Directed Differentiation of iPSCs Into Cardiomyocytes

Directed differentiation of Ctrl- and RYR2^–/–-iPSCs into CMs (Ctrl-iPSC-CMs, RYR2^–/–-iPSC-CMs, respectively) was induced by modulating WNT signaling as previously described (Lian et al., 2013; Cyganek et al., 2018). Briefly, when monolayer cultures of iPSCs on 12-well plates reached 80–90% confluency, differentiation was initiated by changing the E8 medium to cardio differentiation medium, which was composed of RPMI 1640 with Glutamax and HEPES (Thermo Fischer Scientific), 0.5 mg/ml human recombinant albumin (Sigma-Aldrich) and 0.2 mg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich). Cells were first treated with 4 μM of the GSK3β inhibitor CHIR99021 (Millipore) for 48 h and then with 5 μM of the WNT signaling inhibitor IWP2 (Millipore) for additional 48 h. Afterward, cells were cultured in cardio differentiation medium for another 4 days. From day 8, cells were cultivated in cardio culture medium containing RPMI 1640 with Glutamax and HEPES, supplemented with 2% B27 (Thermo Fischer Scientific). At day 20, beating iPSC-CMs were detached from the plate by incubating with 2 ml of 1 mg/ml collagenase B (Worthington Biochemical), dissolved in cardio culture medium, for 1 h at 37°C. Floating iPSC-CM sheet was gently transferred into a falcon tube and dissociated with 3 ml of 0.25% Trypsin/EDTA (Thermo Fischer Scientific) for 8 min at 37°C. Digestion was stopped by adding the double volume of the cardio digestion medium (80% cardio culture medium, 20% FCS, and 2 μM Thiazovivin). Cells were centrifuged at 200 g for 5 min, re-suspended in cardio digestion medium, and replated into Geltrex-coated 6-well plates at a density of 800,000 cells/well. Afterward, iPSC-CMs were cultured in cardio culture medium until 90 days.