Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience

Abstract

Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.

Figure 1. Gene network controlling bone marrow MSC proliferation

We selected 64 transcripts that were downregulated after adipocytic, osteoblastic and chondrocytic differentiation (3). Ingenuity software allowed to determine the network with the highest score (score of 46, including 21 focus molecules effectively detected in the MSCs out of 30 molecules belonging to the theoretical network). Focus molecules are indicated by filled symbols.

Fig 2. Major lineage-determining effectors in MSCs

Lineages are regulated to a considerable extent by members of the TGF-β superfamily of growth factors activating downstream Smad signaling mediators. This is a modified diagram from (44). Abbreviations: BMP: bone morphogenetic protein; C/EBP: CCAAT/enhancer binding protein; GDF: growth and differentiation factor; MRTF: myocardin-related transcription factor; Osx: Osterix; PPAR : peroxisome proliferator-activated receptor- ; Runx2: Runt-related transcription factor 2; Sox5/6/9: SRY (sex determining region Y)-box 5, -box 6, -box 9; SRF: serum response factor; TGF-β: Transforming growth factor-β.

Fig 3. Connective tissue repair by MSCs

A, B: Orthotopic bone repair Segmental critical size bone defect (2 mm) created in femoral midshaft of athymic nude mice; the defect was filled with MSC-ceramic transplant. A: Defect in the absence of grafting; note the presence of fibrous tissue filling the gap B: Bone reconstruction (8 weeks after engraftment) was apparent in place of the fibrous tissue (arrow). C, D: Orthotopic cartilage repair Large size defect was created in the patella of Merinos sheep. Autologous bone marrow MSCs were harvested and expanded in culture for 2 passages, before being seeded in fibrin clots or scaffolds of chitosan + TGFb3. The material was implanted in the patella defect. Animals were left in the field for 8 weeks before sacrifice. C: lesions filled with ovine MSC in fibrin clot D: Lesions filled with ovine MSCs embedded in chitosan scaffolds + TGFβ3. Arrows indicate the junction between endogenous and new tissues E: Heterotopic tendon formation The intramuscular transplantation of adenovirally modified MSCs (C3H10T½ embryonic cell line) expressing Smad8 and Bmp2 leads, 4 weeks after implantation, to the heterotopic formation of tendinous elements (hematoxylin and eosin staining). The tendinous element (shown within the black and white arrowheads) is characterized by a tendon-typical crimp pattern and flattened tenocyte-like cells. Abbreviations: B, bone; M, muscle; T, tendon