Isolation and Culture of Muscle Stem Cells

To obtain purified SCs, primary cells were isolated as described previously (Paris et al., 2016 (link)). Muscles were dissected from mice and digested using collagenase II (Gibco, Carlsbad, California) in Dulbecco's modified Eagles medium (DMEM, Sigma-Aldrich) for 60 min at 37°C with agitation. The suspension was then washed and digested in collagenase II and dispase (Gibco) for 30 min at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: CD31-PECy7 (BD Biosciences 561410, San Jose, California), CD45-PECy7 (BD Biosciences 552848), Sca1-FITC (BD Biosciences 562058), Integrin α7 (ITGA7)-Alexa Fluor 647 (AbLab, Vancouver, Canada) and VCAM-PE (Invitrogen RMCD10604). FACS was performed using a FACSAria II Cell Sorter (BD Biosciences) and SCs were collected according to the following sorting criteria: CD31-/CD45-/Sca1-/ITGA7+/VCAM+. FACs-purified SCs were plated at 3000 cells per well in eight-well Permanox chamber slides (Nunc Lab-Tek, Carlsbad, California) and cultured for five days in DMEM with 10% Horse Serum (Thermo Fisher Scientific, Carlsbad, California) and 5 ng/mL FGF2 (Cell Signaling Technologies, Danvers, Massachusetts).