This methodology was taken from our previous study (45). CSCs were explanted from Wistar-Kyoto (WKY) rat hearts. Myocardial samples were excised from WKY rats and cut into 2-mm3 fragments. These were washed with PBS and partially digested with collagenase (Sigma-Aldrich). They were then cultured in IMDM (Invitrogen, Carlsbad, CA, USA) containing 20% fetal bovine serum (FBS) (Corning, Corning, NY, USA) as cardiac explants on a fibronectin (Corning)–coated surface (0.5 mg/ml). Stromal-like cells and phase-bright cells emerged from the cardiac explant. The explant-derived cells were passaged with TrypLE Select (Gibco). Passaged cells were seeded in Ultra-Low attachment flasks (Corning, Corning, NY) at a density of 2 × 104 cells/ml. The spontaneous aggregation of these explant-derived cells into cardiospheres was observed after 1 week. The cardiospheres were then passaged into fibronectin-coated surfaces to complete the generation of cardiosphere-derived CSCs. All cultures were incubated in 5% CO2 at 37°C.

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