Frédéric Deschaseaux

Human Leukocyte Antigen‐G5 Secretion by Human Mesenchymal Stem Cells Is Required to Suppress T Lymphocyte and Natural Killer Function and to Induce CD4+CD25highFOXP3+ Regulatory T Cells

Adult bone marrow‐derived mesenchymal stem cells (MSCs) are multipotent cells that are the subject of intense investigation in regenerative medicine. In addition, MSCs possess immunomodulatory properties with therapeutic potential to prevent graft‐versus‐host disease (GvHD) in allogeneic hematopoietic cell transplantation. Indeed, MSCs can inhibit natural killer (NK) function, modulate dendritic cell maturation, and suppress allogeneic T‐cell response. Here, we report that the nonclassic human leukocyte antigen (HLA) class I molecule HLA‐G is responsible for the immunomodulatory properties of MSCs. Our data show that MSCs secrete the soluble isoform HLA‐G5 and that such secretion is interleukin‐10‐dependent. Moreover, cell contact between MSCs and allostimulated T cells is required to obtain a full HLA‐G5 secretion and, as consequence, a full immunomodulation from MSCs. Blocking experiments using neutralizing anti‐HLA‐G antibody demonstrate that HLA‐G5 contributes first to the suppression of allogeneic T‐cell proliferation and then to the expansion of CD4+CD25highFOXP3+ regulatory T cells. Furthermore, we demonstrate that in addition to their action on the adaptive immune system, MSCs, through HLA‐G5, affect innate immunity by inhibiting both NK cell‐mediated cytolysis and interferon‐γ secretion. Our results provide evidence that HLA‐G5 secreted by MSCs is critical to the suppressive functions of MSCs and should contribute to improving clinical therapeutic trials that use MSCs to prevent GvHD.

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.

Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype.

Summary. Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony‐forming unit (CFU‐F), osteo‐chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti‐CD49a and/or anti‐CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU‐F were always detected in the small population of CD49a‐positive cells. These CFU retained their differentiation potential and gave rise to osteo‐chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45med,low, CD34low, HLA‐II– cell population. Flow cytometry cell sorting showed that MSC with CFU‐F potential were obtained only from a CD49a+/CD45med,low population. In addition, when cultured, they clearly became CD45–, CD34–, HLA‐II–, CD49a+. These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.

HLA-G is a crucial immunosuppressive molecule secreted by adult human mesenchymal stem cells.

Adult bone marrow-derived mesenchymal stem cells (MSCs) are multipotential cells capable of regenerating injured tissues. In addition to their multipotency, MSCs inhibit natural killer cell cytotoxicity and T-lymphocyte alloproliferation. Several immunosuppressive mechanisms have been described, including indoleamine 2, 3, -dioxygenase-induced depletion of tryptophan from the lymphocyte environment, and the secretion of prostaglandin E2 and other immunosuppressive factors. Here, we review data supporting a new MSC immunoregulation pathway, in which the key molecule is the human leukocyte antigen-G protein. This nonclassical human leukocyte antigen-class I molecule was initially found on trophoblasts, where it contributes to tolerance at the materno-fetal interface. Because trophoblasts are also able to express indoleamine 2, 3, -dioxygenase and prostaglandin E2, MSC immunomodulatory properties are similar to those of trophoblasts. These mechanisms should be explored in relation to induction of tolerance to alloantigens for the prevention of graft rejection after transplantation.

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.

Safety concern between autologous fat graft, mesenchymal stem cell and osteosarcoma recurrence.

Background Osteosarcoma is the most common malignant primary bone tumour in young adult treated by neo adjuvant chemotherapy, surgical tumor removal and adjuvant multidrug chemotherapy. For correction of soft tissue defect consecutive to surgery and/or tumor treatment, autologous fat graft has been proposed in plastic and reconstructive surgery. Principal Findings We report here a case of a late local recurrence of osteosarcoma which occurred 13 years after the initial pathology and 18 months after a lipofilling procedure. Because such recurrence was highly unexpected, we investigated the possible relationship of tumor growth with fat injections and with mesenchymal stem/stromal cell like cells which are largely found in fatty tissue. Results obtained in osteosarcoma pre-clinical models show that fat grafts or progenitor cells promoted tumor growth. Significance These observations and results raise the question of whether autologous fat grafting is a safe reconstructive procedure in a known post neoplasic context.

Human marrow stromal precursors are ?1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non‐human marrow stromal cells (primary cultures and lines) and on human CD34+ hematopoietic normal and leukemic precursors. Differential expression of α1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34+ cells. We used this difference to select, in fresh bone marrow samples, α1‐positive cells. We found that all stromal precursors giving rise to colony‐forming units‐fibroblasts (CFU‐F) were present in the α1‐positive fraction. No colonies were detected in the α1‐negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from α1‐positive cells and grown in long‐term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of α4 and α2 integrin subunits in cultures derived from α1‐positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that α1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while α2 and α4 integrin subunits are expressed primarily by immature cells. J. Cell. Physiol. 184:319–325, 2000.

CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment

Bone marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146−/Low and CD146High cells under clonal conditions and after sorting of the non‐clonal cell population to determine whether this expression is associated with specific functions. CD146−/Low and CD146High bone marrow MSCs did not differ in colony‐forming unit‐fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic‐supportive activity. However, CD146−/Low clones proliferated slightly but significantly faster than did CD146High clones. In addition, a strong expression of CD146 molecule was associated with a commitment to a vascular smooth muscle cell (VSMC) lineage characterized by a strong up‐regulation of calponin‐1 and SM22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed towards a VSMC lineage.

Cell therapy for bone repair.

When natural bone repair mechanisms fail, autologous bone grafting is the current standard of care. The osteogenic cells and bone matrix in the graft provide the osteo-inductive and osteo-conductive properties required for successful bone repair. Bone marrow (BM) mesenchymal stem cells (MSCs) can differentiate into osteogenic cells. MSC-based cell therapy holds promise for promoting bone repair. The amount of MSCs available from iliac-crest aspirates is too small to be clinically useful, and either concentration or culture must therefore be used to expand the MSC population. MSCs can be administered alone via percutaneous injection or implanted during open surgery with a biomaterial, usually biphasic hydroxyapatite/β-calcium-triphosphate granules. Encouraging preliminary results have been obtained in patients with delayed healing of long bone fractures or avascular necrosis of the femoral head. Bone tissue engineering involves in vitro MSC culturing on biomaterials to obtain colonisation of the biomaterial and differentiation of the cells. The biomaterial-cell construct is then implanted into the zone to be treated. Few published data are available on bone tissue engineering. Much work remains to be done before determining whether this method is suitable for the routine filling of bone tissue defects. Increasing cell survival and promoting implant vascularisation are major challenges. Improved expertise with culturing techniques, together with the incorporation of regulatory requirements, will open the way to high-quality clinical trials investigating the usefulness of cell therapy as a method for achieving bone repair. Cell therapy avoids the drawbacks of autologous bone grafting, preserving the bone stock and diminishing treatment invasiveness.

Bone regeneration: the stem/progenitor cells point of view

•  Introduction •  Mesenchymal stem cells as skeletal stem cells ‐  Cultured or expanded MSCs ‐  Native MSCs ‐  The neural crest origin of MSCs •  Vascularization by endothelial progenitor cells as prerequisite process before bone repair ‐  Vascularization and bone healing ‐  Endothelial progenitor cells •  Conclusion