The invention relates to a method for isolating muscle-derived stem cells that can be used in cell therapy, said method comprising the steps of (i) dissociating cells from at least one muscle sample, (ii) plating the cells obtained at the end of step (i) on a non-coated cell container, (iii) isolating the cells present in the supernatant of the non-coated cell container obtained at the end of step (ii), (iv) plating the cells obtained at the end of step (iii) on a coated cell container, (v) isolating the cells present in the supernatant of the coated cell container obtained at the end of step (iv), (vi) repeating, or not, the steps (iii) and (iv) at least one or two times, (vii) plating and culturing the cells isolated from the supernatant of the coated cell container obtained at the end of step (vi) until said cells have reached a confluence level of at least 50%, (viii) isolating, at the end of step (vii), the stem cells which can be used in cell therapy, wherein after expansion (a) at least 95% of said cells express CD44, CD73, (b) at least 95% of said cells express CD29, (c) at least 70% of said cells express CD90, and (d) said cells do not express CD4, CD8, CD34, CD45, CD31, CD1 17, CD144 and CD133. The invention also relates to said isolated stem cells and pharmaceutical compositions containing them.
