Collagenase digestion

Rat SKPs (rSKPs) were isolated from the back skin of P30 male green fluorescent protein (GFP)‐expressing Sprague Dawley rats (Sadaaki Takagi Japan SLC, Hamamatsu, Japan, http://www.jslc.co.jp) after an overdose of sodium pentobarbital (27.3 mg/kg, intraperitoneal injection), according to previously described protocols 21. Primary dermal cells were grown in basic fibroblast growth factor (40 ng/ml; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), platelet‐derived growth factor (PDGF)‐BB (25 ng/ml; R&D Systems, Minneapolis, MN, http://www.rndsystems.com), B27 supplement (2%; Thermo Fisher Scientific Life Sciences, Waltham, MA, http://www.thermofisher.com) and penicillin/streptomycin (1%; Thermo Fisher Scientific Life Sciences) in Dulbecco's modified Eagle's medium (DMEM) low glucose/F12 (3:1; Thermo Fisher Scientific Life Sciences). SKPs were plated at 30,0000 cells per milliliter in suspension culture flasks (Falcon). Following primary colony formation, SKPs were dissociated to single cells using collagenase digestion (2 mg/ml; Worthington Biochemical Corp., Lakewood, NJ, http://www.worthington‐biochem.com) and replated at 30,000 cells per milliliter. SKPs were passaged three or four times in static culture before being introduced to the bioreactor expansion to obtain sufficient numbers of cells to seed into the 500‐ml bioreactors at 30,000 cells per milliliter.

Cardiac-derived cells were isolated from BalbC female mice at 2–3 months of age by collagenase digestion (280 U/mL; Worthington, Lakewood, N.J., USA) through the coronary arteries and myocyte were removed by centrifugation. Cells were cultured in F12:DMEM 1:1 supplemented with 10% fetal bovine serum and expanded. Ckit⁺ cells were selected with immunomagnetic CD117 microbeads, mouse (Miltenyi Biotec) (Supplementary Figure 2) and were expanded in Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin and Erythropoietin (Sigma-Aldrich) 2,5 mg in 500 ml [75 (link)].