Isolation and Culture of Cardiac Progenitor Cells

As previously described by our group7 (link)36 (link), CPCs were acquired from the hearts of two-month-old wild-type male C57BL/6 mice (Yangzhou Laboratory Animal Center). After the CPCs were cultured for approximately 10 days, a layer of fibroblast-like cells migrated from the adherent myocardial tissue. Numerous small, round, phase-bright cells emerged from these fibroblast-like cells. These cells were collected using the Accutase digestion enzyme, which does not influence surface markers. The acquired cells were further separated by magnetic-activated c-kit cell sorting magnetic beads (MiltenyiBiotec Inc., GER) according to the manufacturer’s instructions. These separated cells were seeded at 2 × 10⁴ cells/ml on poly-D-lysine-coated (Sigma, USA) dishes in cardiosphere growing medium (CGM: 65% DMEM-F12 [HyClone, USA] mixture containing 10% foetal calf serum [Gibco, USA], 2 mmol/l L-glutamine [HyClone, USA], 0.1 mmol/l 2-mercaptoethanol [Sigma, USA], 2% B27 [Gibco, USA], 5 ng/ml basic fibroblast growth factor (bFGF) [R&D, USA], 10 ng/ml epidermal growth factor (EGF) [Peprotech, USA], 40 nmol/l cardiotrophin-1 [Peprotech, USA], 1 unit/ml thrombin [Sigma, USA], 100 U/ml penicillin G [HyClone, USA], and 100 mg/ml streptomycin [HyClone, USA]).