Epidermal growth factor (egf)

hTSCs were cultured as previously described (Okae et al., 2018 (link)). Briefly, a 6-well plate was coated with 5 μg/mL Collagen IV (Corning, 354233) at 37°C overnight. Cells were cultured in 2 mL TS medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.2% FBS, 0.5% Penicillin-Streptomycin, 0.3% BSA, 1% ITS-X (Gibco, 51500), 1.5 μg/ml L-ascorbic acid (Wako, 013–12061), 50 ng/ml EGF (Rockland, 009–001 C26), 2 μM CHIR99021 (Stemgent, 04–0004), 0.5 μM A83-01 (BioVision, 1725), 1 μM SB431542 (BioVision, 1674), 0.8 mM VPA (Tocris, 2815), and 5 μM Y-27632] and in 5% CO₂ and 20% O₂. Media were changed every 2 days, and cells were passaged using TrypLE Express every 3 days at a ratio of 1:4. Unless otherwise specified, hTSCs between passage 10 and 20 were used for experiments.

hTSCs were derived from naive hESCs as previously described (Dong et al., 2020 (link)). Briefly, naive hESCs maintained in 5i/L/A or alternative naive media were dissociated into single cells using TrypLE. 0.5-1.0 × 10⁶ cells were seeded in a 6-well plate pre-coated with 5 μg/mL Collagen IV and switched to 2 mL hTSC medium (Okae et al., 2018 (link)) [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.2% FBS, 0.5% Penicillin-Streptomycin, 0.3% BSA, 1% ITS-X (GIBCO, 51500), 1.5 μg/ml L-ascorbic acid (Wako, 013–12061), 50 ng/ml EGF (Rockland, 009–001 C26), 2 μM CHIR99021 (Stemgent, 04–0004), 0.5 μM A83-01 (BioVision, 1725), 1 μM SB431542 (BioVision, 1674), 0.8 mM VPA (Tocris, 2815), and 5 μM Y-27632]. Cells were cultured in 5% CO₂ and 20% O₂ at 37°C, media were changed every 2 days, and passaged upon reaching 80%–100% confluency at a ratio of 1:2 to 1:4 using TrypLE. Cells were analyzed by FACS for the hTSC-specific cell surface markers ITGA6 and EGFR after 5-7 passages.