Cellular therapy for cardiac tissue repair by haematopoietic and mesenchymal stem cells

A conceivable strategy in cell and gene therapy is the use of adult stem cells isolated from bone marrow. We defined the optimal conditions for the culture of human and murine mesenchymal stem cells (hMSC and mMSC). We observed that the MSC of both species had a special morphology thanks to which it was possible to isolate them in flow cytometry on the basis of the size and granulosity parameter only. We analyzed the MSC phenotype by flow cytometry and observed that the hMSC were CD31-, CD34-, CD45, CD80- and HLA-DR- while they expressed CD73, CD90, CD105 and HLA-1 antigens. mMSC phenotype was CD34-, CD45-, CD11b-, CD106+ and Sca-1+. We also wanted to determine the frequency in progenitors among the mMSC amplified in vitro. To do so, we first assessed the enrichment in CFU-F (Colony-Forming Units – Fibroblast) progenitors. This method consisted in a secondary culture in liquid medium optimized for the development of colonies of mesenchymal origin. We were able to observe an increase of the CFU-F during the MSC passages. Then, we developed a method in order to assess the progenitors frequency by culturing MSC at limiting dilutions (CFUF-IC, Colony-Forming Units Fibroblast-Initiating cells). Once again, we were able to notice that the frequency in progenitors increased during the successive passages. The ratio between the number of CFU-F and the frequency in progenitors amounted to ± 10. We also performed differentiation assays. We were able to differentiate the mMSC in fat cells,
chondrocytes and osteoblasts. Finally, we developed a model of left coronary artery ligature in mice as well as immunohistochemical markers showing the antigens CD31, -actinin and connexin 43. With the intent to inject various types of grafts in this animal model, we also studied the cell cycle of the stem cells by Hoechst staining. Fluorescence analysis of mMSC isolated from EGFP transgenic mice revealed that their fluorescence increased from ± 30% in marrow to more than 90% for the mMSC isolated and amplified via an in vitro culture.