Activin A-Derived primed hESC Culture

Two different Activin A-derived primed hESC lines (U-11-4-A3; female and U-12-3-A3, male) [19 (link)] were maintained on a mitomycin-inactivated feeder layer of mouse embryonic fibroblasts (MEFs) in hESC medium (Gibco KnockOut-Dulbecco’s Modified Eagle Medium (KO-DMEM) (Thermo Fisher Scientific, Waltham, MA, USA), 20% Gibco KnockOut-serum replacement (KOSR) (Thermo Fisher Scientific), 1% Gibco Penicillin/Streptomycin (Thermo Fisher Scientific), 1% Gibco non-essential amino acids (NEAA) (Thermo Fisher Scientific), 0.4 mM Gibco L-Glutamine (Thermo Fisher Scientific), 0.1 mM Gibco β-mercaptoethanol (Thermo Fisher Scientific)) supplemented with 4 ng/mL FGF2 (Peprotech, Rocky Hill, NJ, USA) and 20 ng/mL Activin A (R&D Systems, Minneapolis, MN, USA). All cultures were maintained at 37 °C in hypoxic conditions (5% O₂ and 6% CO₂).