NK) Cells

13. CB-Derived Regulatory T Cells

14. Redirecting Specificity of CB-Derived T Cells to Leukemia Antigens

15. Conclusion

Chapter 12. Cord Blood Stem Cells for Clinical Use: Diabetes and Cord Blood

1. Diabetes and Global Challenges

2. Stem Cells in Cord Blood

3. Application of Stem Cell Educator Therapy in T1D

4. Application of Stem Cell Educator Therapy in Type 2 Diabetes

5. Conclusions

Dr Yong Zhao’s Bibliography

Section IV. Regenerative Medicine Applications

Chapter 13. Emerging Uses of Cord Blood in Regenerative Medicine–Neurological Applications

1. Introduction

2. Umbilical CB as a Source of Stem Cells for Neurological Applications

3. Potential Mechanisms of CB as Therapy for Patients with Neurological Diseases

4. Unrelated Donor CB Transplantation for Genetic Brain Diseases in Children

5. Ischemic Injuries

6. Neurodegenerative Diseases

7. Autism

8. Challenges

9. Summary

Chapter 14. Biobanks for Induced Pluripotent Stem Cells and Reprogrammed Tissues

1. Introduction: A Brief History of Induced Pluripotency

2. What Defines Pluripotency and the Pluripotent Stem Cell

3. Reprogramming Human Somatic Cells Toward Induced Pluripotent Stem Cells Using Defined Factors

4. Tissue Differentiation from hiPSCs

5. Potential Applications for hiPSCs in Future Regenerative Medicines

6. Considerations Toward Development of hiPSC-derived Therapies

7. Final Considerations

Section V. Cord Blood Banking: A Current State of Affairs

Chapter 15. Cord Blood Banking: Operational and Regulatory Aspects

1. Introduction

2. Conclusions

Chapter 16. Cord Blood Unit Selection for Unrelated Transplantation

1. Overview: Search and Cord Blood Unit Selection

2. Quality/Potency of the CBU

3. Selection of CB Units for Transplant: Interaction of TNC and HLA

4. Selection of CBU with Permissible HLA Mismatches

5. Approaches to Overcome the TNC Limitations of Single CB Grafts

6. Quality of CBU—Banking Practices

7. Patient Diagnosis, Relapse Risk, and CBU Selection

8. Other Immunological Considerations for CBU Selection

9. Other Graft Characteristics Affecting CBU Quality and Safety

10. Back-up CB Grafts

11. Conclusions—Selection Guidelines

Chapter 17. Quality Management Systems Including Accreditation Standards

1. Introduction

2. Quality Management

3. QM Systems

4. Creating a Documented Quality Management System (Program): Quality Management Plan

5. Standardized Systems

6. Accreditation

7. Conclusion

Abbreviations

Chapter 18. Regulation Across the Globe

1. Introduction

2. Regulation in the EU

3. Cord blood banking in the USA

4. Cord blood banking in Asia, Africa, and Oceania

5. Conclusion

Chapter 19. International Development and Import/Export–WMDA

1. Introduction

2. Issues to Consider when Starting a Cord Blood Bank

3. Listing of Cord Blood Units—Making them Available to Transplant Units

4. Registry—Gateway to the World

5. Search for Cord Blood

6. Challenges Related to the Provision of Cord Blood—Selection

7. Challenges Related to the Provision of Cord Blood—Service Provider

8. Challenges Related to the Provision of Cord Blood—Regulation

9. Cord Blood Banks Worldwide

10. Analyzing the Field—Number of Cord Blood Shipments

11. Future Plans for Cord Blood Banks

12. List of Cord Blood Registries/Banks

Section VI. Cord Blood Banking: Current and Future Outlooks

Chapter 20. Allogeneic and Autologous Cord Blood Banks

1. A Perfect Match?

2. Stem Cell Trans-differentiation and Tissue Repair?

3. Source and Quality of Information

4. Maternal and Paternal Knowledge and Preferences

5. Conclusions: What is the Current Child’s Best Interest?

Conflict of Interest

Chapter 21. The Future of Cord Blood Banks

1. Introduction

2. CB and Fetal Annex Tissues are an Abundant Source of Young Stem Cells

3. CB-derived EPCs

4. UC Wharton’s Jelly MSCs

5. The iPSCs

6. CB and UC Components

7. Conclusion

List of Abbreviations

Section VII. The Viewpoint of Society

Chapter 22. An Introductory Note to the Cord Blood Banking Issues in a European and International Environment

Chapter 23. Ethical and Legal Issues in Cord Blood Stem Cells and Biobanking

1. Introduction

2. Scientific Background: Stem Cells from Cord Blood, the Umbilical Cord, and the Placenta

3. Fundamental Ethical and Legal Issues

4. The Status of the Umbilical Cord and the Placenta

5. Legislative Characterizations of Stem Cells from Cord Blood, the Umbilical Cord, and the Placenta

6. Informed Consent

7. Communication of Information to the Donor of Cord Blood, Umbilical Cord, and Placenta Regarding Likely Diseases

8. The Donation of Cord Blood, Umbilical Cord, and Placenta and Noncommercialization

9. Anonymity—Anonymization of Donation and Protection of Data

10. Biological Safety of the Donor and Biobanking

11. Regenerative Medicine and Mesenchymal Cells of the Umbilical Cord and the Placenta

12. Conclusion

Chapter 24. Industrial Economics of Cord Blood Banks

1. Types of Cord Blood Banks

2. Emergence of Hybrid Models

3. Economic Model of Public Banks

4. Economic Model of Commercial Banks

5. Attitudes and Knowledge of Pregnant Women

6. Cost Analysis for Public Banks

7. Cost Analysis for Private Banks

8. The Emergence of Bioinsurance

9. Cost-utility for Public Health

10. Tissue Economies

11. Rethinking the Business Model of Private Banks

Chapter 25. Public Health Policies in European Union: An Innovation Strategy–Horizon 2020

1. Introduction

2. The Configuration Framework of Guiding Principles of Health Policies

3. The Health Status of the Population in the EU

Appendix 1: Evolution Rate (%) of Life Expectancy for Both Sexes per Decade (1970–2010)

Appendix 2: Life Expectancy at Birth, Males–Females (1970–2010)

Appendix 3: Life Expectancy at the Age of 65 Years, Males–Females (1980–2010)

Appendix 4: Age-Standardized Mortality Rate (SDR-ICD-10-Diseases of All Causes, All Ages), per 100,000 Inhabitants

Appendix 5: Basic Causes of Mortality in the EU-28, EU-15, EU-13 (2010)

Appendix 6: Causes of Death—Standardized Death Rate per 100,000 Inhabitants in the EU-28, EU-15, EU-13 (1980–2010)

Appendix 7: Loss in Life Expectancy Years for Men and Women from Death before the Age of 65 Years (1980–2010)

Appendix 8: PYLLs from All Causes with Comparative Reference to Life Expectancy (100,000 Men–Women Aged 0–69), (1961, 2010)

Appendix 9: Health-adjusted Life Expectancy

4. Redesigning Health Policies in Europe

Appendix 10: The Multidimensional Field of Population-appreciated Health Needs

Appendix 11: Main Objectives of Health Policies: Prolonging Life in Good Health and with Quality Years

5. Health Policies on Cord Blood Stem Cells Banking

Appendix

Index

Copyright

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List of Contributors

Julia Bosch,     Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany

Shijie Cai

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Weatherall Institute of Molecular Medicine, University of Oxford, UK

Lee Carpenter,     NHS Blood and Transplant and Radcliffe Department of Medicine, University of Oxford, Oxford, UK

Keith M. Channon,     Department of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, UK

Dominique Charron,     Laboratoire Jean Dausset, Immunology and Histocompatibility Hôpital Saint-Louis AP-HP, Université Paris Diderot, Paris, France

Theofanis K. Chatzistamatiou,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Athens, Greece

Audrey Cras

Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Cell Therapy Unit, Cord Blood Bank and CIC-BT501, Paris, France

INSERM UMRS 1140, Paris Descartes, Faculté de Pharmacie, Paris, France

Robert Danby

Department of Haematology, Oxford University Hospitals NHS Trust, Churchill Hospital, Oxford, UK

NHS Blood and Transplant, Oxford Centre, John Radcliffe Hospital, Oxford, UK

Francesco Dazzi,     Regenerative Medicine, Department of Haematology, King’s College London, London, UK

Amalia Dinou,     Hellenic Cord Blood Bank, Biomedical Research Foundation of Academy of Athens, Athens, Greece

Dominique Farge,     Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Internal Medicine and Vascular Disease Unit, CIC-BT501, Paris, France

Lydia Foeken,     World Marrow Donor Association, WMDA Office, Leiden, The Netherlands

Antonio Galleu,     Regenerative Medicine, Department of Haematology, King’s College London, London, UK

Marietta Giannakou,     MEP, Head of the Greek EPP Parliamentary Delegation, Former Minister of National Education and Religious Affairs, Former Minister of Health, Welfare and Social Security

Vasiliki Gkioka

Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece

Evaluation Expert, Hellenic Transplant Organization, Athens, Greece

Aspasia Goula,     Organizational Culture in Health Services, Technological Educational Institute of Athens, Greece

Gregory Katz,     ESSEC Business School, Chair of Therapeutic Innovation; Fondation Générale de Santé, Paris, France

Cheen P. Khoo

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Gesine Kögler,     Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany

George Koutitsas,     Process Analysis and Strategy Implementation Expert, National Insurance, Athens, Greece

Joanne Kurtzberg,     The Robertson Clinical and Translational Cell Therapy Program and Carolinas Cord Blood Bank, Duke University, Durham, NC USA

Paul Leeson,     Department of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, UK

Stefanie Liedtke,     Institute for Transplantation Diagnostics and Cell Therapeutics, University of Dusseldorf Medical School, Dusseldorf, Germany

Pascale Loiseau,     Laboratoire Jean Dausset, Immunology and Histocompatibility Hôpital Saint-Louis AP-HP, Université Paris Diderot, Paris, France

Daniel Markeson

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Department of Plastic and Reconstructive Surgery, Stoke Mandeville Hospital, Aylesbury, UK

University College London Centre for Nanotechnology and Regenerative Medicine, Division of Surgery and Interventional Science, Royal Free Hospital, London, UK

Elena Markogianni,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece

Emeline Masson,     Laboratoire Jean Dausset, Immunology and Histocompatibility Hôpital Saint-Louis AP-HP, Université Paris Diderot, Paris, France

Efstathios Michalopoulos,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece

Anna Rita Migliaccio,     Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, USA

Maria Mitrossili

Health Law of Technological Educational Institute of Athens, Greece

Institutional Technological Institute of Athens, Athens, Greece

Cristina Navarrete

Histocompatibility and Immunogenetic Services and NHS-Cord Blood Bank, National Blood and Transplant (NHSBT), England, UK

Division of Infection and Immunity, University College London, London, UK

Laura Newton

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Department of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, UK

Yannis Nikolados,     Economists in Health Management, Technological Institute of Athens, Greece

Amanda L. Olson,     MD Anderson Center, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, Houston, Texas, USA

Paul J. Orchard,     Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA

Daniela Orsini,     World Marrow Donor Association, WMDA Office, Leiden, The Netherlands

Andreas Papassavas,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece

Thalia Papayannopoulou,     Department of Medicine/Hematology, University of Washington, Seattle, WA, USA

George Pierrakos,     Primary Health Management, Technological Educational Institute of Athens, Greece

Sergio Querol,     Barcelona Cord Blood Bank and Haematopoietic Progenitor Cell Unit, Banc Sang i Teixits, Barcelona, Spain

Teja Falk Radke,     Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany

Paolo Rebulla,     Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy

Vanderson Rocha

Department of Haematology, Oxford University Hospitals NHS Trust, Churchill Hospital, Oxford, UK

NHS Blood and Transplant, Oxford Centre, John Radcliffe Hospital, Oxford, UK

Eurocord, Hôpital Saint Louis APHP, University Paris VII IUH, Paris, France

Marcos Sarris,     Health and Sociology and Quality of Life, Technological Educational Institute of Athens, Greece

Aurore Saudemont,     Anthony Nolan Research Institute and University College London, London, UK

Andromachi Scaradavou

National Cord Blood Program, New York Blood Center, New York, NY, USA

Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Markella Serafetinidi,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece

Elizabeth J. Shpall,     MD Anderson Center, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, Houston, Texas, USA

Angela R. Smith,     Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA

Sotiris Soulis,     Health Economics and Social Protection, Technological Educational Institute of Athens, Greece

Stamatia Sourri

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Catherine Stavropoulos-Giokas,     Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Athens, Greece

Jessica M. Sun,     The Robertson Clinical and Translational Cell Therapy Program and Carolinas Cord Blood Bank, Duke University, Durham, NC USA

LingYun Sun,     Department of Immunology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China

Antoine Toubert,     Laboratoire d’Immunologie et Histocompatibilité, INSERM UMR1160, and Université Paris Diderot, Sorbonne Paris Cité, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Paris, France

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

Suzanne M. Watt

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Youyi Zhang

Stem Cell Research Laboratory, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Yong Zhao,     Hackensack University Medical Center, Hackensack, NJ, USA

Foreword

Twenty-Five Years of Cord Blood Transplant

Since the first human cord blood transplant, performed in 1988, cord blood banks have been established worldwide for collection and cryopreservation of cord blood for allogeneic hematopoietic stem cell transplant. Umbilical cord blood (UCB) has now become a commonly used source of hematopoietic stem cells for allogeneic transplantation. Today, a global network of cord blood banks and transplant centers has been established for a common inventory with an estimated 600,000 UCB banked and an estimated 30,000 UCB units distributed worldwide for adults and children with severe hematological diseases. Several studies have shown that the number of cells is the most important factor for engraftment while some degree of HLA mismatches is acceptable. The absence of ethical concern, the unlimited supply of cells explains the increasing interest of using cord blood for stem cell therapy.

Much has been learned in a relatively short time on the properties of cord blood hematopoietic progenitors and their clinical application. Cord blood transplant needs to meet several new challenges. First, several methods of improvement of the speed of engraftment and decreasing transplant-related mortality are investigated such as the increase of donor pool to decrease the number of HLA mismatches or the use of double cord blood transplants. Other methods are currently investigated such as cord blood intrabone infusion, ex vivo expansion with cytokine cocktails or homing factors or addition of mesenchymal stromal cells. More interestingly, nonhematopoietic stem cells have been isolated from cord blood and placenta and could be used for the treatment of auto-immune diseases or for regenerative medicine.

E. Gluckman, MD, FRCP,     Professor Emeritus of Hematology, Eurocord,     Assistance publique des hôpitaux de Paris (APHP),     Institut universitaire d’Hématologie (IUH) Hospital Saint Louis Paris, France

Section I

Introduction

Outline

Chapter 1. Introduction to Cord Blood Stem Cells

Chapter 1

Introduction to Cord Blood Stem Cells

Dominique Charron¹, Catherine Stavropoulos-Giokas²,  and Cristina Navarrete³     ¹Laboratory Jean Dausset, Immunology & Histocompatibility, Hopital Saint Louis, Paris, France     ²Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens (BRFAA), Greece     ³Histocompatibility and Immunogenetic Services and NHS-Cord Blood Bank, National Blood and Transplant (NHSBT), England, UK; Division of Infection and Immunity, University College London, London, UK

Abstract

Hematopoietic stem cell transplantation (HSCT) can be curative for selected malignant and nonmalignant diseases. In recent years, cord blood (CB) has become a standard alternative source of HSC to bone marrow (BM) or peripheral blood (PB) for allogeneic HSCT mainly in patients who lack a human leukocyte antigen (HLA)-matched donor. This transition of CB from a waste, as it was previously considered, to a valuable cell product has been relatively rapid, and it has been accompanied by the establishment of dedicated facilities for the safe storage of these CB units, i.e., cord blood banks (CBBs) and in the development of procedures, standards, and regulations to secure the safe storage of high-quality CB units, i.e., CB banking. CB medicine consolidation is now at stake in transplantation and regenerative therapies.

Keywords

Bone marrow; Cord blood; Hematopoietic stem cell transplantation; Human leukocyte antigen; Mesenchymal stem cells; Peripheral blood

Hematopoietic stem cell transplantation (HSCT) can be curative for selected malignant and nonmalignant diseases. In recent years, cord blood (CB) has become a standard alternative source of hematopoietic stem cells (HSC) to bone marrow (BM) or peripheral blood (PB) for allogeneic HSCT mainly in patients who lack a human leukocyte antigen (HLA)-matched donor. This transition of CB from a waste, as it was previously considered, to a valuable cell product has been relatively rapid, and it has been accompanied by the establishment of dedicated facilities for the safe storage of these CB units, i.e., cord blood banks (CBBs) and in the development of procedures, standards, and regulations to secure the safe storage of high-quality CB units, i.e., CB banking.

The efficacy of CB HSC for allogeneic transplantation has significantly increased over the past years and its use is nowadays widespread in many transplantation centers. Since the first CB transplantation was successfully performed in a child with Fanconi anemia with his HLA-identical sibling, the number of allogeneic unrelated and related transplants carried out for various hematological and nonhematological disorders has increased steadily. To perform these transplants, CBBs were established for the collection, cryopreservation, selection, and release of unrelated CB units for national and international exchanges. More than 25,000 unrelated CB transplants have been performed worldwide, provided by CBBs that have collected more than 800,000 CB units.

CB cells have proliferative advantage and decreased immune reactivity when compared to other sources of hematopoietic stem cells. These properties should give a clear advantage for engraftment and diminution of acute Graft-versus-Host Disease (aGvHD). However due to the reduced volume, total nucleated cells (TNC) and HSC numbers present in a CB unit compared to BM or PB, the engraftment potential of CB has been limited. In order to overcome this issue, clinical protocols for using two CB units have been successfully developed and implemented.

Several studies have shown that TNC dose and HLA matching are important factors for survival after transplantation. Donor search algorithms have now been developed indicating that the best units should contain more than 2  ×  10⁷ TNC and more than 2  ×  10⁵ CD34+ cells per kilogram of the patient’s body weight. If only HLA-A, -B, and DRB1 matching (6/6) are considered, the number of HLA mismatches should not be more than two. Additional advantages of banked CB compared to BM are the absence of risk to the donor, the rapid and direct availability of the cells, and the absence of infectious disease at birth.

CB also contains stem cells that have retained embryonic properties and they can be isolated, and, when cultured in appropriate conditions, they give rise to cell lines that can be used for tissue engineering and regeneration of nonhematopoietic organs or tissues.

In the context of regenerative medicine or tissue engineering, CB is also a suitable source of stem cells such as mesenchymal stem cells (MSCs), and of endothelial cell progenitors. These cells have the potential to differentiate into various cell lineages including hepatocytes, muscle, cardiac myoblasts, pancreatic islets, keratinocytes, and neuronal cells and blood vessels, respectively.

These cells could be used to replace damaged or degenerating tissues or to learn more about development and cellular signaling, in normal cells or tissues or in pathological conditions. Possible applications include vascularization or bone reconstruction, and many more. Also, these cells could be used to deliver missing gene products.

Although the current results are very promising, more research is needed in order for these cells to be used in clinical settings. Considering the availability of CB and the absence of ethical problems associated with their collection, banking, and manipulation, compared to other sources of immature cells, CB could probably become a valuable source of cells to be used in stem cell therapy.

In this book, we have tried to cover the main fields of interest regarding CB. The book starts with elements of the biology behind the product, and continues with a series of articles on current and future clinical applications covering both transplantation and the field of regenerative medicine. There is a section dedicated to the organization and function of the CB banking sector, the current state of regulatory affairs, and the challenges of the future. And last, but not least, the ethical, economical, societal, and public health impact of CB banking is presented.

The presence of relatively mature HSCs in human CB was demonstrated by Knudtzon in 1974. Ten years later, Ogawa and colleagues documented the presence of primitive HSCs in CB. The current knowledge on CB hematopoiesis, the biology of hematopoietic stem and progenitor cells present, and their characteristics that give CB its unique properties are exposed in Chapter 3, that discusses how understanding the cells opens the road to transplantation.

However, it was not until 1989 that experimental and clinical studies were published indicating that human CB could be used in a clinical setting. The first successful cord blood transplantation (CBT) reported in 1988 was made possible by close collaboration among three groups: A.D. Auerbach (Rockefeller University, New York, USA), H.E. Broxmeyer (Indiana University, Indianapolis, USA), and E. Gluckman (Saint-Louis Hospital, Paris, France). Based on the diagnostic test of Auerbach for Fanconi anemia and the basic work on stem cell biology of Broxmeyer, Gluckman performed the first CBT in Saint-Louis Hospital, on a 6-year-old boy from North Carolina with severe Fanconi’s anemia, using cryopreserved CB of his HLA-identical younger sister who was unaffected by the disorder.

Since the initial HLA-matched sibling CB transplant was carried out, the substantial logistics and clinical advantages of CB as a source of HSCs for transplantation have become clear. The proliferative capacity of HSCs in CB is superior to that of cells in BM or PB from adults. Also, the immaturity of lymphocytes in CB dampens the risk of aGvHD which remains the main obstacle to the success of allogeneic transplantation of HSCs. An additional advantage of CB is the low viral (cytomegalovirus and Epstein-Barr virus) load of neonatal/fetal blood compared to adult blood.

The immune cells present in CB have unique properties that confer to CB its characteristics in the context of transplantation. The relative immaturity of these cells could explain the lower incidence of GvHD, but also opens roads for the improvement of the therapeutic potential of CB, by taking advantage of their inherent plasticity and flexibility. An overview of the mechanisms involved in the regulation and function of the immune component of CB is given in Chapter 4, as well as proposed strategies for expanding the clinical scope of CB from an immunological perspective.

The histocompatibility component is equally important. HLA is one of the decisive factors for selecting a CB unit and the impact of HLA matching on outcomes is critical. This affects the immunogenetic typing strategy for banking. In Chapter 6, the impact of both HLA and non-HLA immunogenetic factors is presented and discussed, toward a comprehensive immunogenetic assessment to establish the basis for a successful donor choice for a given patient.

Serious disadvantages of CB are the low number of HSCs compared with BM or mobilized PB, the increased risk of graft failure, the delayed hematopoietic engraftment, and the lack of donor lymphocyte transfusion for immune therapy. Additional possible disadvantages of the CB seem to be the difficulty in searching the large number of existing CBBs for matched grafts, the variation in the required handling, and, finally, uncertainty concerning aspects of post-thawing cell recovery and overall quality of CB among the CBBs.

A number of strategies have been proposed in order to overcome these limitations resulting in experimentation followed by trials. In Chapter 11, these strategies are presented: from administration of multiple units, to special pretransplantation regimens, passing from expansion and further manipulation of CB-derived cell subpopulations. The field of CBT is not static and novel approaches are being evaluated with the goal of optimizing transplantation outcomes.

Hematological malignant and nonmalignant diseases remain the main indications of CB, which is seen as an alternative to BM or peripheral blood stem cells (PBSC). Chapter 7 focuses on allogeneic related and unrelated transplantation in children and adults, giving a comprehensive view of the state of the field.

But other clinical conditions suitable for CBT have emerged. Chapter 8 is dedicated to the treatment of primary immune deficiencies and metabolic diseases by CBT. As these diseases affect children, CB, in some cases, in conjunction with assisted reproductive technology and preimplantation screening, is a precious graft source, especially in cases where the delay in engrafting is of paramount importance. Another application field is the use of CB in the treatment of autoimmune diseases, as described in Chapter 9. Following the successful use of HSCT for the treatment of severe autoimmune diseases, the use of CB with the added benefit of CB-derived MSCs with known immune-modulatory properties is very promising. The use of CB-derived immune-modulating T regulatory cell (Treg)s and MSCs in inflammatory disorders is examined in Chapter 10, and in Chapter 12, an immunomodulation model for treating diabetes with CB-derived cells is described.

Over those years much has been learned about the properties of CB hematopoietic progenitors and their clinical applications. But, CB is a complex tissue containing several subpopulations of nonhematopoietic cells, summarized in Chapter 2, that have unique functional characteristics and distinct differentiation capacity, and have their origins in different stages of fetal development. A whole chapter (Chapter 5) is dedicated to the endothelial cell compartment of CB, and their potential use as therapy. The nonhematopoietic stem cells that have been isolated from CB such as MSCs and endothelial cells, etc., can be grown and differentiated in various tissues and potentially in the near future, the CB cells will be used for the treatment of diseases such as diabetes, arthritis, burns, neurological disorders, and myocardial infarction.

This leads us away from CB transplantation and toward the realm of regenerative medicine. This is as yet an uncharted territory for now most of the work has been carried out using animal models and clinical trials have not yet yielded definitive results. CB, as described in Chapter 13, is being used as a source of cells for trials for neurological diseases and conditions, although routine administration (as in hematological diseases) is not for the near future. Another potential use is as a source of induced pluripotent stem cells (iPSCs). The biology and possible applications of iPSCs are presented in Chapter 14, as well as issues related to their eventual clinical use and ongoing trials.

The first efforts in CB banking were initiated in the laboratory of Broxmeyer at the Indiana University School of Medicine, where 7 of the first 10 units of CB collected for allogeneic transplantation use, were stored. These preliminary results led to the institution of CBBs. Besides many similarities, CB banking and traditional blood banking show a number of important differences.

The first CBBs were established in 1993 at the New York Blood Center (National CB program) by Pablo Rubinstein, in Milan (Milan CBB) by Girolamo Sirchia, and at the Bone Marrow Donor Center in Dusseldorf by Peter Wernet. These first three programs aimed at the implementation of large repositories of cryopreserved CB collected from healthy newborns. National regulatory agencies and transplant centers are aware of the need for international standards in order to promote quality throughout all phases of CB banking, with the production of high-quality CB units for transplantation. These standards, which include all practical aspects of CB banking, such as mother’s informed consent, collection techniques, labeling and identification, infectious and genetic disease testing, HLA typing, methodology of cell processing, cryopreservation, transportation, and release, have been extensively published.

Two major models of CBBs that have emerged: those dedicated to the collection and distribution of CB units from unrelated donors for allogeneic transplantation, routinely called public CBBs; and those dedicated to the storage of CB for related and autologous transplantation, often referred as private banks. Chapter 20 discusses the value of the two models regarding the best interest of the newborn/donor and the factors influencing the parents’ choice. Most of the clinical usage covered by this book concerns unrelated allogeneic banks, but one should not forget that the majority of cryopreserved CB units in the world are in fact stored for personal use. There is a whole industry centered on this, as is very thoroughly explained in Chapter 23: on the one hand publicly funded public banks and on the other for-profit commercial banks; both business models are analyzed for their efficacy, cost-effectiveness, and also their perspectives from a fiscal point of view.

A whole section of this book is dedicated to the regulatory and practical aspects of CB banking. Part of the attraction of CB as a source of HSCs for transplantation comes from the fact that it is a readily available and fully traceable product, of known quality, thoroughly tested and prepared according to stringent standards. In addition, CB collection poses no risk to the donor and provides a unique opportunity for recruiting donors from ethnic minorities. These donors, who are traditionally underrepresented in unrelated volunteer donor registries, can provide a higher frequency of rare haplotypes, thus reducing the time from initial donor search to final selection to a median time of 25–36  days, rather than months, typically required for an unrelated donor.

The whole process of banking is explained in successive chapters, starting with an overview of the CB banking process in Chapter 15 that takes the reader from the initial donor recruitment and the collection of CB after obtaining maternal consent, to the processing that takes place in dedicated laboratories, the testing and HLA typing to the subsequent selection and administration of a CB unit for transplantation. It continues with a clinician’s point of view in Chapter 16: how the selection of the most suitable available unit for a given patient is made, and how the quality indices influence this choice. A large part of this chapter is dedicated to the importance of HLA matching and the influence a particular strategy regarding HLA compatibility can have on transplantation outcome.

The need for standards in order to ensure the quality of the administered product was recognized early on, with the first accreditations taking place in the 1990s. Today, accreditation standards are but a part of integrated quality management schemes that regiment all aspects of CBB operation and extend to the transplantation centers, as described in Chapter 17. In addition, legislators have set some general sets of rules for CB banking establishments (whatever their character: public or private) in the developed world, with developing emerging nations following, as CB is seen as a national resource that can in part ensure the well-being of the population. Chapter 18 is dedicated to the regulations governing those establishments, with emphasis on EU and North American legislation, and a general overview is given for the rest of the world.

Emphasis is also placed on the international cooperation and the existence of organizations like WMDA that regulate the distribution of CB units. Their operation is described in Chapter 19, and it encompasses all stages from the initial search from donor to the feedback from the end user.

CBBs are relatively new entities, and their role is most probably not fully developed. Regulatory and financial issues aside, the existence of CBB has some impact on society as a whole. Although CB appears to be conflict-free from ethical and moral viewpoints, the development of CBBs, both public and private, and their potential use in the broader field of regenerative medicine as opposed to the well-defined scope of HSCT, raises ethical dilemmas that will need to be contemplated in a rapidly advancing scientific context in a diverse but globalized environment. These are discussed in Chapter 22, while Chapter 24 discusses the place of CBB in Public Health Policy planning in the EU. Chapter 21 gives some insight into what could be the future of CBBs, with regenerative medicine applications, such as those previously described, being integrated in their operations.

The future of CBBs will not be limited to HSCT, but will include the fields of immunotherapy and regenerative medicine. Researchers and clinicians could take advantage of the whole spectrum of stem cells that make up CB, the cells’ plasticity and scientific advances in the fields of induced differentiation (as with endothelial progenitors), pluripotency (CB as a source of iPSC), or immunomodulation. CBBs have the advantage of already satisfying a number of quality and regulatory requirements for clinical administration, as they already follow stringent protocols with an emphasis on safety and traceability, and already adhere to Good Manufacturing Process standards.

In this book, we have tried to give an up-to-date description of the field of CB in human medicine. We believe that we are at the beginning of an era when the concept of advanced therapies and regenerative medicine will finally be translated into everyday practice, and that CB could very well be at the center of this: this is reflected by the title of the book which emphasizes the importance CB could achieve in medicine.

Section II

Cord Blood Cells Biology

Outline

Chapter 2. Cord Blood Content

Chapter 3. Cord Blood Hematopoiesis: The Road to Transplantation

Chapter 4. Immunobiology of Cord Blood Cells

Chapter 5. Cord and Cord Blood-derived Endothelial Cells

Chapter 6. HLA and Immunogenetics in Cord Blood Transplantation

Chapter 2

Cord Blood Content

Gesine Kögler, Julia Bosch, Stefanie Liedtke,  and Teja Falk Radke     Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany

Abstract

During the last decade, cord blood (CB) has been under intense investigation in in vitro differentiation models and in preclinical animal models ranging from bone to cartilage regeneration, cardiovascular diseases including myocardial disease, neuronal disorders, and liver failure. Several CB subpopulations as endothelial cells, mesenchymal stromal cells (CB MSC), unrestricted somatic stromal cells (USSC) as well as cells derived from the cord (umbilical cord mesenchymal-like fibroblasts) were approached already in distinct assays. The results clearly reveal that the immunophenotype shared by the adherent populations from CB and cord (beside endothelial cells) are more similar to each other, but functionally different to bone marrow mesenchymal stromal cells (BM MSC). Special attention should be paid to the genes associated to the process of osteogenesis and cartilage formation: In quantitative PCR experiments, unlike BM MSC, the CB stromal cells expressed this bone signature on a lower level. Thus, USSC and CB MSC exhibit a more immature status than BM MSC with respect to the genetic control of bone and cartilage formation. In contrast, the fibroblast-like stromal cells derived directly from cord tissue, though being of similar morphology and immunophenotype, failed to differentiate into osteoblasts, chondrocytes, and adipocytes.

CB-derived cells also demonstrate notable paracrine effects resulting in the regeneration of cardiomyocytes and neural cells as well as support of hematopoiesis in vivo. In addition, these cells provide a defined source for reprogramming into induced pluripotent stem cells.

Keywords

Cartilage; Cord blood; ECFC; Endothelial cells; HOX code; Mesenchymal stromal cells; Unrestricted somatic stromal cells (USSC)

Chapter Outline

1. Biological Background of Cord Blood Cells—Development of Hematopoietic and Nonhematopoietic Cells 9

2. Endothelial Cells in CB 9

3. Stromal Cells in CB and Cord Tissue as Compared to BM 10

4. Isolation, Expansion, and Characterization of CB-derived Adherent Cells from CB 12

5. Generation of Cell Clones and Clonal Populations 12

6. USSC and CB MSC 12

7. Generation of Adherent Cells from the Wharton’s Jelly (Cord) 12

8. Gene Expression Profiles 13

9. Correlation of HOX-gene Expression and Regenerative Potential 13

10. Bone and Cartilage Forming Potential of Cord Blood Stromal Cells 16

11. Why Do We Have These Progenitors or Elusive Cells in CB? 16

12. USSC and MSC from CB Support Hematopoietic Cells 17

13. Liver Regeneration and Potential of CB-derived Stem Cells to Undergo Hepatic Differentiation 20

14. Cardial Regeneration In vivo 21

15. In vitro Differentiation Potential Toward Cardiomyocytes 21

16. CB Subpopulations for Neuronal Regeneration 22

17. Reprogrammed Subpopulations from CB 22

18. Conclusion 23

List of Acronyms and Abbreviations 23

References 24

1. Biological Background of Cord Blood Cells—Development of Hematopoietic and Nonhematopoietic Cells

Cord blood (CB) is characterized by a unique richness in hematopoietic stem and progenitor cells, particularly those early cells which are detected in in vitro assays like the long-term culture-initiating cell (LTC-IC) assay and high proliferative potential-colony forming cell (HPP-CFC) assay or in vivo due to their potential to repopulate nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (SRC—SCID repopulating cells). It has been extensively shown that hematopoietic stem cells (HSC) develop during embryogenesis and fetal life in a complex process involving multiple anatomic sites and niches (yolk sac, the aorta–gonad–mesonephros region, placenta, and fetal liver),¹ before they colonize the bone marrow (BM), as summarized recently.² As fetal and neonatal hematopoietic cells are markedly different from adult HSC with respect to their proliferative capacity, it was conceivable that different mechanisms and/or niches control engraftment and self-renewal of HSC during fetal and adult life. Since fetal blood is formed in close association with organs, the search for cell functions as niches similar to cell types present in adult BM environment (osteoblasts, endothelial cells, fibroblasts, reticular cells) was a logical consequence.

Although the HSC contained are currently the most relevant cells in CB with regard to clinical application, it also contains nonhematopoietic cell types which bear interesting properties that can potentially be utilized in regenerative medicine.

2. Endothelial Cells in CB

Endothelial progenitor cells (EPCs) have been investigated as a potential source of cells for vascular repair. First described in 1997,³ EPCs have been characterized by many investigators based on their morphology and surface antigen expression,⁴ but frequently without stringent in vivo analysis of function.⁵

By the group of Mervin Yoder,⁶ endothelial cord forming cells (ECFCs) in CB have been shown to be the only circulating cells that possess all the characteristics of an endothelial cell progenitor, including distinct functions. To isolate ECFCs, CB-derived mononuclear cells (MNC) or CD34+/CD45− cells are plated on a collagen-coated surface and form adherent colonies with a cobblestone-like morphology between day 7 and 14.⁶ ECFCs are rare cells, found at a concentration of about 0.05–0.2  cells/ml in adult peripheral blood. They are enriched in human umbilical CB, being found at a concentration of about 2–5  cells/ml. ECFC can be enriched from each CB sample (fresh or cryopreserved) applying the isolated CD34+-subpopulation as a basis. ECFC progeny express the cell surface antigens CD31, CD105, CD144, CD146, von-Willebrand factor, and kinase insert domain receptor (KDR), but do not express the hematopoietic or monocyte/macrophage cell surface antigens CD14, CD45, or CD115.⁶ Additionally, they are characterized by uptake of acetylated-low-density lipoprotein (AcLDL). Functionally ECFC progeny form tubes when plated alone and form de novo functionally active human blood vessels in vivo. One potential clinical use of ECFCs is in the treatment of patients with ischemia and defective wound healing due to impaired neoangiogenesis.⁷ The authors state that the ability of implanted endothelial cells to form a vascular network when the host’s angiogenic response is inhibited suggests that this strategy could be useful in treating patients with impaired wound healing. These and other reports suggest that ECFCs represent an excellent cell source for vascular engineering strategies. While there are not so many data available of the use of ECFCs in human clinical trials, the results with preclinical rodent studies provide some hope for patients who suffer from poor vascular function. Moreover, based on their growth kinetic, they are interesting candidates for tissue engineering in combination with MSC, induced pluripotent stem (iPS) cells, or mature tissue cells.

3. Stromal Cells in CB and Cord Tissue as Compared to BM

The heterotopic transplantation of BM results in the formation of ectopic bone and marrow.⁸ This osteogenic potential is associated with nonhematopoietic stromal cells coexisting with HSC in the BM.⁹,¹⁰ Friedenstein and colleagues originally called these cells osteogenic or stromal stem cells,¹⁰,¹¹ in the following years the terms mesenchymal stem cells, mesenchymal stromal cells, or skeletal stem cells have been widely used in the literature.¹² In the present article, the BM-derived nonhematopoietic cells are referred to as bone marrow mesenchymal/multipotent stromal cells (BM MSC). Several groups proved the in vivo osteogenic potential—including the recruitment of hematopoietic cells of recipient origin—of BM MSC after transplantation on a hydroxyapatite scaffold.¹³,¹⁴ After the original reports by Friedenstein, many other sources of MSC-like cells were described, for example adipose¹⁵ or fetal tissue. In 2000, the first adherent cells (also termed MSC) from CB (as summarized in Koegler et al. ¹⁶) or in the umbilical cord tissue,¹⁷,¹⁸ which revealed an immunophenotype (CD45−, CD13+, CD29+, CD73+, CD105+) similar to BM MSC, however without any in vivo reconstitution studies. This was accompanied by many other publications, as summarized by Kogler et al.¹⁸ In 2004, our group was able to detect cells in CB with a different proliferative potential, the so-called unrestricted somatic stromal cells (USSC),¹⁹ and in the following years these data were confirmed by other groups (Figure 1).²⁰–²²

Several markers described in the literature for defined subtypes of MSC from BM, e.g., CD271, CD140b, STRO-1, GD2, or NG2 could not be correlated to functional subpopulations in CB.

In the publication of 2004,¹⁹ we described USSC as a homogenous cell population with respect to their phenotype. During the following years, further detailed characterization in vitro and in vivo applying clonal cell population isolated and expanded from CB, clearly revealed distinct cell populations.²³,²⁴ We termed them according to the revisited MSC concept:²⁵ USSC and CB mesenchymal/multipotent stromal cells (CB MSC).²⁶ We could also define that clonal USSC and CB MSC lines differ most likely in their developmental origin reflected by a distinct homeobox (HOX) gene expression and expression of the delta-like 1 homolog (DLK-1), resulting in a completely different differentiation and regeneration in vivo applying specific models for neural, cardial, liver, and mesodermal (skeletal) regeneration. About 20 (out of 39) HOX-genes are expressed in CB MSC (HOX-positive), whereas native USSC (HOX-negative) reveal no HOX-gene expression.²⁷ In addition, USSC display a lineage-specific lack of the adipogenic differentiation potential along with the expression of DLK-1 (Figure 2).²³,²⁴,²⁸

Figure 1  Neonatal stromal cells.

Neonatal stromal cells were isolated from cord tissue/Wharton’s Jelly (UC MSC) or cord blood, respectively. In cord blood, two distinct nonhematopoietic stromal cell populations were described so far: USSC and CB MSC. UC MSC, umbilical cord mesenchymal stromal cells; USSC, unrestricted somatic stromal cells; CB MSC, cord blood mesenchymal stromal cells.

During recent years more than 300 cell lines were generated, characterized, and cryopreserved. Moreover, it was clearly documented that neonatal CB cells delivered at birth (>36  weeks of gestation) do not contain any OCT4A positive embryonic-like cells, and that the majority of data published by other groups were misleading due to artifacts based on OCT4A pseudogenes.²⁹ Due to the developmental status, it is unlikely that OCT4A is found in fetal, neonatal CB of 36  weeks gestation.³⁰ Moreover, more than 25,000 allogeneic CB transplantations have been performed to date without any germ cell tumor formation (including teratoma). In contrast to iPS cells (also from CB cells)³¹ that form teratomas, the absence of tumor formation in clinically relevant models (nude mice) is one of the most important features indicating why native neonatal CB subpopulations are promising candidates in regenerative cell-based approaches.

Beside the cell populations described above, other cells with embryonic characteristics were claimed to be present in CB. These so-called very small embryonic-like (VSEL) cells were described as being pluripotent and positive for expression of OCT4.³² As of today, when nearly every cell can be reprogrammed to a pluripotent state with a defined set of transcription factors resulting in a true pluripotent nature, this puts the idea of pluripotency in CB to a break. In addition, recent reports doubt the existence of these cells.³³ This resulted in a report in Nature News, summarizing studies refuting the existence of VSEL cells in tissue, including CB.³⁴ Since there are many publications about the regenerative ability of CB cells, we will try to focus on the ones that, in addition to the ECFC already highlighted above, could have therapeutic applicability based on cell numbers generated as well as functional data available in vitro and in vivo.

Figure 2  Discrimination of cord blood-derived stromal cell types.

The nonhematopoietic stromal cell types from cord blood—USSC and CB MSC—can be distinguished on the basis of differentiation potential and gene expression. (A) In vitro adipogenic differentiation potential (21  days). Lipid vacuoles were visualized by Oil Red O staining. Scale bar: 100  μm. (B) RT-PCR analyses to evaluate the expression of DLK-1 in USSC and CB MSC (Kluth et al. 2010, Stem Cells Dev). (C) RT-PCR of HOX-genes in the indicated cell types (black: absent (−); green: present, different expression levels are reflected by the color-intensity). UC MSC, umbilical cord mesenchymal stromal cells; USSC, unrestricted somatic stromal cells; CB MSC, cord blood mesenchymal stromal cells; BM MSC, bone marrow mesenchymal stromal cells; DLK-1, delta-like one homolog; HOX, homeobox.

4. Isolation, Expansion, and Characterization of CB-derived Adherent Cells from CB

Cultures of USSC and CB stromal cells are generated by the same method. Briefly, blood is collected from the umbilical cord vein directly after clamping, with informed consent of the mother. MNC are obtained by density-based Ficoll gradient separation and are subsequently cultured in plastic culture flasks containing serum-enriched medium supplemented with defined amounts of dexamethasone. As soon as colonies of adherently growing cells are observed, usually after 7–21  days in culture at 37  °C with 5% CO2 and humidified atmosphere, the dexamethasone is omitted to avoid unwanted triggering of osteogenic differentiation.

Classification of the adherent cells into USSC and CB MSC can be assessed only after generation by determining the adipogenic differentiation potential, expression of DLK-1 as well as HOX-gene expression. Over recent years, we initiated adherent cell cultures from 1009 CB samples, from which 40% (n  =  394) gave rise to an average of 1–11 colonies per individual blood sample. Due to their very low frequency in CB (1–100 per 1  ∗  10−⁸ MNC),²³,²⁴,²⁸ other fetal sources (cord) were also used,³⁵,³⁶ however, resulting in stromal cells had a completely different functionality, as discussed below.

5. Generation of Cell Clones and Clonal Populations

The availability of clones as well as clonal populations is mandatory to characterize stromal populations derived from CB as compared to those derived from BM.

Colony-clonal populations were obtained during generation of cell lines by applying special cloning cylinders. In this case, cell lines were generated as described before and, if distinct, separate colonies were observed, a cloning cylinder was attached on a single colony and cells were trypsinated according to the standard protocol. While these cells could be regarded as clonal in being derived from one single developing colony, expansion of single cells is mandatory for true clonality. Therefore, cells of one colony or of already established cell lines, respectively, were plated at low density into six-well cell culture plates. Employing an AVISO CellCelector™, single cells were picked and transported to a defined destination well of a 96-well cell culture plate. For verification, pictures were taken before and after each picking process to document successful single cell selection. By subsequent cultivation, initially with preconditioned medium, clonal lines were established which then could be expanded under standard conditions. In the case of cells picked from primary colonies, the remaining cells were expanded as bulk culture and referred to as initial cell line.

6. USSC and CB MSC

USSC and CB MSC exhibit a comparable proliferative potential (Figure 3(A)³⁶), and can be distinguished on the basis of gene expression and differentiation potential (Figure 2(A)). Both cell populations can be expanded under conditions that conform to clinical requirements (good manufacturing practice; GMP).³⁷ Here, the target cell number to be reached is more than 1.5  ×  10⁹ cells within 30 cumulative population doublings (corresponding to passage 4–5).

Applying in vitro adipogenic differentiation assays, CB MSC, but not USSC, can differentiate into adipocytes (Figure 2(B)). Former results indicated a correlation of this absent adipogenic potential and the expression of DLK-1 in USSC, since USSC but not CB MSC express DLK-1. Therefore, this marker can be applied to discriminate the CB stromal cells on a transcript but not protein level.²³

Applying microarray- and PCR-analyses, the presence of HOX-genes was defined as a distinguishing feature between USSC and CB MSC besides the expression of DLK-1: USSC do not express HOX-genes, while CB MSC are HOX-positive (Figure 2(C)²⁷).

In addition to the discriminating features presented in Figure 2, further differences between USSC and CB MSC were detected. USSC possess a higher hematopoiesis-supporting capacity in coculture experiments.²⁴ With respect to the immunophenotype, CB MSC reveal a stronger expression of CD146 (melanoma adhesion molecule, MCAM) than USSC. During flow cytometric analysis, no endothelial (CD31negative), leukocytic (CD45negative), epithelial (CD326negative), or antigen-presenting (HLA-DRnegative) phenotype was detected in any cell type analyzed.³⁶

7. Generation of Adherent Cells from the Wharton’s Jelly (Cord)

The umbilical cord consists of one vein and two arteries, which are embedded in a connective tissue, the so-called Wharton’s Jelly (Figure 1). Many protocols to isolate stromal cells from Wharton’s Jelly (often referred to as umbilical cord mesenchymal stromal cells, UC MSC), with or without an enzymatic digestion step, have been published over time. In our group, UC MSC were isolated as described in³⁶ by the outgrowth of stromal cells from umbilical cord tissue cut into small pieces and cultivated in standard culture medium. Commonly, in vitro differentiation assays were performed to prove the identity of the isolated cells as being MSC-like with the potential to give rise to cells of skeletal tissues (osteoblasts, chondroblasts, adipocytes).³⁸ In accordance with our results, further studies reported a restricted differentiation potential of so-called UC MSC concerning in vitro³⁹,⁴⁰ and in vivo⁴¹ assays. Compared to USSC and CB MSC from CB as well as to BM MSC, these cells derived from the cord tissue demonstrated the highest proliferation potential (Figure 3(A)), but the lowest (or completely absent) differentiation potential toward the osteogenic, chondrogenic, and adipogenic lineage.³⁶ Therefore, it also has to be questioned whether the term mesenchymal or multipotent does apply at all to stromal cells from cord tissue.

Figure 3  Characterization: growth kinetics and immunophenotype.

(A) Representative growth kinetics. (B) Flow cytometric analysis of CD146 and the corresponding isotype controls. Foreskin fibroblasts were used as negative control. CPD, cumulative population doublings; UC MSC, umbilical cord mesenchymal stromal cells; USSC, unrestricted somatic stromal cells; CB MSC, cord blood mesenchymal stromal cells; BM MSC, bone marrow mesenchymal stromal cells.

8. Gene Expression Profiles

Microarray gene expression analysis is a useful tool in comparing different cell types and provided further insight into the gene expression profile of CB-derived USSC and CB MSC in comparison to BM MSC.³⁵

The expression of the majority of genes was shared by USSC, CB MSC, and BM MSC (Figure 4(A)). The expression pattern of USSC and CB MSC was more similar to each other than to BM MSC, which reflects the common CB origin.

These arrays also gave hints for differences in regard to differentiation capacity, and subsequent analysis of specific gene expressions was carried out. Special attention was paid to genes associated with the process of osteogenesis and in according quantitative PCR experiments, a stronger expression of bone sialoprotein (BSP), osterix (OSX, correctly SP7), bone morphogenetic protein 4 (BMP4) and osteocalcin (OC) in BM MSC compared to the CB-derived cell types was shown (Figure 4(B)³⁵). Unlike BM MSC, the CB stromal cells lacked the typical bone signature.

Thus, USSC and CB MSC exhibit a more immature status than BM MSC with respect to the genetic control of bone formation.

9. Correlation of HOX-gene Expression and Regenerative Potential

The vertebrate skeleton consists of elements which vary regarding their embryonic origin. The craniofacial skeleton is derived from the cranial neural crest, the axial skeleton from the paraxial mesoderm, and the limb skeleton from the lateral plate mesoderm.⁴² Depending on the embryonic origin, a different HOX-gene expression pattern can be detected. While most parts of the skeleton—including the corresponding tissue-derived progenitor cells—are HOX-positive, the craniofacial skeleton is HOX-negative.⁴³,⁴⁴

Figure 4  Gene expression profile.

(A) The gene expression of USSC, CB MSC, and BM MSC (each in triplicate) was analyzed applying PrimeView™ Human Gene Expression Arrays. The Venn-diagram illustrates the common and unique expression of genes. The gene expression was filtered for each cell type: A probe set had to be expressed above the background (twentieth and hundredth percentile of the raw signal distribution) in at least two out of three replicates. This resulted in 39,519 transcripts for the group USSC, 39,493 transcripts for CB MSC, and 39,454 transcripts for BM MSC which were compared. (B) Quantitative RT-PCR analyses of genes expressed differentially in USSC, CB MSC, and BM MSC. Illustrated are the arithmetic means and standard deviations of at least three different cell lines per cell type. ∗p  =  0.01–0.05, significant; ∗∗p  =  0.001–0.01, very significant (unpaired t-test). RPL13A was used as housekeeping gene. USSC, unrestricted somatic stromal cells; CB MSC, cord blood mesenchymal stromal cells; BM MSC, bone marrow mesenchymal stromal cells; BSP, bone sialoprotein; OSX (SP7), osterix; BMP4, bone morphogenetic protein 4; OC (BGLAP), osteocalcin; RPL13A, ribosomal protein L13A.

Leucht and coworkers performed transplantation experiments in mice and analyzed the influence of the Hox-gene expression.⁴⁴ As illustrated in Figure 5(A), skeletal progenitor cells derived from the mesodermal tibia (Hoxpositive) were transplanted in defects of the ectodermal mandible (Hoxnegative), which resulted in cartilage formation instead of bone regeneration. On the contrary, the transplantation of mandibular cells (Hoxnegative) in a defect of the Hox-positive tibia resulted in effective bone repair (Figure 5(A)). The Hox-positive cells retained their Hox-gene expression after transplantation in a Hox-negative tissue. In contrast, Hox-gene expression was adapted in the previously Hox-negative cells following transplantation in a Hox-positive tissue. These results led to the conclusion, that the Hox-negative status of the mandibular cells and the potential to adapt a Hox-gene expression after transplantation may be regarded as beneficial concerning the regenerative potential.

As described above, CB-derived USSC lack the expression of HOX-genes, while CB MSC are HOX-positive (Figure 2(C)). As illustrated in Figure 5(B), we transferred the in vivo model described by Leucht et al. to an in vitro coculture model.²⁶ USSC were tagged using the green fluorescent protein (GFP) and cocultivated with HOX-positive cells (UC MSC, CB MSC, or BM MSC) for 5  days. After coculture and cell sorting, the previously HOX-negative USSC expressed HOX-genes (Figure 6(A)). Analogous to the Hox-negative mandibular cells, which started to express Hox-genes after transplantation in the tibial defect (Figure 5(A)), USSC were able to adapt a HOX-gene expression after cocultivation with HOX-positive