Cell preparation

Experimental procedures and protocols were approved by the Animal Experiment Ethics Committee of the Kyoto Prefectural University of Medicine. Murine CMPs were isolated from wild-type C57BL/6 mouse hearts (10- to 16-week-old).⁹ Briefly, the mice were killed by deep anesthesia with pentobarbital. The hearts were excised, and atria were used in this study. The minced tissue fragments were digested twice for 30 min at 37 °C with 0.2% (w/v) type II collagenase and 0.01% (w/v) DNase I (Worthington Biochemical, Lakewood, NJ, USA). After digestion, cells were passed through a 70-μm filter to remove debris and transferred to Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% (v/v) fetal bovine serum (FBS) (Life Technologies). The cells were collected and size fractionated on a 30–70% Percoll gradient to obtain CMPs expressing the Sca-1 antigen. CMPs were seeded on 60-mm collagen I-coated dishes (Asahi Glass, Tokyo, Japan) in DMEM/F12 supplemented with 10% (v/v) FBS and 20 ng/ml bFGF. The medium was changed every 3 days.