Cardiomyocyte Differentiation and Characterization from hiPSCs

All hiPSC lines were expanded as adherent cultures in feeder-free conditions on Matrigel-coated dishes in the presence of chemically defined medium (E8 Essential Medium, Life Technologies). Differentiation of hiPSC to cardiomyocytes (CMs) was performed over a period of 30 days following a previously reported protocol48 (link) based on small molecules-mediated canonical Wnt pathway modulation (see Supplementary methods for more detail). Subsequently we enriched for CMs by switching the culture medium to DMEM supplemented with lactic acid (4 mmol/L) in substitution of glucose for 6 days as previously reported49 (link). Cultures of hiPSC-CMs were then enzymatically dissociated into single cells using Elastase (Serva) and Liberase (Roche Chemicals) as described previously31 (link). CMs were stained with anti-NKX-2.5 and cardiac TroponinT antibodies as described in the Supplementary methods. To allow for single-cell electrophysiological measurements, dissociated cells were plated at a low density on Matrigel-coated coverslips. Electrophysiological measurements were performed 9–14 days after dissociation.