Adult muscle ‘stem’ cells can be sustained in culture as free-floating myospheres

The effectiveness of cell-based therapy to treat muscle disease has been hampered by difficulties in isolating, maintaining and propagating the stem cells that are needed for treatment. Here we report the isolation of muscle-derived stem cells from both young and old mice and their propagation over extended periods of time in culture as “free-floating” myospheres. Analysis of these sphere-forming cells showed that they express stem cell antigen-1 (Sca-1), β1 integrin (CD29), Thy-1 (CD90), and CD34, but did not express CD45, CD31, or myogenic markers (Pax7, Myf5, and MyoD). We found that cells derived from myospheres and then grown adherently (MDACs) behaved similar to primary myoblasts, in that these cells expressed myogenic markers and were able to easily form multinucleated myotubes. Unlike the parental myospheres but analogous to primary myoblasts, MDACs expressed Pax7, Myf5, and MyoD, indicating that the parent myosphere cells were a more primitive type of cell. In support of this we demonstrated that myospheres were also able to differentiate into adipogenic and osteogenic cells in culture, as well as being able to contribute to injured muscle in vivo. In summary, we report that primitive adult muscle stem cells can be easily isolated and sustained in culture as myospheres.