Derivation of Human Trophoblast Stem Cells

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.