Isolation and Differentiation of Murine Skeletal Muscle Stem Cells

Skeletal muscle stem cells were isolated from hind limb muscles of 6- to 8-week-old mice (kindly provided by P. Muñoz Cánoves of the Pompeu Fabra University, Barcelona) as described in [75 (link)]. After mechanical and enzymatic dissociation with 1 % Pronase protease (Calbiochem), the filtered digest was centrifuged through an isotonic Percoll (Amersham) gradient (60 % overlaid with 20 %) and cells were collected from the gradient interface, resuspended in growth medium (GM: Ham’s F-10 plus 20 % FBS, 5 ng/ml bFGF, 100 U/ml penicillin and 100 μg/ml streptomycin) and grown on tissue culture dishes coated with 0.05 mg/ml collagen I from rat tail (Becton and Dickinson) to amplify the myoblast population. To induce myotube formation, confluent proliferating primary myoblasts were grown on matrigel coated culture dishes (Basement Membrane Matrix, Becton and Dickinson) and switched to differentiation medium the next day (DM: DMEM plus 2 % horse serum, 100 U/ml penicillin and100 μg/ml streptomycin (Life Technologies)) for 4 days. Myofibres directly isolated from EDL muscles were kindly provided by S. Gutarra. Two biological replicates of each experiment were performed and analysed. Mouse protocols were approved by the Animal Care and Use Committee of the PRBB, and the Ethical Committee for Animal Experimentation of the Government of Catalonia.