Directed Differentiation of hESCs

The neuroectoderm differentiation was based on Douvaras and Fossati (2015 (link)). In brief, 90% confluent hESCs were subjected to neural induction for 8 days using 100 nM RA (Sigma-Aldrich), 10 μM SB431542 (Tocris), and 250 nM LDN193189 (STEMCELL Technologies). The induction of cardiac progenitor differentiation was based on Lin and Zou (2020 ). hESCs at 80%–90% confluence were treated with 5 mM CHIR99021 (Tocris) for 24 h, after which the cells were cultured in medium with 0.6 U/mL heparin (Sigma-Aldrich) for 24 h. Subsequently, the medium was supplemented with 0.6 U/mL heparin and 3 mM IWP2 (Tocris) for another 3 days. The 8-day depatoblast differentiation was based on Boon et al. (2020 (link)). Differentiation was initiated at 40%–50% confluency, and the hESCs were treated with with 50 ng/mL activin A (STEMCELL Technologies), 50 ng/mL WNT3A (PeproTech), and 6 μL/mL DMSO (Sigma-Aldrich) for 48 h. The cells were incubated for an additional 48 h in the same medium without WNT3A. Then, the medium was changed to contain 50 ng/mL BMP4 (STEMCELL Technologies) and 6 μL/mL DMSO for the following 4 days. More details on the differentiation and the media composition can be found in the supplemental information.