Activin a

hiPSC differentiation was initiated when cells reached 80%–90% confluency, by temporal modulation of the Wnt/ß‐catenin signaling following a protocol previously described by the authors.^[93, ^94] Briefly, expansion medium (TeSR‐E8) was replaced by differentiation media (RPMI 1640 (Gibco, ThermoFisher Scientific) with B27 without insulin (ThermoFisher Scientific) (RPMI + B27‐I), supplemented with CHIR99021 (12 × 10⁻⁶ m) (Tocris Bioscience), Activin A (80 ng mL^–1) (Tebu‐bio), and ascorbic acid (50 µg mL^–1) (Sigma Aldrich). After 24 h, the medium was replaced by differentiation medium supplemented with IWR1 (5 × 10⁻⁶ m) (Selleckchem) and ascorbic acid (50 µg mL^–1). At day 3 (72 h after differentiation induction), medium was exchanged for differentiation medium supplemented with IWR‐1 (5 × 10⁻⁶ m). At day 6, medium was replaced by maintenance medium [RPMI 1640 supplemented with B27 with insulin (RPMI + B27)].

In this study two hPSC lines were used namely, the hiPSC line DF19-9-11T.H (WiCell) and the hESC line (HES3 reporter line NKX2-5(eGFP/w)^{22 (link)}) kindely provided by Dr. David Elliott. hPSCs were routinely expanded in Synthemax II-SC (corning) coated plates in mTESR1 medium (STEMCELL Technologies). Before cardiac differentiation induction, hPSCs were replated on Matrigel® (Corning) coated plates and cultured in mTESR1 medium until reaching 80%-90% confluency. Briefly, expansion medium was replaced by RPMI Medium (ThermoFisher Scientific) supplemented with B27 without insulin (RPMI/B27, ThermoFisher Scientific), 12 µM CHIR99021 (Biogen Cientifica SL), 80 ng/mL Activin A (Tebu-bio) and 50 µg/mL Ascorbic acid (Sigma-Aldrich). After 24 hr, the medium was completely replaced by RPMI/B27 supplemented with 5 µM IWR-1 (Sigma-Aldrich) and 50 µg/mL Ascorbic acid (Sigma-Aldrich). At day 3, i.e. 72 hr after day 0, medium was exchanged for RPMI/B27 supplemented with 5 µM IWR-1 until day 6. Medium was then exchanged every two days, until day 15. At day 15, cell preparations containing > 80% cardiac troponin T-positive CMs (confirmed by flow cytometry) were obtained.