Cardiac Differentiation of Human iPSCs

Using a previously established protocol, cardiac differentiation of human iPS cells was initiated in 90% confluent cell monolayers by replacing the mTeSR™1 medium with CDM3 (chemically defined medium) with 3 components: RPMI Medium 1640 (1×, Gibco), 500 μg mL⁻¹ of recombinant human albumin (Sigma-Aldrich) and 213 μg mL⁻¹ of l-ascorbic acid 2-phosphate (Sigma-Aldrich), supplemented with 1% penicillin/streptomycin.^{61 (link)} Medium was changed every 48 hours. For the first 48 hours, the medium was supplemented with 3 mM of glycogen synthase kinase 3 inhibitor CHIR99021 (Tocris). On day 2, the culture was switched to CDM3 medium supplemented with 2 mM of the Wnt inhibitor Wnt-C59 (Tocris). After day 4 of differentiation, the medium was changed to CDM3 with no supplements. Contracting cells were noted around day 10, when medium was changed to RPMI 1640 supplemented with B-27™ (50×; Gibco), and were used in experiments without selection for cardiomyocytes.