Ebook: Regenerative Medicine and Cell Therapy

Most human tissues do not regenerate spontaneously, which is why “cell therapy and tissue engineering ” are promising alternative treatments. The principle is simple: patients' or donors' cells are collected and introduced into the injured tissues or organs directly or in a porous 3D material, with or without modification of their properties. Their use in new therapeutic areas will depend on advances in biology, materials science and engineering.

There is an extraordinarily wide range of opportunities for clinical applications of regenerative medicine. Among possible medium-term therapeutic applications are, cardiac insufficiency, preparation of small diameter arteries, treatment of atherosclerosis, cartilage defects, bone repair, burns, diabetes, liver or bladder regeneration, and neurodegenerative disorders. This concept of regenerative medicine is an emerging multidisciplinary field involving surgery, medicine, biology, chemistry, mechanics and engineering which can be defined as “the way to improve health and quality of life by restoring, maintaining, or enhancing tissue and organ functions”.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate in different tissues. Although initially it may seem best to choose an autologous cell, these cells are generally not easily available, they are frequently in a pathological state and expansion cannot be extended to all tissues and organs. This explains the growing interest in stem cells that are produced during the development of the embryo, then of the foetus and finally in adult tissues and organs. Different stem cells (SC) with different potential can be isolated and characterised (totipotent, mesenchymal of different origins, especially those present in tissues...). It is undeniable that cells like bone marrow, adipose tissue or Wharton Jelly stem cells, which have limited potential, are of potential interest for applications because they are easily separated and prepared and no ethical problems are involved in their use.

Adult or embryonic stem cells are incontestably major subjects of research and for the development of therapeutic applications but are a sensitive subject which can sometimes trigger highly emotional reactions. Embryonic stem cells can apparently self-renew without limit in culture, but the mechanisms underlying this capacity are still not completely understood. However, they should lead to new knowledge and current research on the mechanisms behind intercellular communications, differentiation, and proliferation are based on the idea that, by mastering their regeneration potential, new clinical applications will become possible.

Despite the acknowledged promise of embryonic stem cells, in many cases adult stem cells provide a more interesting approach to clinical applications. In other respects, some lineages of adult stem cells are capable of greater plasticity than is assumed based on their tissue origin. Nowadays, mesenchymal stem cells, which were originally described in bone marrow, represent an enormous potential value for regenerative medicine. During the last 10 years, these multipotent cells have generated considerable interest, and mesenchymal stem cells in particular have been shown to escape allogenic immune response and be capable of immunomodulatory activity. These properties may be of great interest in regenerative medicine in the future, but today it is probably better to consider mesenchymal stem cells as a mixed population of progenitors rather than as homogeneous stem cells.

Recently, cells isolated from amniotic fluid also appeared to be of potential interest in regenerative medicine. These cells express some markers also expressed by embryonic stem cells and appear to be similar to stem cells isolated from Wharton Jelly.

The full potential of these stem cells is not yet known and it is possible that all the types of cell needed for regenerative medicine could be obtained from umbilical cord, placenta and amniotic fluid.

The regeneration of tissue is and will remain a challenge for the future development of cell therapy and tissue engineering. Many problems remain to be solved and scientific and technical knowledge is lacking that could lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardised product, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations. It can be also noted that recently (since 2005–2006) tissue engineering had been replaced by cell therapy. The focus has switched from organ growth to cell therapy where cells are implanted to restore damaged or diseased tissues (in vivo engineering).

This book does not claim to be a handbook which covers all aspects of regenerative medicine or cell therapy. For those purposes, a much larger volume would be needed.

In this volume, the first part (11 chapters), is devoted to basic knowledge of stem cells. The second part (20 chapters) is dedicated to potential clinical applications (hematology, cardiac, vascular, osteoarticular, liver, skin, etc.) without having been able to envisage all the applications that will doubtless be developed in the coming decade. We hope that this book will provide a stimulus for basic researchers and clinicians involved in regenerative medicine or cell therapy.

The editor would like to thank all the authors for their outstanding contributions which enabled the publication of this volume.

J.F. Stoltz

June, 2012

The Academy recently discussed the topic of stem cells in the framework of the comments it made about the recommendations attached to the parliamentary commission's mission on the upcoming revision of bioethics laws [1]. The document below results from commission I's work (biology) and brings additional information to a subject in full scientific change, and whose expected medical applications still remain broadly prospective. The results achieved so far underline however, the huge advantage of that research area and encourage to renew our request to provide researchers working in France with the necessary freedom for their research, including on human embryo stem cells.

Stem cells are valuable tools for the study of developmental biology, drug discovery and modeling and understanding disease, and they also show great potential for the development of stem-cell-based therapies. Stem cells and their derivatives do indeed form the basis of therapies for treating a select number of diseases, including hematopoietic stem cell transplants for bone marrow diseases and the use of epithelial stem-cell-based treatments for corneal disorders and burns. Recent studies reveal that regenerative medicine is in transit to clinical trials include the use of human embryonic stem cell (hESC) derivatives to treat macular degeneration, stroke and amyotrophic lateral sclerosis. The hESC have two essential characteristics: the unlimited proliferation capacity and pluripotency, which was the ability to differentiate into any cell type of the human body. By their self-renewal capacity and the ability to differentiate for example into neurons or keratinocytes, it can be imagine to prepare a bank of allogenic cells derived from hESCs to treat patients. The current challenge is the transition from bench to bedside.

In this chapter we review the criteria that allow to define the Mesenchymal Stem Cell as a stem cell. The methods implemented for this demonstration are well documented. This chapter will provide guidelines for the study of clonogenicity, self-renewal ability, phenotype, differentiation potential and regeneration capacity of human MSCs.

Mesenchymal stem cells, now commonly called mesenchymal stromal cells (MSCs), represent a rare and heterogeneous population of stem/progenitor cells that reside primarily in the bone marrow (BM) but can also be isolated from different sites such as adipose tissue, peripheral blood, cord blood, liver and fetal tissues. They are described as non-hematopoietic, clonogenic, plastic-adherent cells and capable of differentiating into multiple mesodermal lineages. They lack the inherent ethical consideration of embryonic stem cells and thanks to their capacity to home and engraft into injured tissues and modulate immune response, cell survival and angiogenesis, mesenchymal stromal cells represent a potential novel candidate for regenerative medicine. MSC immune-modulating properties make also them tools for the treatment of graft-versus-host disease (GVHD) as well as severe auto-immune diseases. This clinical interest needs to be substantiated by a clear understanding of the MSC molecular and cellular mechanisms, leading to their use in human therapeutics in the best conditions of security and efficacy. This chapter first presents what is actually known about MSC phenotypic and functional characteristics, focusing on their differentiation, trophic and immune-modulatory capacities. It then briefly describes the prerequisites for MSC culture in vitro for clinical applications.

Cellular therapy involves the isolation, expansion and transplantation of human cells for the replacement or regeneration of injured tissues. It is one of the most significant repair strategies in Regenerative Medicine today. Mesenchymal stem cells represent a versatile, multimodal cellular therapy for the treatment of a broad spectrum of diseases. They stimulate a host repair response by paracrine mechanisms involving immunomodulatory, cytoprotective and pro-angiogenic mechanisms. It also appears that they may be tolerated as a fully allogeneic therapy.

Mesenchymal stem cells (UC-MSC) are characterized by their multipotency, immunosuppression and hypo-immunogenicity. MSC transplant raises hope for treatment of various diseases including graft versus host disease, myocardial infarction and autoimmune disease. Human umbilical cord tissue is an abundant and accessible resource of MSC with no ethical concern, which takes great advantage in clinical application. We are dedicated in banking of clinical grade umbilical cord MSC (UC-MSC) and large-scale manufacturing of cell injection to insure the safety, efficiency and reproduction of UC-MSC as a cell drug.

Umbilical cord blood is a readily available source of hematopoietic stem cells used with increasing frequency as an alternative to bone marrow or peripheral stem cells for transplantation in the treatment of malignant and nonmalignant conditions in children and adults. The recent interest in stem cell research and public fascination with promises of stem cell-based therapies, fueled by the media, have led researchers to explore the potential of UCB stem cells in therapy for regenerative medicine applications.

Adipose derived mesenchymal stromal cells or adipose stem cells (ASCs) are undifferentiated progenitor cells residing in various locations of the fat tissue in the body. The increasing interest towards these adult stem cells relies on the ease of harvest in large quantities and the robust expansion rate that make them attractive sources of cells for regenerative medicine. These cells show phenotypic, differentiation and paracrine characteristics similar to adult mesenchymal stem cells (MSCs) isolated from bone marrow. These properties are shared by culture-expanded MSCs and ASCs. However, the phenotype as well as the migration potential of native and culture-expanded ASCs has been recently shown to differ. In the current review, we focus on the characterization of native ASCs, their functional properties and their relevance to adult stem cell-based regenerative medicine applications.

One of the greatest challenges in cell therapy is to deliver viable stem cells to injured tissues with a high engraftment efficiency. Some studies have shown that under physiological conditions, stem cells may be mobilized by cytokines or growth factors which allow them to leave their niche and home into peripheral tissues. To improve this phenomenon, recombinant proteins or pharmacological agents are now widely used. In this review we will focused on the three main types of circulating stem cells: hematopoietic stem cells, endothelial progenitor cells and mesenchymal stem cells.

Cancer Stem Cells (CSCs) represent a small minority of cells that have properties of tumor initiation, self-renewal, unlimited proliferation, and have the ability to differentiate to give rise to progeny cells that are more differentiated and non-tumorigenic. CSCs can be derived from somatic stem cells, progenitors or even differentiated cells that have reacquired properties of self-renewal. Such populations of cells have been identified in various cancers. Here, we will focus on leukemic stem cells (LSCs) as a paradigm for CSC biology. We will discuss on the hierarchical and clonal organization of the leukemic clone, the frequency of LSCs as well as their fundamental properties and murine models used to study LSCs. Furthermore, we will describe several potential mechanisms that could be modulated to eradicate these LSCs, a therapeutic strategy which is believed to represent a major step forward in leukemia therapy.

Almost all cells in the human body are subjected to mechanical stresses. These forces can vary from a few Pascals (shear stress) to some Mega Pascals (on hip cartilage). It is now well known that mechanical forces have a decisive effect on cellular physiology. However, although the main biological effects of mechanical forces are well documented, the relation between mechanical stress and physiological phenomena is mainly unknown (mechanotransduction phenomenon). In this chapter, some effects of mechanical stresses on different cells (endothelial cells, vascular or muscular cells, chondrocyte, etc.) are summarized.

Myofibroblasts are responsible for contraction of granulation tissue in skin wounds. They are also present in many pathological situations, derived either from local cells in the tissue or from circulating precursor or stem cells. Because of their important role in pathological scarring and fibrosis, as well as a role in for cancer-associated myofibroblasts in tumor growth, much interest centres on the factors that regulate their differentiation and survival. Myofibroblast phenotype is regulated by the microenvironment of damaged tissue, including growth factors, the extracellular matrix, mechanical stress and mechanical signaling to which they are subjected.

Viral infections represent one of the main causes of morbidity and mortality following Hematopoietic Stem Cell Transplantation. Anti-viral treatment failure may be explained by absence of specific immune reconstitution. In the 1990s, anti-viral immunotherapy initially consisting in total donor lymphocyte infusion presented efficiency but was often associated with adverse effects. Specific antiviral immunotherapy emerged and relied on isolation of mono or multi-virus donor-derived-specific T cells with or without in vitro expansion. During the last ten years, such an adoptive transfer has been proved feasible, and helpful in specific anti-viral immune reconstitution, and rarely associated with advers events. Two main evolutions contributed to allow a good reactivity to propose immunotherapy in case of anti-viral treatment failure: development of allogeneic CTL banks and improvement of CTL isolation methods using immunomagnetic technology which presents the advantage to be fast.

Post-infarction heart failure is closely associated with a major loss of differentiated cardiomyocytes consecutive to ischemia, which cannot be overcome by the poor renewal of cardiac cells in the heart. This has led many investigators to develop various strategies for cell-based therapy to improve defective contractile performance and heart function. Although indirect beneficial effects can be expected from various mechanisms, such as improvement of tissue perfusion, tissue contractility recovery or paracrine secretions, a definitive treatment requires neocardiogenesis in order to repopulate the ischemic area. The concept of cell plasticity opened new perspectives in this field, suggesting a possible use of different adult cells with the aim to achieving cell transdifferentiation towards cardiomyocytes when placed in an appropriate in vivo context. Among the sources of adult stem or progenitor cells for cardiomyocyte regeneration, cells derived from adipose tissue are now being investigated, including at the clinical level. According to several reports, the transplantation of these cells is associated with a functional benefit, which however cannot only be explained by transdifferentiation regardless of the underlined mechanisms. The purpose of this chapter is to discuss, in a critical view, the direct and indirect effects of stromal cells derived from adipose tissue on heart regeneration.

Cardiomyocyte deficiency is the leading cause of heart failure. The recent discovery of resident adult cardiac progenitor cells together with the rapid evolution of the stem cell research area have begun to re-evaluate the clinical relevance of regenerative cardiac therapies. Uncovering the developmental origins of adult cardiac progenitors, understanding how these populations are set aside and characterizing the molecular mechanisms regulating their fate during development are of crucial importance for regenerative medicine and will bring new insights into cardiac repair strategies.

Cell therapy is considered a potential therapeutic avenue for the treatment of skeletal muscle diseases. Heterologous approaches have been attempted in the context of Duchenne muscular dystrophy, a generalized degenerative disease. Cell transplantation trials, however, have been first hampered by the poor survival and the limited migratory ability of the cells, and a first wave of optimizations defined conditions of cell injection and recipient immunosuppression, allowing the achievement of a second set of clinical trials. Further investigations identified anoikis, oxidative stress, fusion inability and some administration methodologies as causes of early massive cell death. New concepts have emerged contemporaneously regarding the correction of gene expression (gene supplementation, exon skipping, gene surgery...), and new myogenic cell types have been identified, mainly in the family of perivascular cells, that can be administered systemically. Then, preclinical models were proposed to adapt the injection strategies, to combine them with genetic modifications of the cells or with pharmacological interventions on the environment to improve the success of implantation. In the meantime, autologous approaches have been attempted in the context of localized repairs. Therefore the continuous identification of new stem cell candidates, the invention of new strategies to restore correct gene expression, the technical and pharmacological improvements of transplantation success, are justified by yet unmet social and medical needs.

With decades of research completed, rAAV-based gene therapies for muscle diseases such as DMD have finally progressed to clinical trials. Multiple approaches, such as gene replacement with mini-dystrophin, gene correction by exon skipping, and reducing chronic inflammation, show promise for the future. Obstacles encountered during human trials have led to novel strategies, including the generation of rAAV pseudo-types, the construction of modified muscle specific promoters, and targeted capsid modifications.