Directed Cardiac Differentiation of hiPSCs

hiPSCs (>p20) were split at 1:12 to 1:15 ratios using EDTA as above and grown for 3–4 days at which time they reached ~75% confluence. Media was changed to CDM3^{6 (link)}, consisting of RPMI 1640 (10–040–CM, Corning), 500 µg ml⁻¹Oryza sativa–derived recombinant human albumin (Oryzogen Sciencell), and 213 µg ml⁻¹L–ascorbic acid 2–phosphate (Sigma–Aldrich). Media was changed every other day (48 h). For days 0–2, media was supplemented with 6 µM CHIR99021 (MedChem Express)^{19 , 20} . On day 2, media was changed to CDM3 supplemented with 2 µM Wnt–C59 (Biorbyt). Media was changed on day 4 and every other day for CDM3. Contracting cells were noted from day 7. At day 10, media was changed to CDM3L made with using RPMI 1640 no glucose (11879–020, Life Technologies), 500 µg ml⁻¹ recombinant human albumin, and 213 µg ml⁻¹L–ascorbic acid 2–phosphate supplemented with 4 mM L–lactic acid (Sigma–Aldrich). At day 15, media was changed to CDM3M consisting of RPMI 1640 no glucose, 500 µg ml⁻¹ recombinant human albumin, 213 µg ml⁻¹L–ascorbic acid 2–phosphate supplemented with 10 mM D–galactose (Sigma–Aldrich)^{21 (link)}, 4 mM L–lactic acid, 1 mM sodium pyruvate (Life Technologies), 20 µg ml⁻¹ insulin (Life technologies), 1 × chemically defined lipid concentrate (Life Technologies), and 200 ng ml⁻¹ tri–iodo–L–thyronine (Sigma–Aldrich)^{22 (link)}.