Cardiotrophin 1

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]).

C-kit-positive murine CSCs were isolated using our previously described methods [15 (link)]. Briefly, CSCs were obtained from the hearts of 2-month-old wild-type male C57BL/6 mice (Yangzhou Laboratory Animal Center). After the CSCs were cultured for 1 to 2 weeks, a layer of fibroblast-like cells migrated from the adherent myocardial tissue. Some small, round, phase-bright cells emerged from these fibroblast-like cells and were collected using the digestion enzyme Accutase (Millipore, USA). Enriched c-kit-positive cells were further isolated using c-kit magnetic-activated cell sorting (MACS) magnetic beads (Miltenyi Biotec Inc., Germany) according to the manufacturer’s instructions. These separated cells were seeded on poly-d-lysine-coated (Sigma, USA) dishes at a density of 2 × 10⁴ cells/ml in cardiosphere growth medium (CGM: 65% Dulbecco’s modified Eagle’s medium (DMEM)-F12 (HyClone, USA) containing 10% fetal calf serum (Gibco, USA), 2 mmol/l l-glutamine (Gibco, USA), 0.1 mmol/l 2-mercaptoethanol (Sigma, USA), 2% B27 (Gibco, USA), 5 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, 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 (Gibco, USA), and 100 mg/ml streptomycin (Gibco, USA)). The culture medium was changed every 3 days.