Cardiomyocyte Differentiation Protocol

We began the cardiomyocyte differentiation protocol when cells were 80% confluent using the protocol developed by Feaster et al [28 (link)]. Briefly, differentiation was initiated (day 0–2) by replacing TeSR E8 medium with RPMI 1640 medium (Lonza, cat 12-702F) supplemented with B27 (minus insulin, Gibco, cat A1895601) and CHIR99021 (6 μM, LC Laboratories, cat C-6556), a GSK3 inhibitor. On days 3–4, CHIR99021 was removed and replaced with RPMI 1640 medium supplemented with B27 (minus insulin) and IWR-1 (500 μM, Sigma, cat I0761), a Wnt signaling inhibitor. On days 5–9, cells were maintained in RPMI 1640 medium supplemented only with B27 (minus insulin). From days 10–15, a metabolic selection protocol was employed using RPMI 1640 without glucose (Life Technologies, cat 11879) plus B27 without insulin. Following metabolic selection, cells were maintained in RPMI 1640 supplemented with B27 (Gibco, cat 17504-044) and 1% pen strep (Gibco, cat 10378-016). Beating cardiomyocytes were fed daily until day 20 when functional assays were carried out as described below.