Blood cord-derived human induced pluripotent stem cells (hiPSCs) were obtained from Life Technologies (Episomal human iPSC line, A18944, Warrington, UK). hiPSC colonies were expanded on Geltrex (Gibco, Warrington, UK) coated plates in Essential 8 cell culture medium (Thermo Scientific, Warrington, UK) until at least passage 20 before differentiation.
Chemically-defined differentiation to cardiomyocytes was achieved following a previously described protocol [24 (link)]. Briefly, when ~90% confluent hiPSCs were stimulated with differentiation medium containing RPMI 1640 + HEPES + GlutaMax + B27 supplement—insulin (Invitrogen, Warrington, UK), along with 6 µM GSK3 inhibitor Chir99021 (Sigma, Gillingham, UK), for 48 h. The media were then replaced with a differentiation medium containing 2 µM Wnt inhibitor C59 (Selleckchem, Houston, TX, USA) for a further 48 h. Cells were maintained in differentiation medium from days 4 to 8, before changing to a maintenance medium (RPMI/HEPES/GlutaMax/B27 + insulin supplement) once spontaneously-beating colonies were established for prolonged culture. Cardiomyocytes were selected through the addition of 5 mM sodium lactate (Sigma, Gillingham, UK) to the media from day 15, before plating for individual assays.
Chemically-defined differentiation to cardiomyocytes was achieved following a previously described protocol [24 (link)]. Briefly, when ~90% confluent hiPSCs were stimulated with differentiation medium containing RPMI 1640 + HEPES + GlutaMax + B27 supplement—insulin (Invitrogen, Warrington, UK), along with 6 µM GSK3 inhibitor Chir99021 (Sigma, Gillingham, UK), for 48 h. The media were then replaced with a differentiation medium containing 2 µM Wnt inhibitor C59 (Selleckchem, Houston, TX, USA) for a further 48 h. Cells were maintained in differentiation medium from days 4 to 8, before changing to a maintenance medium (RPMI/HEPES/GlutaMax/B27 + insulin supplement) once spontaneously-beating colonies were established for prolonged culture. Cardiomyocytes were selected through the addition of 5 mM sodium lactate (Sigma, Gillingham, UK) to the media from day 15, before plating for individual assays.
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