Human ESC Line Maintenance and Differentiation

Human ESC line used in these studies was approved by the Ethics Commission of the TUM Faculty of Medicine (# 447/17S). Authorization to use the hESC-TN was granted by the Central Ethics Committee for Stem Cell Research of the Robert Koch Institute to AM (AZ 3.04.02/0131). Generation of the hESC-TN line was described in Zawada et al. (2023) (link). hESCs were maintained on Matrigel-coated plates (Corning, 354277) in Essential 8 medium (Thermo Fisher Scientific, A1517001) containing 0.5% penicillin/streptomycin (Thermo Fisher Scientific, 15140-122) under standard culture conditions (37°C, 5% CO₂); medium was refreshed every day. Cells were passaged every 4 days with 0.5 mM EDTA (Invitrogen, AM92606) in PBS without Ca²⁺ and Mg²⁺ (PBS^−/−; Thermo Fisher Scientific, 10010023). To promote better cell survival, 2 µM ROCK inhibitor Thiazovivin (Sigma-Aldrich^®, SML1045) was added for 24 h after passaging. Cells were differentiated according to differentiation protocols described below.