Gibco tryple select

Cardiac differentiation was performed as published (Fischer et al. 2018 (link)) with slight modifications. Briefly, hiPSC were harvested from culture dishes with good colony morphology (day 5–6 after split) by removing medium and adding Gibco™ TrypLE™ Select (Thermo Fisher Scientific) for 1–2 min. As soon as single cells appeared at the borders of the colonies, TrypLE was removed by suction and the cells harvested as single cells in mTeSR-ROCK (mTeSR1 with 10 µM Y27632 dihydrochloride). Cells were counted and 5 × 10⁴ cells/ml were seeded at 100 µl per well into a 96-well Polystyrene Conical Bottom MicroWell™ Plate (249,952, Thermo Fisher Scientific). The plates were centrifuged at 500g for 5 min at room temperature. After 20 h of incubation, EBs had formed (one EB per well) and cardiac differentiation was induced by exchanging 80 µl medium to D0 medium. All medium recipes are included in supplementary Table 1. After 24 ± 2 h, 80 µl medium was exchanged to TS medium. After 24 ± 2 h, 80 µl medium were exchanged to Wnt medium. After 24 ± 2 h, 80 µl medium was exchanged with TS medium. Three days later (72 ± 2 h), 60 µl medium was exchanged with 80 µl fresh TS medium added per well. The following day beating of the cardiomyocyte containing EBs was scored.

Human-induced pluripotent stem cells were differentiated into cardiomyocytes as described in Lauschke et al. (2020 (link)). Essentially, hiPSC were harvested as single cells by incubation in Gibco™ TrypLE™ Select (Thermo Fisher Scientific) for 1–2 min. A single cell suspension of 5 × 10⁴ cells/ml was seeded at 100 µl per well into a 96-well Polystyrene Conical Bottom MicroWell™ Plate (249952, Thermo Fisher Scientific) in mTeSR-ROCK. The plates were centrifuged at 500g for 5 min at RT and incubated over night at 37 °C and 5% CO₂. After 20 h, medium was exchanged by removing 80 µl/well old medium and adding 80 µl/well D0 medium. After this, medium was exchanged daily (24 h ± 2 h) with respective medium on the following days: TS-medium on day 1, Wnt-medium on day 2, TS-medium on day 3 and TS-medium on day 6. 80 µl/well old medium was exchanged for 80 µl/well new medium, except for day 6, where only 60 µl/well were removed. All media components are listed in Supplementary Table 4.