OBJECTIVETo establish the methods for purification, culture, and identification of adult human skeletal muscle stem cells in vitro and to explore the biological properties of the cells.

METHODSMuscle stem cells were obtained by reformed enzymatic digestion of muscle tissue from the consenting donors and cultured in serum-free medium. The morphology was inspected by an inverted phase contrast microscope. Phenotypic characteristics of the cells and expression of cell-specific markers were determined using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The growth of single cells in suspension culture was observed and recorded continuously. The cells were analyzed for their multi-lineage differentiation potential into osteoblastic, adipocyte, and smooth muscle cell lineages.

RESULTSPrimary cultured human skeletal muscle stem cells proliferated and formed the big spheres when cultured with serum free medium. Immunofluorescence staining displayed Pax7 and ALDH1 positive expression in the cell spheres. Furthermore, Myod and Desmin showed positive expression in the monolayer cells derived from the spheres. The gene expressions of Oct3/4, Nanog, Sox2 and Pax7 in the cells were determined by RT-PCR. The cell clones formed from single cells grew well. In addition, they were capable of spontaneous differentiation into myotubes in differentiation medium and into other mesodermal cell lineages in induction medium.

CONCLUSIONSHuman muscle stem cells with properties of self-renewal capacity and multi-differentiation could be successfully isolated and expanded in vitro.