Guided Cardiac Differentiation

Two different cardiac myogenic differentiation protocols were used, as follows.

At 90% cell confluency, on day 3 or 4 after SMiPSC generation, cardiac differentiation was induced by adding 25 ng/mL BMP4 (Life Technologies, Carlsbad, USA) and 5 μM CHIR99021 (http://Selleckchem.com, Houston, TX, USA) in RPMI1640 medium (Life Technologies, Carlsbad, USA), which activated the WNT pathway, and 3 days later, 10 μM IWR1 (Sigma-Aldrich, St. Louis, USA) was added to inhibit this signaling. After 7 days of cardiac differentiation, insulin-depleted medium was exchanged with insulin-supplemented medium to promote further cell proliferation. On day 12, the differentiated cell population was metabolically selected via a 4-day incubation with 4 mM lactate-supplemented DMEM w/o glucose (Thermo Fisher, Waltham, USA). After day 16, enrichment medium was exchanged with basal medium (RPMI+B27+glutamine). The differentiation scheme is presented in Supplementary Figure 2.

When iPSCs reached 70% confluency on day 4, cardiac differentiation was induced by applying a 2-day incubation in Medium A provided in a PSC Cardiomyocyte Differentiation Kit (Life Technologies, Carlsbad, USA). Next, medium B was added for another 2 days and exchanged with Cardiomyocyte Maintenance Medium (M) every other day. Additionally, from day 12 to day 16, cells were subjected to metabolic selection and maintained for 4 days in enrichment medium – DMEM w/o glucose supplemented with 4 mM lactate. A scheme of the protocol is presented in Supplementary Figure 3.