Luc Sensebé

Human Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: A User's Guide

Mesenchymal stem cells (MSCs) are adult stem cells that hold great promise in the field of regenerative medicine. They can be isolated from almost any tissue of the body and display, after expansion, very similar properties and minor differences, probably due to their microenvironment of origin. Expansion in vitro can be obtained in cytokine-free, serum-enriched media, as well as in serum-free, basic fibroblast growth factor-enriched media. A detailed immunophenotypic analysis is required to test the purity of the preparation, but no unique distinguishing marker has been described as yet. Functional assays, that is, differentiation studies in vitro, are needed to prove multilineage differentiation of expanded cells, and demonstration of pluripotency is necessary to identify most immature precursors. MSCs show powerful immunomodulative properties toward most of the cells of the immune system: this strengthens the theoretical rationale for their use also in an allogeneic setting across the major histocompatibility complex (MHC) immunological barriers. Systemic intravenous injection and local use have been tried: after systemic injection, MSCs show a high degree of chemotaxis based on pro-inflammatory cytokines, and localize at inflamed and neoplastic tissues; local regeneration has been improved using synthetic, as well as organic scaffolds. On the other hand, inadequate heterotopic in vivo differentiation and neoplastic transformation are potential risks of this form of cell therapy, even if evidence of this sort has been collected only from studies in mice, and generally after prolonged in vitro expansion. This review tries to provide a detailed technical overview of the methods used for human bone-marrow (BM)-derived and adipose-tissue (AT)-derived MSC isolation, in vitro expansion, and characterization for tissue repair. We chose to use BM-MSCs as a model to describe techniques that have been used for MSC isolation and expansion from very different sources, and AT-MSCs as an example of a reliable and increasingly common alternative source.

Defining the risks of mesenchymal stromal cell therapy

Abstract We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.

Mechanisms of bone repair and regeneration.

Bone problems can have a highly deleterious impact on life and society, therefore understanding the mechanisms of bone repair is important. In vivo studies show that bone repair processes in adults resemble normal development of the skeleton during embryogenesis, which can thus be used as a model. In addition, recent studies of skeletal stem cell biology have underlined several crucial molecular and cellular processes in bone formation. Hedgehog, parathyroid hormone-related protein, Wnt, bone morphogenetic proteins and mitogen-activated protein kinases are the main molecular players, and osteoclasts and mesenchymal stem cells are the main cells involved in these processes. However, questions remain regarding the precise mechanisms of bone formation, how the different molecular processes interact, and the real identity of regenerative cells. Here, we review recent studies of bone regeneration and repair. A better understanding of the underlying mechanisms is expected to facilitate the development of new strategies for improving bone repair.

Immunological characterization of multipotent mesenchymal stromal cells--The International Society for Cellular Therapy (ISCT) working proposal.

Cultured mesenchymal stromal cells (MSCs) possess immune regulatory properties and are already used for clinical purposes, although preclinical data (both in vitro and in vivo in animal models) are not always homogeneous and unequivocal. However, the various MSC-based clinical approaches to treat immunological diseases would be significantly validated and strengthened by using standardized immune assays aimed at obtaining shared, reproducible and consistent data. Thus, the MSC Committee of the International Society for Cellular Therapy has decided to put forward for general discussion a working proposal for a standardized approach based on a critical view of literature data.

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

Good Manufacturing Practices Production of Mesenchymal Stem/Stromal Cells

Because of their multi/pluripotency and immunosuppressive properties mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs has led to production processes that need to be in accordance with Good Manufacturing Practice (GMP). In cellular therapy, safety remains one of the main concerns and refers to donor validation, choice of starting material, processes, and the controls used, not only at the batch release level but also during the development of processes. The culture processes should be reproducible, robust, and efficient. Moreover, they should be adapted to closed systems that are easy to use. Implementing controls during the manufacturing of clinical-grade MSCs is essential. The controls should ensure microbiological safety but also avoid potential side effects linked to genomic instability driving transformation and senescence or decrease of cell functions (immunoregulation, differentiation potential). In this rapidly evolving field, a new approach to controls is needed.

The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.

We previously identified multipotent stem cells within the lamina propria of the human olfactory mucosa, located in the nasal cavity. We also demonstrated that this cell type differentiates into neural cells and improves locomotor behavior after transplantation in a rat model of Parkinsons disease. Yet, next to nothing is known about their specific stemness characteristics. We therefore devised a study aiming to compare olfactory lamina propria stem cells from 4 individuals to bone marrow mesenchymal stem cells from 4 age- and gender-matched individuals. Using pangenomic microarrays and immunostaining with 34 cell surface marker antibodies, we show here that olfactory stem cells are closely related to bone marrow stem cells. However, olfactory stem cells also exhibit singular traits. By means of techniques such as proliferation assay, cDNA microarrays, RT-PCR, in vitro and in vivo differentiation, we report that when compared to bone marrow stem cells, olfactory stem cells display (1) a high proliferation rate; (2) a propensity to differentiate into osseous cells; and (3) a disinclination to give rise to chondrocytes and adipocytes. Since peripheral olfactory stem cells originate from a neural crest-derived tissue and, as shown here, exhibit an increased expression of neural cell-related genes, we propose to name them olfactory ectomesenchymal stem cells (OE-MSC). Further studies are now required to corroborate the therapeutic potential of OE-MSCs in animal models of bone and brain diseases.

Risk of tumorigenicity in mesenchymal stromal cell–based therapies—Bridging scientific observations and regulatory viewpoints

In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.

Specific Lineage‐Priming of Bone Marrow Mesenchymal Stem Cells Provides the Molecular Framework for Their Plasticity

Lineage‐priming is a molecular model of stem cell (SC) differentiation in which proliferating SCs express a subset of genes associated to the differentiation pathways to which they can commit. This concept has been developed for hematopoietic SCs, but has been poorly studied for other SC populations. Because the differentiation potential of human bone marrow mesenchymal stem cells (BM MSCs) remains controversial, we have explored the theory of lineage‐priming applied to these cells. We show that proliferating primary layers and clones of BM MSCs have precise priming to the osteoblastic (O), chondrocytic (C), adipocytic (A), and the vascular smooth muscle (V) lineages, but not to skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineages. Priming was shown both at the mRNA (300 transcripts were evaluated) and the protein level. In particular, the master transactivator proteins PPARG, RUNX2, and SOX9 were coexpressed before differentiation induction in all cells from incipient clones. We further show that MSCs cultured in the presence of inducers differentiate into the lineages for which they are primed. Our data point out to a number of signaling pathways that might be activated in proliferating MSCs and would be responsible for the differentiation and proliferation potential of these cells. Our results extend the notion of lineage‐priming and provide the molecular framework for inter‐A, ‐O, ‐C, ‐V plasticity of BM MSCs. Our data highlight the use of BM MSCs for the cell therapy of skeletal or vascular disorders, but provide a word of caution about their use in other clinical indications. Stem Cells 2009;27:1142–1151

Bone Marrow Mesenchymal Stem Cells: Biological Properties and Their Role in Hematopoiesis and Hematopoietic Stem Cell Transplantation

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that are present in practically all tissues as a specialized population of mural cells/pericytes that lie on the abluminal side of blood vessels. Originally identified within the bone marrow (BM) stroma, not only do they provide microenvironmental support for hematopoietic stem cells (HSCs), but can also differentiate into various mesodermal lineages. MSCs can easily be isolated from the BM and subsequently expand in vitro and in addition they exhibit intriguing immunomodulatory properties, thereby emerging as attractive candidates for various therapeutic applications. This review addresses the concept of BM MSCs via a hematologist’s point of view. In this context it discusses the stem cell properties that have been attributed to BM MSCs, as compared to those of the prototypic hematopoietic stem cell model and then gives a brief overview of the in vitro and vivo features of the former, emphasizing on their immunoregulatory properties and their hematopoiesis-supporting role. In addition, the qualitative and quantitative characteristics of BM MSCs within the context of a defective microenvironment, such as the one characterizing Myelodysplastic Syndromes are described and the potential involvement of these cells in the pathophysiology of the disease is discussed. Finally, emerging clinical applications of BM MSCs in the field of hematopoietic stem cell transplantation are reviewed and potential hazards from MSC use are outlined.