Culture of Human Trophoblast Stem Cells

The culture of human trophoblast was performed as described previously^{47 (link)}. Briefly, the plate was coated with 5 μg ml⁻¹ Collagen I (Corning) at 37 °C at least for 1 h. hTSCs were cultured in hTSCs medium (DMEM/F12 (Gibco) supplemented with 0.1 mM 2-mercaptoethanol (Gibco), 0.2% FBS (Gibco), 0.5% Penicillin–Streptomycin (Gibco), 0.3% BSA (Sigma-Aldrich), 1% ITS-X supplement (Gibco), 1.5 μg ml⁻¹l-ascorbic acid (Sigma-Aldrich), 50 ng ml⁻¹ Epidermal Growth Factor (EGF, MedChemExpress), 2 μM CHIR99021 (MedChemExpress), 0.5 μM A83-01 (MedChemExpress), 1 μM SB431542(MedChemExpress), 0.8 mM valproic acid (VPA, Wako) and 5 μM Y27632 (MedChemExpress)). hTSCs were dissociated with TrypLE (Gibco) for 8 min at 37 °C, and the cells were passaged to a new Collagen I-coated plate. hTSCs were routinely passaged every 4–5 d at a 1:4–1:6 ratio.