Mesenchymal Stromal Cells as Tumor Stromal Modulators

Mesenchymal stromal/ stem cells (MSCs) represent a heterogeneous cell population with immunomodulating, tissue repairing, differentiating, migratory and angiogenic abilities, making them important tools for clinical and translational research. An understanding of the role of MSCs in modulating tumor growth provides a glimpse into their role in non-pathological tissue remodeling and potential regenerative tissue therapies.

Mesenchymal Stromal Cells as Tumor Stromal Modulators is a comprehensive source for the understanding of the role of MSCs as ubiquitous connective tissue cell components, which may have both direct and indirect effects on the tumor microenvironment and potential for regenerative therapeutics for various diseases. Using cancer as a model disease, this book explores the transformative role MSCs play in the recruitment of disease cells, cell repair and immunological defenses.

• Explores the biology of mesenchymal stromal cells (MSCs) and tissue related function
• Discusses the bidirectional communication between tumor stroma and MSCs derived from bonemarrow, from adipose tissue and from other tissue types
• Provides in-depth analysis of the effects of MSCs on key processes that regulate disease progression,such as angiogenesis, metastatic potential, invasion, proliferation, tumor immune privileges

• Language: English
• Publisher: Academic Press
• Release date: Oct 24, 2016
• ISBN: 9780128031032

Book preview

Mesenchymal Stromal Cells as Tumor Stromal Modulators

Marcela F. Bolontrade

Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

Mariana G. García

Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET, Facultad de Ciencias Biomédicas, Universidad Austral, Pilar, Buenos Aires, Argentina

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

1. What Are Mesenchymal Stromal Cells? Origin and Discovery of Mesenchymal Stromal Cells

Introduction

The Discovery of Mesenchymal Stromal Cells in Bone Marrow

How to Isolate Mesenchymal Stromal Cells

The Essential Characteristics of Mesenchymal Stromal Cells

The Biological Functions of Mesenchymal Stromal Cells

Mesenchymal Stromal Cells Do Not Reside Exclusively in the Bone Marrow

Concluding Remarks

Glossary

List of Acronyms and Abbreviations

2. Mesenchymal Stem/Stromal Cells From Adult Tissues

Introduction

Mesenchymal Stem/Stromal Cells Derived From Bone Marrow

Mesenchymal Stem/Stromal Cells Derived From Adipose Tissue

Mesenchymal Stem/Stromal Cells Derived From Menstrual Blood

Mesenchymal Stem/Stromal Cells Derived From Dental Pulp

Mesenchymal Stem/Stromal Cells Derived From Skeletal Muscle

Conclusion

Glossary

List of Acronyms and Abbreviations

3. Mesenchymal Stem/Stromal Cells From Neonatal Tissues

Introduction

Development of Neonatal MSC Tissue Sources

Placental Mesenchymal Stem Cells (P-MSCs)

Umbilical Cord Stroma Mesenchymal Stem Cells (UCS-MSCs)

Umbilical Cord Blood Mesenchymal Stem Cells (UCB-MSCs)

Clinical Perspectives

Conclusion

Glossary

List of Acronyms and Abbreviations

4. Mesenchymal Stem/Stromal Cells Derived From Pluripotent Stem Cells

Introduction

Pluripotent Stem Cells

Biological Pathways Involved in Mesoderm Formation

Derivation of Mesenchymal Stem/Stromal Cells From Pluripotent Stem Cells

Pluripotent-Derived Mesenchymal Stem/Stromal Cell Characterization

Experimental Therapy With PD-MSCs

Concluding Remarks and Future Directions

Glossary

List of Acronyms and Abbreviations

5. Mesenchymal Stem/Stromal Cells as Biological Factories

Introduction

MSCs as Factories of Active Soluble Molecules

MSCs as Factories of Extracellular Vesicles

Glossary

List of Acronyms and Abbreviations

6. MSC Recruitment From Distant and Local Tissues in Homeostasis and Tissue Remodeling

Introduction

MSC Distribution and Engraftment After Transplantation

Who Are the Mesenchymal Stem Cells In Vivo?

A Model for the Action of Local MSCs (and Pericytes) During Tissue Repair

Strategies for the Use of MSCs to Treat Injuries

Conclusions and Remarks

Glossary

List of Acronyms and Abbreviations

7. Mesenchymal Stem/Stromal Cell Trafficking and Homing

Introduction

MSC Migration, Homing, and Therapeutic Potential

MSCs and Tumor Microenvironment

Discrepancies in Pro/Antitumor Promoting Roles of MSC Homing to Tumors

Conclusion

Glossary

List of Acronyms and Abbreviations

8. Tumor-Secreted Factors That Induce Mesenchymal Stromal Cell Chemotaxis

Introduction

Peptide Signaling Molecules

Nitric Oxide: A Nonpeptide Signaling Molecule

Other Signaling Mechanisms

Hypoxic Tumor Microenvironment

Irradiated Tumor Microenvironment

Conclusions

Glossary

List of Acronyms and Abbreviations

9. Mesenchymal Stromal Cell Recruitment by Gastrointestinal Carcinomas

Introduction

Cellular and Tissue Sources of Mesenchymal Stromal Cells

Mesenchymal Stromal Cell Biodistribution upon Systemic Administration

Migratory Axis in Cancer

Mechanisms and Factors Involved in MSC Migration Toward Gastrointestinal Carcinomas

MSC Effect on Tumor Growth and Metastasis

MSCs for the Treatment of GIC

Conclusions

List of Acronyms and Abbreviations

10. Mesenchymal Stem/Stromal Cell Recruitment by Central Nervous System Tumors

Introduction

Mesenchymal Stem/Stromal Cells Are Recruited Into Brain Tumors

Mechanisms Underlying Tropism of Mesenchymal Stem/Stromal Cells for Central Nervous System Tumors

The Role of Endogenous, Naturally Recruited Mesenchymal Stem/Stromal Cells in Glioma Biology

Exogenous Mesenchymal Stem/Stromal Cells as Therapeutic Delivery Vehicles of Antiglioma Agents

Conclusions and Prospects for Clinical Use of Bone Marrow-Mesenchymal Stem/Stromal Cells in Glioma Therapy

Glossary

List of Acronyms and Abbreviations

11. Mesenchymal Stem Cell Transition to Tumor-Associated Stromal Cells Contributes to Cancer Progression

Introduction

Origins of Tumor-Recruited Stroma

Tumor-Associated Fibroblasts

Cellular Origins of Tumor-Associated Stroma

Tumor-Associated Fibroblast Markers

Conclusion

Glossary

List of Acronyms and Abbreviations

12. Mesenchymal Stromal Cells and Tumor Angiogenesis

Introduction

MSCs in Normal and Neoplastic Microenvironments

Tumor Tropism of MSCs

MSCs Are Involved in Tumor Angiogenesis and Lymphangiogenesis

Role of MSCs Residing in Tumors of Specific Organs: A Summary With Emphasis on the Angiogenic Modulation

Differential Effects of MSCs in Tumor Angiogenesis According to MSC Source and Culture Conditions

Molecular Mechanisms Involved in MSC-Mediated Tumor Angiogenesis

Antitumor Effects of MSCs

Conclusions

Glossary

List of Acronyms and Abbreviations

13. Role of MSCs in Antitumor Drug Resistance

Introduction

Resistance Mechanisms Revealed Upon Direct Contact With Tumor Cells

Mechanisms of Resistance Related to the Secretion of Soluble Factors

Increased Expression of Antiapoptotic Proteins

Effect of Mesenchymal Stem/Stromal Cells on Gene Expression

Discussion

List of Acronyms and Abbreviations

14. Multifunctional Roles of Tumor-Associated Mesenchymal Stem Cells in Cancer Progression

Introduction

Biological Influences of Mesenchymal Stem/Stromal Cells on Cancer Cells

Multiple Forms of Mesenchymal Stem/Stromal Cell–Cancer Cell Crosstalk in Malignancy

Cancer Cell MSC Mimicry

Mesenchymal Stem/Stromal Cells as Cells-of-Origin for Sarcomas and Carcinomas

Mesenchymal Stem/Stromal Cells in Cancer Therapy

Conclusions

Glossary

List of Acronyms and Abbreviations

15. Mesenchymal Stem Cells as Regulators of the Bone Marrow and Bone Components

General Concepts of Bone Marrow Mesenchymal Stem Cells

Hematopoiesis

Bone Marrow and Osteolineage Cells

MSCs and Other Stromal Cells

Different Functional Characteristics of Cells Residing in Different Bone Locations

The Hematopoietic Niche

Skeletal and Hematopoietic Stem Cell Niches in the Bone

Role of Osteoblasts and MSCs in the Osteoclastogenesis Process

Signaling Pathways That Govern Osteoblast Turnover

Osteoclasts and Bone Resorption

Osteoclasts in the Formation of the Hematopoietic Niche in the Bone Marrow/Bone

Morphologycal, Phenotypical, and Functional Characteristics of Bone Marrow MSCs: Importance in the Regulation of Osteogenesis, Osteoclastogenesis, and Bone Resorption Processes

Tumor Cell Interactions With the Bone Marrow/Bone Microenvironment: Premetastatic and Metastatic Niches

Bone Marrow/Bone Premetastatic Niche: Finding a Fertile Soil for Breast Cancer Cells. Importance of MSCs in Untreated Advanced Breast Cancer Patients

Concluding Remarks

List of Acronyms and Abbreviations

16. The Bone Marrow Microenvironment as a Regulator of Tumor Dormancy

Introduction

Tumor Microenvironment as a Niche for Disseminated Tumor Cells

Clinical Relevance of Disseminated/Circulating Tumor Cells

Molecular Characteristics of Disseminated/Circulating Tumor Cells

Concept of Dormancy of Disseminated/Circulating Tumor Cells

Regulation of Cellular Dormancy by the Tumor Microenvironment

Role of Fibroblasts and Extracellular Matrix in Disseminated Tumor Cell Dormancy

Role of Bone Marrow Mesenchymal Stem/Stromal Cells and Exosomes in Disseminated Tumor Cell Dormancy

Clinical Applications and Future Directions

Glossary

List of Acronyms and Abbreviations

17. Mesenchymal Stem/Stromal Cells and the Tumor Immune System

Introduction

Mesenchymal Stem/Stromal Cells: Overview

Polarization of Mesenchymal Stem/Stromal Cells and Immune Function

Overview of the Immune System

Mesenchymal Stem/Stromal Cell Effect on the Immune System Modulation

Immune-Dependent Propagation of Tumors

The Interaction of Macrophages and Mesenchymal Stem/Stromal Cells During Tumorigenesis

Mesenchymal Stem/Stromal Cells and Tumor Survival

Immunomodulation and Mesenchymal Stem/Stromal Cells

Conclusions

List of Acronyms and Abbreviations

18. The Inflammatory Environment and Its Effects on Mesenchymal Stem/Stromal Cells

Introduction

Mesenchymal Stem/Stromal Cells and Their Microenvironment

Conclusion

Glossary

Abbreviations

19. All Aboard: Mesenchymal Stem/Stromal Cells as Cell Carriers for Virotherapy

Introduction

Characteristics of Mesenchymal Stem/Stromal Cells

Therapeutic Uses of Mesenchymal Stem/Stromal Cells

Mesenchymal Stem/Stromal Cell–Loadable Viruses

Loading of Virus on Mesenchymal Stem/Stromal Cells

Current Research Progress With Mesenchymal Stem/Stromal Cell-Loaded Viruses

Clinical Trials

Limitations of and Means to Improve Mesenchymal Stem/Stromal Cell Virotherapy

Conclusions

Glossary

List of Acronyms and Abbreviations

20. Engineered Mesenchymal Stem/Stromal Cells for Cellular Therapies

Introduction

Engineering Strategies of Mesenchymal Stromal/Stem Cell Modification

Glossary

List of Acronyms and Abbreviations

21. Extracellular Vesicles From Mesenchymal Stem Cells and Their Potential in Tumor Therapy

Introduction

Potential of Mesenchymal Stem/Stromal Cell’ Extracellular Vesicles in Tumor Treatment

Tumor Targeting: Mesenchymal Stem/Stromal Cell Extracellular Vesicles in Drug Delivery Systems

The Future: EV-based Therapies in Clinical Development

Concluding Remarks

Glossary

List of Acronyms and Abbreviations

22. Therapeutic Purposes and Risks of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells

Introduction

Clinical Application of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells

Risks of Ex Vivo Expansion of Mesenchymal Stem/Stromal Cells

Risks Related to Immunosuppression and Infection From Mesenchymal Stem/Stromal Cells

Risks Related to Hypoimmunogenic Properties of Mesenchymal Stem/Stromal Cells

Risks Associated With the In Vivo Protumorigenic and Proangiogenic Potential of Mesenchymal Stem/Stromal Cells

Risks Related to Mesenchymal Stem/Stromal Cell-Induced Chemoresistance

Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting: Advantages and Disadvantages

Biodistribution and Long-term Safety of Mesenchymal Stem/Stromal Cells: What Is the Fate of MSCs In Vivo?

Concluding Remarks and Future Perspectives

Glossary

List of Acronyms and Abbreviations

23. Concluding Remarks

Index

Copyright

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List of Contributors

M.A. Amorós,     Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

A.B.B. Angulski,     Instituto Carlos Chagas, Fiocruz-Paraná, Curitiba, Paraná, Brazil

K. Anton,     Geisinger Medical Center, Danville, PA, United States

K.D. Asensi,     Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

G. Bassi,     University of Verona, Verona, Italy

M.F. Bolontrade,     Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

M.N. Bouchlaka,     University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

K.M. Bussard,     Wake Forest University, Winston–Salem, NC, United States

A. Can,     Ankara University School of Medicine, Ankara, Turkey

C.M. Capitini,     University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

A.L. Chang,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

N.A. Chasseing,     Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

H. Choi,     Central Texas Veterans Research Foundation, Temple, TX, United States

A. Correa,     Instituto Carlos Chagas, Fiocruz-Paraná, Curitiba, Paraná, Brazil

B. Couderc

University of Toulouse III Paul Sabatier, Toulouse, France

Cancer Research Center of Toulouse (CRCT), Toulouse, France

Institut Universitaire Du Cancer, Toulouse, France

L. da Silva Meirelles,     Lutheran University of Brazil, Canoas, Rio Grande do Sul, Brazil

J. Domenech

François Rabelais University, Tours, France

University Hospital of Tours, Tours, France

M. Duroux,     Aalborg University, Aalborg, Denmark

V.B. Fernández-Vallone,     Université Libre de Bruxelles (ULB), Brussels, Belgium

M.G. García,     Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET, Facultad de Ciencias Biomédicas, Universidad Austral, Pilar, Buenos Aires, Argentina

J. Glod,     National Institutes of Health, Bethesda, MD, United States

R.K. Goel,     University of Saskatchewan, Saskatoon, Canada

R.C.S. Goldenberg,     Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

J.M. Gudbergsson,     Aalborg University, Aalborg, Denmark

P. Hematti,     University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

S.C. Hung

China Medical University, Taichung, Taiwan, ROC

Academia Sinica, Taipei, Taiwan, ROC

P.T. Kamga,     University of Verona, Verona, Italy

J.R. Kane,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

D. Kanojia,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

A.E. Karnoub

Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States

Harvard Stem Cell Institute, Cambridge, MA, United States

Broad Institute of MIT and Harvard, Cambridge, MA, United States

J.W. Kim,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

M. Krampera,     University of Verona, Verona, Italy

V. Labovsky,     Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

F.F. Lang,     The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States

A. Le Naour

University of Toulouse III Paul Sabatier, Toulouse, France

Cancer Research Center of Toulouse (CRCT), Toulouse, France

Institut Universitaire Du Cancer, Toulouse, France

M.S. Lesniak,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

J. Lucas,     Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

E. Lukong,     University of Saskatchewan, Saskatoon, Canada

C. Luzzani,     Fundación FLENI, Belén de Escobar, Pcia. de Buenos Aires, Argentina

F.C. Marini,     Wake Forest University, Winston–Salem, NC, United States

L.M. Martinez,     Instituto de Biología y Medicina Experimental (IBYME), CONICET, CABA, Buenos Aires, Argentina

G.D. Mazzolini,     Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET, Facultad de Ciencias Biomédicas, Universidad Austral, Pilar, Buenos Aires, Argentina

D.B. Mello,     Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

S.G. Miriuka,     Fundación FLENI, Belén de Escobar, Pcia. de Buenos Aires, Argentina

J.M. Muller,     University of Poitiers, Poitiers, France

J. Munoz

Rutgers New Jersey Medical School, Newark, NJ, United States

Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

J. Murphy,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

L.A. Mutkus,     Wake Forest University, Winston–Salem, NC, United States

G.R. Nahas,     Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

N.B. Nardi,     Lutheran University of Brazil, Canoas, Rio Grande do Sul, Brazil

K. Nemeth

Medical College of Wisconsin, Milwaukee, WI, United States

Semmelweis University, Budapest, Hungary

A.H. Nwabo Kamdje,     University of Ngaoundere, Ngaoundere, Cameroon

T. Ochiya,     National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan

M. Ono,     National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan

B.C. Parker Kerrigan,     The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States

C. Phillips,     Central Texas Veterans Research Foundation, Temple, TX, United States

P. Pobiarzyn

Rutgers New Jersey Medical School, Newark, NJ, United States

Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

S. Ramakrishnan,     University at Buffalo, The State University of New York, Buffalo, NY, United States

P. Rameshwar

Rutgers New Jersey Medical School, Newark, NJ, United States

Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

A. Rashidi,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

D. Sarkar,     University at Buffalo, The State University of New York, Buffalo, NY, United States

P.F. Seke Etet,     Qassim University, Buraydah, Saudi Arabia

E. Spaeth,     Stem Cell Reserve, Houston, TX, United States

D.A. Spencer,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

M.A. Stimamiglio,     Instituto Carlos Chagas, Fiocruz-Paraná, Curitiba, Paraná, Brazil

K.A. Stumpf,     Wake Forest University, Winston–Salem, NC, United States

L. Vecchio,     Qassim University, Buraydah, Saudi Arabia

N.D. Walker

Rutgers New Jersey Medical School, Newark, NJ, United States

Graduate School of Biomedical Sciences at New Jersey Medical School, Newark, NJ, United States

Z. Yigman,     Ankara University School of Medicine, Ankara, Turkey

J.S. Young,     Northwestern University Feinberg School of Medicine, Chicago, IL, United States

Preface

This book is focused on mesenchymal stromal cells (MSCs) and on their relationship with tumor stromal development. It aims at covering the advances reached on the field of MSCs, particularly related to tumor progression, but also pointing out their very well-known role in regenerative medicine. Due to their immunosuppressive capacities, MSCs are increasingly being used in tissue engineering and cell-based therapies, principally focusing in decreasing inflammatory responses. However, the therapeutic application of these cells against tumors is still a promising tool.

The current volume attempts to readdress the role of MSCs as tumor modulators, giving a comprehensive overview on the physiological role of MSCs as cellular elements in the tumor stroma, on the cross-talk between other tumor components and MSCs, and on the possibility of therapeutically approaching tumors by modulating MSCs or manipulating a tumor microenvironment by delivering genetically modified MSCs.

This book was divided into six main sections: an introduction, biodistribution of MSCs, the cross-talk established between MSCs and the tumor, MSCs and metastatic niches, MSCs and the immune system, and, finally, the potential use of MSCs for tumor treatments. The first section comprises the general aspects concerning the biology of MSCs since their early discovery in 1867 by the German pathologist J. Conheim, going through the pioneer work of A. Friedenstein and the establishment of the relationship of MSCs with hematopoiesis. The evolution of the nomenclature used to name these plastic cells reflects the pathway of research that uncovered the role of MSC as niche establishers as well as the historical aspects related to the first steps in the MSC field. Besides their role as hematopoietic niche supporters, this section covers different anatomical adult and embryonic sources of MSCs as well as MSCs obtained from induced pluripotent stem cells, MSC differentiation capacity, and their biological properties that convert them into potential cell factories in their niche of residence, or in their new niche on arrival. The second section covers aspects related to MSC biodistribution and migratory capacity, taking into account this property in both physiological and pathological conditions including cancer. The third section is related to the cross-talk established between MSCs and tumors, covering different aspects of this interaction such as MSC ability to home into tumors and the effect of MSCs on tumor development, with an in-depth analysis of their role as tumor-associated fibroblastic cells, and MSC modulation of key tumor-development processes such as angiogenesis and acquisition of drug-resistance traits. Section 4 is focused on the current knowledge on MSCs and their role on metastatic processes, as well as their role as modulators of tumor dormancy. Section 5 analyzes immune responses in the tumor microenvironment and modulation by MSCs, as well as a special consideration of the microenvironmental inflammatory traits that likewise modulate MSCs. Finally, the last section is devoted to a discussion on the potential use of MSCs as therapeutic tools for tumors, considering viral approaches to introduce therapeutic genes in MSCs, as well as enzymatic engineering of MSCs for targeting purposes. This section also includes an in-depth discussion of the therapeutic properties of MSCs–extracellular vesicles for tumor treatment approaches, taking also into account current clinical trials, products, therapeutic concerns, and valid normative. Since past ISSCR president Irving Weissman urged scientists and physicians to acquire a critical view and raise concern to avoid the indiscriminate and unapproved use of stem cell therapeutics, we consider this aspect was very well covered in this last section and throughout the book, raising a particular note of caution but also emphasizing the promise that stem cell research poses, particularly MSCs for the development of tumor treatment strategies and regenerative medicine.

We thank the Elsevier editorial team for their help on this process, particularly Lisa Eppich. We also thank Mariana Amorós for artistic editing of the figures. We would like to thank all the contributors for their valuable input, which turned this book into a resourceful guide on relevant aspects of MSC research and current and potential applications particularly in the oncology field. And a final and special note of thanks is given to our families, who endured our involvement in this project from the beginning.

Marcela F. Bolontrade,  and Mariana G. García

1

What Are Mesenchymal Stromal Cells? Origin and Discovery of Mesenchymal Stromal Cells

J. Domenech¹,²     ¹François Rabelais University, Tours, France     ²University Hospital of Tours, Tours, France

Abstract

The concept of the mesenchymal stromal cell (MSC) has emerged following a number of studies starting from the middle of the 19th century that led to the discovery of the intrinsic osteogenic potential of bone marrow (BM) cells and their capacity to support hematopoiesis. Thus, a relationship was established between bone formation and the development of hematopoiesis. A mesenchymal precursor cell was identified in BM that displays differentiation potential towards osteocytic, chondrocytic, and adipocytic lineages and hematopoiesis-supporting capacity. Currently, mesenchymal stromal cell is the most commonly accepted term for this cell type. Thereafter, the concept of hematopoietic stem cell (HSC) niche was developed. This is a specific place where BM stromal cells are in intimate contact with HSCs to control their behavior. Very recently published data have helped to better characterize in vivo the cellular composition and anatomical localization of the HSC niche. Initially described in BM, MSCs have been found in almost all pre- and postnatal tissues. The actual interest for MSCs is mainly due to their multipotency ability with and thus their potential use for tissue repair, as well as their contribution to tumoral niches that could represent new antitumoral therapy targets.

Keywords

Bone marrow; Function; Hematopoietic stem cell; History; Mesenchymal stromal cell; Microenvironment; Niche; Phenotype

Chapter Outline

Introduction

The Discovery of Mesenchymal Stromal Cells in Bone Marrow

The Osteogenic Potential of Bone Marrow Cells, a Feature Already Described in the 19th Century

The Emerging Concept of Hematopoietic Stem Cells

The Discovery of a Common Mesenchymal Precursor in Bone Marrow

The Mesenchymal Precursor Also Displays Stromal Cell Function, Which Is Essential for the Control of Hematopoiesis in Bone Marrow

Different Hematopoietic Inductive Microenvironments Are Present in Hematopoietic Organs

In Vitro Modeling of a Bone Marrow Stroma With Hematopoietic Stem Cell Supporting Activity

The Concept of a Hematopoietic Stem Cell Niche

Towards a Unified Definition of Mesenchymal Precursors

How to Isolate Mesenchymal Stromal Cells

The Essential Characteristics of Mesenchymal Stromal Cells

Mesenchymal Stromal Cell Immunophenotype: A Homogeneous Profile That Hides a Heterogeneous Population

Mesenchymal Stromal Cell Differentiation Potential

Multipotency: A Key Property of Mesenchymal Stromal Cells

Pluripotency: The Optional Potential of Mesenchymal Stromal Cells

Plasticity: A Common Property of Mesenchymal Stromal Cells

The Biological Functions of Mesenchymal Stromal Cells

Stromal Function: Mesenchymal Stromal Cells as the Main Organizer of the Hematopoietic Niche

Mesenchymal Stromal Cells Display Vascular Smooth Muscle Cell Features

The Physiological Role of Mesenchymal Stromal Cells in Bone Marrow

The Contribution of Osteolineage Cells in the Niche

The Contribution of Vascular/Perivascular Cells in the Niche

The Mesenchymal Stromal Cells–Nervous System Association

Integrating the Different Actors of the Hematopoietic Stem Cell Niche: Where Are the Mesenchymal Stromal Cells?

Role in the Immune System: Mesenchymal Stromal Cells as Immunomodulatory Cells

Mesenchymal Stromal Cells Do Not Reside Exclusively in the Bone Marrow

Concluding Remarks

Glossary

List of Acronyms and Abbreviations

References

Introduction

The concept of the mesenchymal stromal cell (MSC) has gained importance in recent years, after a number of studies that led to the discovery of the osteogenic potential of bone marrow (BM) cells and their capacity to support hematopoiesis. Thus, a hematopoietic niche has been characterized in vivo in terms of cellular composition and anatomical localization in BM. MSCs, which represent the main organizer of the niche, have been used as an outstanding model of adult stem cells to study cell stemness and mesenchymal differentiation. Initially described in BM, MSCs have been found in almost all pre- and postnatal tissues. The actual interest for MSCs is mainly due to their multipotency that could be exploited for clinical applications in tissue repair and to their contribution to the formation of niches for tumor cells both of hematopoietic and nonhematopoietic origin (that could constitute new antitumoral therapy targets). This chapter will present the history of MSC discovery and the evolution of the concept, their key features (phenotype and function), the description of the BM niche and the other tissues in which they reside.

The Discovery of Mesenchymal Stromal Cells in Bone Marrow

The Osteogenic Potential of Bone Marrow Cells, a Feature Already Described in the 19th Century

In the middle of the 19th century, a nonhematopoietic cell population within the hematopoietic bone marrow (BM) was described by the German pathologist Julius Friedrich Cohneim.¹ Using animal experiments, he demonstrated that adherent fibroblastoid cells migrated from the BM toward sites of tissue injury, suggesting the existence of BM mesenchymal precursor cells. Almost at the same time, Emile Goujon demonstrated the intrinsic osteogenic potential of BM cells² by performing heterotopic autologous transplantation experiments in rabbits and chickens. These experiments showed that bone tissue could form in skeletal muscles in which red marrow was locally transplanted.

The Emerging Concept of Hematopoietic Stem Cells

Almost one century later, Till and McCulloch’s seminal works showed that the different hematopoietic cell lineages originate from BM multipotent hematopoietic stem cells (HSC) rather than from lineage-specific stem cells.³,⁴ Specifically, they observed regeneration nodules in the spleen of mice transplanted with BM cells after exposure to a lethal dose of radiation. Some of these nodules contained erythrocytic, granulocytic, and megakaryocytic cells and their clonal nature was confirmed by the presence of the same chromosomal alterations induced by low-dose irradiation in the donor BM cells before transplantation. The cells giving rise to these nodules were named colony-forming units in spleen (CFU-S).

The Discovery of a Common Mesenchymal Precursor in Bone Marrow

It took many years to elucidate the cellular origin of the BM nonhematopoietic fraction described by Emile Goujon. In 1968, Tavassoli and Crosby confirmed his results by transplanting autologous BM fragments in various extramedullary sites.⁵ They showed that in these transplant sites, bone originated from surviving reticular cells that differentiated into osteoblasts (OBs) responsible for bone formation. In addition, these reticular cells participated in the reconstruction of BM microcirculation before hematopoietic repopulation. However, these studies were performed with whole BM fragments and did not allow identifying the precise nature of the putative bone cell progenitor. A few years later, Alexander Friedenstein provided the first evidence of an OB and fibrous tissue precursor in rodent BM that displayed a fibroblastoid phenotype and clonogenic potential in vitro.⁶,⁷ Such precursor cells could be separated from hematopoietic cells in BM and spleen tissues due to their ability to rapidly adhere to plastic tissue culture dishes. After 1–2  weeks, such cells seeded at low density in basic serum-containing culture medium (without any growth-stimulating factor) generated discrete colonies consisting of spindle-shaped cells with fibroblastic morphology at the approximate frequency of 10−⁵. These colony-forming cells were named colony-forming unit fibroblastic (or CFU-F) and their clonal origin was demonstrated based on the linear relationship between colony frequency and number of plated cells and by using chromosomal markers. ³H-thymidine incorporation assays showed that most of these cells were quiescent before culturing, but could go through extensive expansion after multiple passages. Moreover, fibroblast-like cells from BM (but not from spleen) could differentiate spontaneously into bone, and occasionally cartilage, in diffusion chambers.⁸ This feature was preserved after extensive culture and passaging, demonstrating the high self-replicating capacity of these cells. Based on these characteristics, Friedenstein called these cells osteogenic stem cells.

The Mesenchymal Precursor Also Displays Stromal Cell Function, Which Is Essential for the Control of Hematopoiesis in Bone Marrow

Another fundamental discovery made by Friedenstein was that these precursors could differentiate in vivo and favor the emergence of hematopoietic tissue, when each CFU-F colony was transplanted under the renal capsule of rodents.⁹,¹⁰ After about 2  weeks, 15% of the colonies produced bone, adipose, and reticular tissue. Another 15% contained the same tissues (of donor origin), but were associated with hematopoietic cells (of host origin). The remaining colonies formed either fibrous tissue or nothing. These data clearly demonstrated that mesenchymal precursors allowed the reconstruction of a fully active BM organ that could host HSCs and support hematopoiesis. They also confirmed the existence of BM stromal cell activity, previously reported by other authors,¹¹ and supported the hypothesis of the HSC niche proposed by Schofield.¹² In this context, Maureen Owen, who actively collaborated with Friedenstein, proposed, by analogy with the hematopoietic system, a hierarchical organization of the stromal lineage with stem cells, committed progenitors, and maturing cells.¹³ The last group included reticular, fibroblastic, osteocytic, and adipocytic cells. Owen and Friedenstein used, thereafter, the term BM stromal stem cells.¹⁴

Different Hematopoietic Inductive Microenvironments Are Present in Hematopoietic Organs

Late in the 1960s, John Trentin’s pioneering works clearly demonstrated that stromal cells play a critical role in HSC differentiation towards all blood cell lineages.¹¹ Stromal cells were considered as the nonhematopoietic component of the hematopoietic organs (BM and spleen in rodents). Considering the previous discoveries by Till and McCulloch³ on CFU-S development that introduced the concept of HSCs, Trentin suggested that the interaction of stromal cells with HSCs constitutes an inductive event to promote HSC commitment and differentiation. He then proposed the term hematopoietic inductive microenvironments (HIMs),¹⁵ based on the observation that lethally irradiated mice injected with BM cells displayed predominantly granulocytic colonies in BM and erythroid colonies in spleen. This indicates that, in rodents, granulopoiesis occurs mainly in BM and erythropoiesis in spleen. Trentin confirmed this observation by implanting total BM trocar biopsies from transplanted mice directly in the spleen of irradiated secondary recipients. Seven days later, he observed that the type of colonies in the spleen depended on the stroma type with which the cells were in contact. Thus, within the implanted BM stroma (that was easily distinguished in spleen by the presence of bone tissue), most colonies were granulocytic, while within the rest of the spleen stroma the main colony type was of an erythroid nature. Later, other HIMs were identified as inducers of HSC differentiation towards other lineages, such as for megakaryocytopoiesis,¹⁶ erythropoiesis,¹⁷ or B lymphopoiesis.¹⁸

In Vitro Modeling of a Bone Marrow Stroma With Hematopoietic Stem Cell Supporting Activity

The HSC-supporting activity of BM stroma was first modeled in vitro by T. Michael Dexter by establishing long-term cultures (LTCs) of mouse marrows.¹⁹ This system (which does not contain any additional growth factor) allows both the development of a stromal layer and the growth of hematopoietic cells, including CFU-S, which are maintained over the stromal layer. In Dexter-type LTCs, HSCs tend to migrate under the stromal layers where they proliferate and differentiate, resulting in the formation of cobblestone areas. Thereafter, Rob Ploemacher demonstrated that the number of HSCs in mouse BM tissue could be quantified by using LTCs in which cells were plated at limiting dilution densities. He named these cells cobblestone area-forming cells (CAFCs)²⁰ and showed that the time needed for cobblestone area formation in culture from these cells positively correlated with their immaturity state. This emphasized the relevance of such in vitro models to explore the hierarchy and heterogeneity of the hematopoietic system. The LTC technique was also adapted later to human BM cells seeded at high cell densities²¹ as well as at limiting dilution densities.²² A recent study demonstrated, in human LTCs, that the cells localized underneath the stromal layer display the most immature features and are more quiescent.²³

The Concept of a Hematopoietic Stem Cell Niche

In recent years, the HIM concept, which initially considered a whole tissue, has evolved towards the notion of a stem cell niche, in which various subpopulations of stromal cells possess specific roles in the control of HSC fate. In 1978, Schofield introduced the notion of a niche in which the HSC is fixed in a specific place and its behavior is determined by the surrounding/neighboring cells.¹² This intimate contact of HSC with stromal cells is essential to preserve its stemness, by favoring its continuous proliferation without commitment and thus its self-renewal capacity. However, if the HSC (or its progeny) leaves the stem cell niche, it can lose its stemness and start differentiating. Several years later, the niche concept was expanded to a variety of stem cells present in other adult tissues and also during development.²⁴ Spradling and colleagues stressed that stem cells receive tissue-specific signals from a given niche cell that functions as a hub to determine the stem cell behavior and generate differentiated cells with specific functions. Therefore, the stem cell niche represents a structural unit where cell fate decisions are spatiotemporally controlled by crosstalk signals, of which some are shared by several tissues.

Towards a Unified Definition of Mesenchymal Precursors

Since the studies by Friedenstein and Owen in the 1970s, several names have been attributed to the BM cell with mesenchymal precursor function. This name variety has its root in the desire to highlight a particular property of this cell. Friedenstein first used the term osteogenic stem cell based on its bone formation capacity. However, he and Owen decided, later, to use stromal stem cell to underline its HSC-supporting activity. In the 1990s, Arnold Caplan demonstrated that BM mesenchymal precursors give rise not only to stroma and bone tissue, but also to cartilage and fat and introduced for the first time the term mesenchymal stem cell (MSC).²⁵ Additionally, he showed that MSCs could also differentiate into myoblasts and tenocytes, which represent nonskeletal lineages.²⁶ Considering MSC multipotency, Caplan proposed a hierarchical model for the genesis of mesodermal tissues where MSCs would generate most mesenchymal cell types. Thereafter, the acronym MSC was widely used in the literature. Nevertheless, Dennis and colleagues found that the differentiation potential of immortalized MSC clones from adult mouse BM displayed considerable heterogeneity, ranging from monopotential to quadripotential (for osteogenesis, chondrogenesis, adipogenesis, and stromagenesis) clones.²⁷ Therefore, they challenged the stem cell nature of such cells and opted for mesenchymal progenitor cells. Similar heterogeneity was found also in human BM samples.²⁸ Moreover, Paolo Bianco and colleagues remarked that differentiation of these cells towards nonskeletal lineages (striated skeletal muscle and tendons) was not really proven at the clonal level in vitro. Hence, they proposed to call these cells skeletal stem cells to indicate that their differentiation capacity is restricted to the osteocytic, chondrocytic, and adipocytic lineages.²⁹ MSC involvement in myogenesis and tendogenesis, particularly in vivo, could be attributed in part to their capacity to produce a considerable number of trophic factors, including cytokines or growth factors which can act both in an autocrine and paracrine manner, as highlighted by Caplan and Dennis.³⁰ This mechanism could also explain the apparent differentiation capacity of MSCs towards nonmesodermal lineages (such as neural cells and hepatocytes), leading to the expansion of possible rare populations. However, these findings might also reflect the plasticity of these cells (discussed in The Essential Characteristics of Mesenchymal Stromal Cells section).

Ultimately, there is no doubt about MSC multipotency. Indeed, they can give rise to bone, fat, cartilage, and hematopoietic-supporting stroma and exhibit high proliferative capacities. Although MSC pluripotency remains to be demonstrated, they could not be considered to be stem cells unless their self-renewal capacity is proven. This was formally confirmed only in 2007 by Paolo Bianco’s team³¹ by showing that a subpopulation of human BM cells that express the CD146 antigen (also expressed by subendothelial cells) had hematopoietic-supporting activity. This activity could be transferred in vivo by subcutaneous transplantation of BM cells expanded from CD146+ CFU-Fs in immunocompromised mice. Moreover, it could be reproduced in secondary recipients, which received cells expanded from a single CD146+ CFU-F colony obtained in the primary recipient. Therefore, these data strongly support MSC self-renewal capacity. Paolo Bianco named these cells mesenchymal stromal cells. This name is the most commonly accepted term today and maintains the same acronym as mesenchymal stem cell.

Finally, due to the confusion created by the multiple names for mesenchymal precursors, the International Society for Cellular Therapy (ISCT) decided to clarify their nomenclature. They proposed to call the fibroblast-like plastic-adherent cells in standard culture conditions multipotent mesenchymal stromal cells, while reserving the name of mesenchymal stem cells to the cells that meet the specific stem cell criteria.³² Thereby, a cell that adheres to plastic can be defined as a multipotent mesenchymal stromal cell only if it meets the following precise minimal criteria³³: (1) expression of the membrane markers CD105, CD73, and CD90 without endothelial and leukocyte markers (particularly macrophage markers); and (2) in vitro differentiation into osteocytes, adipocytes, and chondrocytes.

Due to their special differentiation capacity, MSCs isolated from the BM stromal fraction became an outstanding model to study stem cell biology and are potentially interesting cells for regenerative medicine.³⁴

How to Isolate Mesenchymal Stromal Cells

According to the technique initially described by Friedenstein and colleagues,⁷ BM MSCs can be relatively easily isolated from primary cultures initiated with BM cells harvested by aspiration (particularly for humans) or using BM plugs (in animals) and seeded at low (clonal) or higher (nonclonal) densities (Fig. 1.1). Although total BM cells can be used directly without any prior separation, cells can be more or less separated by density gradient centrifugation (eg, in Percoll or Ficoll solutions) or by immunophenotypic selection using various cell subset markers (see below). The expansion medium (αMEM or DMEM) is usually enriched with prescreened fetal bovine serum (FBS) at lower concentration (10%) than for hematopoietic cells in order to eliminate most of the nonadherent hematopoietic cells and to avoid contamination by adherent hematopoietic cells (ie, monocytes). The expansion medium may be supplemented with basic FGF (or FGF2) to increase MSC growth capacity.³⁵ The medium is replaced twice-weekly and adherent cell growth is regularly monitored (Fig. 1.1). When layers become subconfluent (80–90% confluent) after about 2  weeks, cells are detached by trypsin treatment and plated at approximatively 1–10  ×  10³ cells/cm², or at limiting dilution cell densities (at 10–40/cm²) to obtain discrete CFU-F-derived colonies. Standardized MSC culture conditions have been proposed in the framework of the European FP6 research program Genostem for the generation of undifferentiated MSCs that retain a restricted differentiation potential towards the osteocytic, adipocytic, chondrocytic, and vascular smooth muscle (VSM) lineages.³⁶ Indeed, it is crucial to control MSC culture conditions, such as cell seeding density, type of selected cells, serum batch, and oxygen rate, because these parameters can determine the quantity and especially the quality of the produced MSCs. In addition, when MSCs are cultured for therapeutic purposes, the use of FBS-supplemented culture medium raises safety problems. Therefore, several authors have proposed substitutes, such as autologous or allogeneic pooled human platelet lysates,³⁷ that allow good MSC production without significant alteration of their qualitative properties.

Figure 1.1  Standard technical procedures for expansion and CFU-F cultures of bone marrow (BM)-derived mesenchymal stromal cells (MSCs).

The Essential Characteristics of Mesenchymal Stromal Cells

The characteristics of human MSCs have been extensively described by Mark Pittenger and colleagues.²⁸ In this paper, they detailed the culture conditions to obtain human MSCs, their morphology, immunophenotype, and differentiation potential.

Mesenchymal Stromal Cell Immunophenotype: A Homogeneous Profile That Hides a Heterogeneous Population

Currently, there is no true specific marker to characterize the MSC population within the BM tissue. Three antibodies (SH2, SH3, and SH4) were thought to recognize human MSC surface antigens without crossreacting with hematopoietic cells and mature OBs.³⁸ However, it was later shown that SH2 binds also to endoglin (CD105), a type III TGFβ receptor that is present also on macrophages and endothelial cells. Similarly, SH3 and SH4 recognize also the ecto-5′-nucleotidase CD73, an antigen widely distributed on subsets of lymphocytes, macrophages, dendritic cells, endothelial cells, and epithelial cells. However, these antibodies represent useful tools for MSC characterization when combined with antibodies against endothelial markers (CD31, CD34), monocyte/macrophage markers (CD14), or a pan-leukocyte marker (CD45), to exclude the corresponding cells. In addition, an immature subset of BM MSCs can be distinguished by expression of tissue nonspecific alkaline phosphatase, a cell-surface glycoprotein usually present in osteoblastic cells. Moreover, Pittenger and colleagues reported that the whole population of human BM MSCs uniformly express CD73, CD105, CD29 (Integrin β1), CD44 (homing-associated cell adhesion molecule [HCAM]/hyaluronic acid receptor), CD71 (transferrin receptor 1), CD90 (Thy-1), CD106 (vascular cell adhesion molecule-1 [VCAM-1]), CD120a (tumor necrosis factor receptor 1 [TNFR1]), and CD124 (interleukin-4 receptor α [IL-4 Rα]), while it remains negative for CD14, CD34, and CD45 antigens.²⁸ The identification of mouse MSCs is based on the detection of the equivalent antigens, with the exception of stem cell antigen-1 (Sca-1) that is expressed in both murine MSCs and HSCs.³⁹

Overall, MSC morphological and immunological features suggest the homogeneity of this cell population, a characteristic that is maintained through repeated passages (Fig. 1.2 shows an example of MSC immunophenotypical analysis). However, MSCs in nonclonal primary cultures are, in fact, a mixture of cells with variable proliferation capacity, multipotency, and stemness. Therefore, they should be considered as the progeny of clonogenic cells (ie, CFU-Fs). This is consistent with the observation that MSC clones show variable differentiation potential.²⁷,²⁸ The discovery of surface markers expressed only by a fraction of the whole MSC population allowed better understanding of the real composition of BM MSCs and suggested their possible hierarchical organization. In addition, they might help selecting a more homogeneous immature MSC subset. Several reports showed that selected MSCs based on the expression of some of these markers exhibit several immaturity features and the widest differentiation capacity. For instance, the anti-STRO-1 antibody⁴⁰ recognizes a cell-surface antigen expressed by human BM stromal cells. About 10% of mononuclear cells and more than 95% of nucleated erythroid precursors are STRO-1+, while committed hematopoietic progenitor cells (HPC) are STRO-1–. Isolation of an STRO-1+/glycophorin A- (a marker of erythroid precursors) population in human BM leads to a 100-fold increase of the CFU-F fraction. In Dexter-type LTCs, STRO-1+ cells can form a stromal layer that includes adipocytes, smooth muscle cells, and fibroblast-like cells and supports the generation of clonogenic and mature hematopoietic cells from LTC-initiating cells (LTC-IC).²² STRO-1+ cells sorted again from these layers retain their initial ability to form a new complete stromal layer, suggesting that this marker can select stromal cell precursors. Dennis and colleagues demonstrated that besides their hematopoiesis-supportive activity, STRO-1+ cells are really multipotent cells by inducing their differentiation towards the adipocytic, osteocytic, and chondrogenic lineages.⁴¹ On the other hand, Deschaseaux and Charbord used CD49a/α1 integrin to select the BM stromal fraction, because this marker is expressed by BM stromal cells, but not by CD34+ cells.⁴² They reported that CD49a+ cells contained all the CFU-Fs and could generate stromal cells, suggesting that this subpopulation is also enriched in stromal cell precursors, as previously described with the STRO-1+ cell population.⁴⁰

Figure 1.2  Phenotype of BM MSCs obtained after expansion. Representative images of (A) flow cytometry analysis of classical membrane markers, (B) western blot analysis of lineage-specific (osteogenic, adipogenic, and vascular smooth muscle, VSM) antigens, and (C) immunofluorescence analysis of the expression of the mesenchymal intermediate filament vimentin, the VSM-specific cytoskeleton α smooth muscle actin (ASMA), the BM extracellular matrix molecule fibronectin, and (D) the nuclear embryonic-associated SOX2 and BMI-1.

Furthermore, Cattoretti and colleagues reported that a rare population of human BM stromal cells expresses neural growth factor receptor (NGFR/CD271).⁴³ Compared to the negative fraction, CD271+ cells (which represent about 2% of all BM mononuclear cells) concentrate almost all CFU-F activity (like STRO-1+ cells) and have higher expansion and osteogenic and adipogenic differentiation capacities.⁴⁴ It should be specified that CD271 expression is totally lost after MSC expansion in cultures containing FGF2.

A few years later, Sacchetti and colleagues demonstrated that selected CD146+ cells from human BM support BM stromal function and can be serially transferred in mice,³¹ a proof of their self-renewal capacity. In addition, these CD146+ cells could generate bone, adipocytes, sinusoids, and adventitial reticular cells, a cell population shown to be associated in vivo with blood vessel walls. Moreover, the selected CD146+ population displayed the highest proliferative activity, contained all the CFU-Fs, and retained tri-lineage differentiation capacity. The CD146 antigen (or melanoma cell adhesion molecule [MCAM]) was already considered a marker of VSM cells, myofibroblasts, pericytes, and endothelial cells not only in the BM, but also in many other tissues.⁴⁵ These data indicate that CD146 also represents a good marker of the immature and multipotent subset of MSCs.

More recently, Bruno Delorme and colleagues focused on identifying molecules that could specifically distinguish native MSCs from hematopoietic and endothelial cells and from fibroblasts belonging to nonhematopoietic tissues.⁴⁶ They found 17 proteins that are MSC- specific, including eight surface proteins that could be used for cell selection. The molecules that allowed the highest CFU-F enrichment were CD73, CD130 (gp130; common chain of the IL-6 receptor family), CD146, CD200 (OX-2 membrane glycoprotein), and integrin αvβ5 (a vitronectin receptor), as compared to CD49b (integrin α2), CD90 (Thy-1), and CD105. Moreover, only CD73, CD146, and CD200 were downregulated when the selected cells were induced to differentiate. This indicates that these three antigens might represent the best tools to isolate the immature MSC population. However, it must be mentioned that, in this study, CD271 was not tested because it was not retained initially as a BM MSC-specific marker.

Finally, all the MSC subpopulations described so far do represent immature stages, as indicated by their ability to support clonogenic activity and, for some of them, by their multipotency. However, it is not clear whether and to what extent these MSC subpopulations may overlap and whether and how they are hierarchically organized. Another important question is their localization within the BM tissue and their respective contribution in vivo to the hematopoietic niche organization. In a recent study, Tormin and colleagues described in human BM, two CD271+ MSC populations (lineage-/CD45–) that displayed comparable phenotypes, gene expression profiles, clonogenic efficiencies, as well as stroma and bone formation capacities in vivo.⁴⁷ They could be differentiated only based on CD146 expression and in situ localization within BM. Specifically, CD271+/CD146+ cells have the morphology of reticular cells and are observed in perivascular regions, while CD271+/CD146− cells are bone-lining MSCs close to endosteal regions. Their different in vivo localizations could indicate different roles within the endosteal and perivascular niches, respectively (as detailed in The Biological Functions of Mesenchymal Stromal Cells section).

Mesenchymal Stromal Cell Differentiation Potential

First, it is important to stress that MSCs expanded in proliferation medium without any differentiation inducer already coexpress intracellular molecules that are specific of several differentiation pathways. For instance, they can express simultaneously osteoblast (parathyroid hormone receptor, PTHR), adipocyte (peroxisome proliferator-activated receptor γ [PPARγ] and leptin) and VSM (alpha-smooth muscle actin [ASMA] and caldesmon) markers, together with vimentin, an intermediate filament expressed in mesenchymal tissues, and important BM extracellular matrix molecules, such as fibronectin (Fig. 1.2B and C). This is consistent with the observation that some stromal cells simultaneously display differentiation structures typical of both adipocyte and VSM lineages⁴⁸ and supports the hypothesis of the lineage priming model, recently proposed for MSC differentiation.⁴⁹

Multipotency: A Key Property of Mesenchymal Stromal Cells

It is now widely accepted that MSC multipotency describes their ability to differentiate into skeletal lineages, such as the osteogenic, chondrogenic, and adipogenic pathways.⁸,²⁵,²⁸,⁴⁹ MSC skeletal lineage differentiation capacity is a key property of MSCs because fibroblasts from nonhematopoietic tissues are devoid of such capacity.²⁸,⁵⁰ The classical procedures to induce BM MSC differentiation into osteoblasts, chondrocytes, and adipocytes are shown in Fig. 1.3.

Figure 1.3  Standard technical procedures for osteogenic, adipogenic, and chondrogenic differentiation of BM MSCs. The classical specific induction media are indicated. After 2–3   weeks, the presence of calcium deposits in osteocytes is revealed by Alizarin Red staining, cellular neutral lipid vacuoles by Nile Red staining (in yellow), and chondrocyte-specific glycosaminoglycans in cell pellets by Toluidine-blue staining and by immunofluorescence analysis of type II collagen expression (in green).

Besides these three lineages, MSCs can also differentiate into VSM, even without any previous induction (Fig. 1.2C), a feature strongly associated with MSC stromal function.⁵¹ Moreover, expression of VSM lineage markers by MSCs suggests a significant role in postnatal angiogenesis or vascular repair, whereas their role in endothelium formation remains more controversial. For instance, the circulating endothelial progenitor cells (EPCs) detected in humans, based on the coexpression of surface markers shared by HSCs (CD34) and angioblasts (vascular endothelial growth factor receptor 2 [VEGFR2]/fetal liver kinase 1 [FLK1]) during embryonic development, were shown to be, in fact, of adult BM origin.⁵² These findings suggest the possibility of postnatal endothelial generation from specific progenitors, in addition to the classical remodeling of differentiated endothelial cells derived from preexisting blood vessels. It is tempting to hypothesize that endothelial cells can originate also from MSCs, as demonstrated for HPCs. However, only one report has provided consistent data in favor of this hypothesis⁵³ and does not allow considering this differentiation pathway as preferential in MSCs. Nevertheless, a significant MSC contribution to vascular reconstruction is still likely, either directly by providing the cells of the muscular layer, or indirectly by constituting a local environment that promotes the migration and tubular organization of endothelial cells.⁵⁴

Pluripotency: The Optional Potential of Mesenchymal Stromal Cells

The question of whether MSCs can differentiate into nonskeletal mesodermal lineages (and even into nonmesodermal lineages) was raised many years ago and is still a matter of debate. In other words: are adult MSCs true pluripotent cells?

Controversies have been often the result of confusion due to data misinterpretation without taking into account the precise experimental procedures. For instance, it is usually difficult to discriminate the indirect and the direct effects of injected MSCs in different tissues in vivo. On the other hand, for in vitro studies, it is important to distinguish between experiments using standard culture media supplemented with classical growth factors and studies in which MSCs are exposed to factors that might induce cell reprogramming, such as DNA-demethylating agents (eg, 5-azacytidine), or in which gene expression is directly modified by transfection. Indeed, the possibility to reprogram fibroblast cells is now undoubtedly established through the generation of induced pluripotent cells (iPS) from mouse embryonic and adult fibroblasts,⁵⁵ human dermal fibroblasts,⁵⁶ and also human⁵⁷ and mouse⁵⁸ MSCs. Moreover, only the study of individual MSC clones allows evaluating the real differentiation capacity of each stem cell clone that must be separated from eventual misleading effects, including trophic effects, cell fusion, transdifferentiation from a fully differentiated cell to another or selection of a rare population.

Nevertheless, during the last 15  years, hundreds of studies have been published on the multiple differentiation capacities of MSCs, particularly in view of their use for cellular therapy in regenerative medicine. These reports evaluated MSC differentiation potential into skeletal lineages (bone, cartilage, and tendons) (see for review Ref. 59), but also into nonskeletal pathways. For instance, it has been shown that in vivo, MSCs promote the regeneration of injured muscles.⁶⁰ However, this effect could be simply explained by cell fusion,⁶¹ although MSCs can truly differentiate into skeletal muscle in vitro when cultured with several factors, including forskolin, an adenylyl cyclase activator that increases cAMP intracellular levels.⁶² Likewise, in vitro generation of beating cardiomyocytes can easily be obtained by culturing murine MSCs with 5-azacytidine⁶³ and locally delivered total BM cells can repair infarcted myocardium in mouse models.⁶⁴ Similarly, intravenous injection of MSCs in rats after acute myocardial infarction improves heart function through a mechanism that favors both angiogenesis and myogenesis.⁶⁵

The ability of murine and human BM MSCs to differentiate in vitro into cells that express neural-specific proteins was first reported in 2000⁶⁶ using a combination of nonspecific (EGF) and neural-lineage-specific (BDNF) growth factors. Neuron-like cells were also obtained from MSCs after coculture with neural cells. Such neural induction of mouse and human MSCs can be clearly improved by transfection of Notch intracellular domain.⁶⁷

BM MSCs could also have a role in hepatocyte differentiation. Several studies reported the transdifferentiation of BM-derived cells into hepatocyte-like cells after in vitro culture⁶⁸–⁷⁰ or in vivo transplantation.⁷¹ It was shown that MSCs are the main BM-derived cell population contributing to this property.⁷¹ However, some authors,⁷² but not all of them,⁷¹ indicated that cell fusion is a frequent event in this experimental setting.

Moreover, BM MSCs’ involvement in the repair of heat-shocked small airway epithelium was demonstrated in vitro.⁷³ Although this phenomenon has been attributed mainly to the many cell fusions occurring in this system, some MSCs really differentiated into epithelial cells. Similarly, transplantation of human MSCs has proven its efficacy in promoting skin wound healing after extensive irradiation.⁷⁴

Finally, it is remarkable that MSCs express simultaneously both lineage-specific markers and markers of undifferentiated cells, as shown in Fig. 1.2A, B, C, and D. For instance, sex determining region Y-box 2 (SOX2) and polycomb complex protein BMI-1, two proteins present at early developmental stages, are concomitantly expressed in adult BM MSCs. BMI-1 is required to inhibit gene transcription (including HOX genes) and has been associated with MSC self-renewal.⁷⁵ Previous studies have reported the expression of embryonic-associated proteins in expanded and undifferentiated MSCs, including octamer-binding protein 4 (OCT4), homeobox protein NANOG, SOX2, and stage-specific embryonic antigen (SSEA) -3 and -4.⁷⁶,⁷⁷ While SOX2 plays a role in MSC proliferation and multipotency,⁷⁸ OCT4 and SOX2 expression appears to be lost after osteogenic induction.⁷⁹ These data are consistent with a latent pluripotent capacity of MSCs that could be required in exceptional (physiological or pathological) circumstances that remain to be identified.

Plasticity: A Common Property of Mesenchymal Stromal Cells

In summary, it is difficult to present a consensus opinion about MSC pluripotency considering their known plasticity. Generally, this property refers to the capacity of adult MSCs to overcome lineage barriers and to adopt the phenotype and the function that are considered specific of other tissues. In fact, plasticity can encompass several processes, including true pluripotency (ie, the ability to differentiate into tissues deriving from other germ layers), transdifferentiation (and reprogramming), or even cell fusion. The only demonstration of true pluripotency in adult BM cells was provided by Verfaillie’s group who described the existence of multipotent adult progenitor cells (MAPCs), isolated from both human and mouse BM mononuclear cells.⁸⁰ This particular cell population displays a fibroblastic morphology similar to MSCs and, after in vitro expansion, can contribute in vivo to all three germ layers. Unfortunately, this property has not been confirmed definitively yet by other authors.

Concerning transdifferentiation, it is now accepted that in some situations, fully differentiated adult cells can differentiate into cells belonging to other lineages.⁸¹,⁸² An example of this process is given by the epithelial–mesenchymal and mesenchymal–epithelial transitions occurring during tumor development.⁸³ Within mesodermal tissues, transdifferentiation is a classical, frequently described process. In particular, fully differentiated OBs, adipocytes, and chondrocytes from MSCs can each be induced to differentiate towards the two other mesenchymal lineages.⁸⁴ Similarly, cloned MSCs that have differentiated into VSM cells retain the capacity to give rise to adipocytes, OBs, and chondrocytes.⁴⁹ Such lineage conversion could be, at least in part, the result of a sequential process of cell dedifferentiation followed by a new differentiation towards another lineage. Such a reprogramming mechanism may account for MSC differentiation into cardiomyocytes⁶³ or skeletal muscle⁶² that can be induced by addition in the differentiation medium of 5-azacytidine or forskolin. A similar effect can be obtained by transfecting MSCs with embryonic pluripotency genes that promote, for instance, their neuronal differentiation,⁶⁷ or their transformation into embryonic-like stem cells (ie, iPS).⁵⁷,⁵⁸

Cell fusion has been evoked to explain some cases of MSC differentiation into skeletal muscle cells,⁶¹ hepatocytes,⁷¹ or epithelial cells.⁷³

These potential mechanisms do not exclude additional in situ trophic effects provided by MSCs administered in vivo on possible resident tissue-specific immature cells, as suggested by Caplan and Dennis.³⁰ An overview of the many soluble mediators secreted by adult BM MSCs, assessed by cytokine array, is provided in Fig. 1.4.

Finally, expression analysis of a wide number of genes and proteins has clarified the differentiation capacity profile of primary layers and clones of BM MSCs.⁴⁹ Undifferentiated MSCs do express simultaneously osteoblastic, chondrocytic, adipocytic, and VSM markers, but not skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineage markers. After induction of differentiation, MSCs express (or overexpress) mainly the markers of the lineage into which they were induced to differentiate, while the other markers are downregulated.

Figure 1.4  Concentration of 66 soluble factors (growth factors, other cytokines, and chemokines) secreted in supernatants of confluent BM MSC cultures at passage 2 (mean and SEM; n   =   3). MSCs were prestimulated or not with TNFα and the cytokines evaluated with the RayBio Human Cytokine Antibody Array.

The Biological Functions of Mesenchymal Stromal Cells

Stromal Function: Mesenchymal Stromal Cells as the Main Organizer of the Hematopoietic Niche

The discovery of HIMs by Wolf and Trentin showed that nonhematopoietic components within hematopoietic tissues influence hematopoiesis, particularly HSC commitment.¹¹ Friedenstein⁹,¹⁰ clearly demonstrated that BM has a hematopoiesis-supporting activity. This function is associated with the development of bone and vascular tissues and can be transferred upon BM transplantation in other animals.³¹

Mesenchymal Stromal Cells Display Vascular Smooth Muscle Cell Features

Analysis of BM stromal cells with hematopoietic-supporting activity obtained from Dexter-type LTCs revealed that they have VSM cell features,²⁵,³⁴,⁴⁸ as attested by the expression of cytoskeletal markers (ASMA and caldesmon) and smooth muscle markers (smooth muscle-specific myosin heavy chain).⁵¹ Within BM, ASMA+ cells are mainly found in the perivascular region surrounding endothelial cells (ECs), where they correspond to capillary-associated pericytes and to VSM cells of the sinus and arteriolar media. They also line trabecular bone in endosteal regions and are dispersed among hematopoietic cell foci.⁵¹ As mentioned above, unselected²⁸ and STRO-1+⁴¹ BM stromal cells expressing VSM lineage markers can differentiate towards the adipocytic, osteocytic, and chondrocytic lineages.

The Physiological Role of Mesenchymal Stromal Cells in Bone Marrow

BM stromal cell multipotency indicates a close relationship (or possibly identity) with mesenchymal stem cells. It is conceivable that this MSC feature, besides a direct hematopoiesis-supportive function, has an important role in preserving the integrity of the local microenvironment. Indeed, differentiated into VSM cells, MSCs can be involved in the development and repair of the vascular network, which is essential for the establishment and maintenance of hematopoiesis.³¹ Moreover, due to their osteogenic potential, MSCs contribute to the physiological bone turnover and the repair of bone fractures and constitute a stem cell reservoir for osteogenesis.⁸⁵ Although MSC chondrogenic potential seems more relevant for joint cartilage repair, it could be important also in BM because endochondral ossification (that proceeds through a cartilage intermediate) is required for adult HSC niche formation.⁸⁶ Likewise, the adipogenic capacity of BM MSCs could be involved in hematopoiesis regulation, because adipocytes within the adult BM microenvironment act like negative regulators of hematopoiesis.⁸⁷ This could explain the inverse correlation observed in BM between hematopoietic activity and the number of adipocytes.

The Contribution of Osteolineage Cells in the Niche

About 10  years ago, several works suggested a predominant role of BM osteolineage cells in HSC control. Specifically, in vitro studies indicated that cultured OBs obtained from trabecular bone could support LTC-IC growth.⁸⁸ In vivo, the number of HSCs and OBs is concomitantly increased in the BM of transgenic mice in which OBs overexpress parathyroid hormone (PTH) or PTH-related protein receptor (PPR).⁸⁹ Likewise, in mice in which the bone morphogenic protein receptor 1A gene (Bmpr1a) had been deleted the number of OBs and quiescent HSCs, considered as long-term reconstituting HSCs (LT-HSCs), is increased.⁹⁰ These authors also found that a subset of immature OBs was overrepresented. These cells, named spindle-shaped N-cadherin+ osteoblastic cells (SNO), line the bone surface and are in contact with LT-HSCs through homophilic adhesive interactions that involve N-cadherin (neural-cadherin, nonepithelial cadherin, CDH2), which is expressed by SNOs and LT-HSCs. N-cadherin presence was associated with the expression of β-catenin that acts as an intracellular signal transducer in the Wnt signaling pathway. N-cadherin belongs to a family of calcium-dependent adhesion molecules, is expressed by osteolineage cells, and plays an essential role in bone formation and in HSC regulation by the BM niche.⁹¹ Indeed, overexpression of N-cadherin in HSCs inhibits the cell cycle and helps maintain HSC self-renewal capacity after repeated transplantations.⁹² Conversely, transient N-cadherin knockdown (by RNA interference) inhibits HSC long-term engraftment.⁹³ The hypothesis of OB involvement in HSC niche function has been strengthened by the discovery that osteopontin (OPN), a phosphorylated glycoprotein mainly produced by OBs in BM, can decrease the HSC pool⁹⁴ and induce HSC quiescence through β1 integrin-mediated adhesion.⁹⁵ Moreover, artificial depletion of the OB population in genetically modified mice leads to a dramatic decrease in HSC number and strongly promotes extramedullary hematopoiesis.⁹⁶ All these data led to the notion of an osteoblastic niche within the endosteal region. In this niche, OBs are considered as the key constitutive element for controlling HSC stemness. However, conflicting reports challenged OB’s central role. First, no N-cadherin expression could be detected in highly purified murine HSCs.⁹⁷ Second, although transient N-cadherin downregulation (by RNA interference) inhibits HSC long-term engraftment,⁹² conditional knockout of the N-cadherin/Cdh2 gene in mice did not clearly alter hematopoiesis. The HSC pool size and cell cycle status remained unchanged as well as their self-renewal capacity and long-term reconstitution capacity.⁹⁸–¹⁰⁰ Therefore, the term osteoblastic niche has been progressively replaced by endosteal niche.

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