B27 complete supplement

Skin fibroblasts were obtained from a healthy volunteer and reprogrammed into pluripotent stem cells by standard methods. HiPSCs from a patient with LQT3 were previously described^{40 (link)}. Human iPSCs were maintained in Essential 8 media and passaged every 3–5 days as single cells on Geltrex-coated plastic dishes (Invitrogen). Differentiation was performed as described elsewhere by sequential inhibition of GSK3 and the Wnt signaling pathways^{49 (link), 50 (link)}. Spontaneously beating CMs between days 20 and 30 were dissociated with 0.25% Trypsin-EDTA (GIBCO), re-plated on Geltrex-coated 96-well plates and maintained in RPMI 1640 medium with B27 complete supplement (Invitrogen) as described previously^{50 (link)}.

iPSCs were cultured on Matrigel (GFR, BD Biosciences, Franklin Lakes, NJ, USA) coated plates in mTeSR1 medium (StemCell Technologies, Vancouver, Canada) at 37 °C. Cardiomyocyte differentiation was performed by modulating Wnt/β-catenin signaling, applying small molecules as previously described by Lian et al. [47 (link)]. iPSCs were dissociated mechanically or enzymatically with Versene solution (Invitrogen, Life Technologies, Woburn, MA, USA) and were seeded in 6-well or 12-well plates containing mTeSR1 medium supplemented in some cases with 5 μmol/L ROCK inhibitor (Cayman Chemical, Ann Arbor, MI, USA). Cells were maintained for additional 1–4 days (with mTeSR1 medium replaced every 1–3 days) before the beginning the differentiation protocol (day 0). On day 0, medium was changed to RPMI supplemented with B27 minus insulin (In vitrogen, Life Technologies, Woburn, MA, USA) and 8 or 10 μmol/L CHIR99021. On the next day (day 1), the medium was changed to RPMI supplemented with B27 minus insulin. On day 3, the medium was changed to RPMI supplemented with B27 minus insulin and 5 or 10 μmol/L IWP-4. On day 5, the medium was changed to RPMI supplemented with B27 minus insulin. From day 7 onwards, cells were cultured in RPMI supplemented with B27 complete supplement (Invitrogen), and medium was replaced 2–3 times a week.