Ex vivo expansion of human circulanting CD133+promising tool for cell-based therapeutic approaches in muscular dystrophy

The use of stem cells in regenerative medicine and cell-based therapies offers immense potential in diseases which have currently no treatment such as Duchenne muscular dystrophy. A possible limitation to the use of CD133+ for a therapeutic application is the relatively low number of cells that can be recovered from peripheral blood mononuclear cells. The goal of ex vivo expansion is to induce proliferation of CD133+ cells while maintaining their primary functional characteristic, namely, their ability to engraft and differentiate. In this work, we expanded in culture stem cells with different combinations of cytokines, to explore possibility of ex vivo expansion of CD133+ cells from peripheral blood. All experiments cultured with different combinations of cytokines supported the growth of CD133+ cells compared with the group without cytokines, but only a cocktail including SCF, bFGF, EGF, VEGF, LIF, TEPA, IL6 showed a significant expansion of stem cells in culture. In this condition we were able to expand the CD133+ cells for more than 50 passages over a 2-month period and we observed no indication of replicative senescence or significant changes in cellular division time during expansion period. Proliferating cells still had the capacity to form hematopoietic and endothelial colonies in semisolid media. Furthermore, we showed that expanded populations of CD133+ cells derived from blood maintain the capacity to differentiate into myogenic cells in vitro and in vivo. Human circulating CD133+ cells were also cultured at 5- or 20-percent oxygen in liquid culture in presence of the better cocktail of cytokines and we analysed and compared their expansion capacity and their vitality. The total number of cells increased 6-fold at 5-percent oxygen and could result in a better maintenance of the balance between primitive progenitor cell renewal and clonogenic progenitor expansion, thus representing a tool of remarkable therapeutic interest.