Résumé :
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Peripheral blood is a promising alternative source of stem cells for transplantation for the treatment of different malignancies. Using CD133, as a marker of stemness, we identified a subpopulation of purified blood-derived stem cells which differentiated in vitro and in vivo into muscle and endothelial cell type. Our results indicate that CD133+ blood-derived stem cells can be expanded for more than 50 in our specific proliferation media and we observed no indication of replicative senescence or significant changes in cellular division time. Furthermore, we did not find any difference by flow cytometry (FACS) analysis between different passages in expanded CD133+ stem cells except for the decrease in the expression of stem cell marker CD133 and we demonstrated that these cells retain their in vitro capacity to differentiate into myogenic cells. CD133 gene is structurally complex spanning over 152 kb on chromosome 4 and its finely regulated by 5 different promoters. It contains at least 37 exons, and produces a protein of 865 amino acids with a MW of 90 kDa. member of a family of 2 known cell surface glycoproteins carrying 5 transmembrane domains. The function of CD133 is not known, and its ligand has not yet been identified. For these reasons we decided to evaluate its expression in proliferating CD133+ stem cells as a measure of their stemness in culture. We demonstrated that the decrease in the expression of CD133 is due to monoclonal antibodies that in native conditions recognize only cells expressing glycosilated CD133 epitopes and therefore cannot be used to determine the total CD133 expression. By western blot analysis we demonstrated that our CD133+ stem cells maintain the expression of CD133 antigen for a long time in culture (21 days), although it's no possible to see the positive fraction by FACS analysis. The aim of ex vivo expansion is to induce cell division and proliferation of CD133+ cells while maintaining their primary functional characteristic, namely, their ability to engraft and differentiate. A better understanding of molecular mechanism regulating CD133 expression would be a powerful tool to improve ex vivo proliferation in order to isolate and expand a stem cell population useful for future clinical studies.
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