Culturing and Differentiating Human Embryonic Stem Cells

H9 hESCs were cultured on irradiated mouse embryonic fibroblast (MEF) as described previously (13 (link), 14 (link)). hESCs were fed daily with DMEM/F12 medium supplemented with 20% Knockout serum replacement (Life Technologies, Seoul, Korea), 0.1 mM 2-Mercaptoethanol, 1% Non-essential amino acid, 1 mM glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 4 ng/ml basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA). Feeder-free culture method was performed as described previously (13 (link), 15 (link)). Briefly, 10 μM of Y27632 (Sigma-Aldrich, Seoul, Korea) were treated for 1 hour prior to detaching hESCs, the cells were harvested with trypsin-EDTA. After elimination of MEF cells on gelatin coated tissue culture dish, hESCs were cultured in conditioned medium supplemented with 10 μM of Y27632 on tissue culture dish coated with Matrigel (BD Biosciences, Seoul, Korea). For differentiation of hESCs, cells were treated 10⁻⁵ M retinoic acids (RA) in culture medium for 10~14 days. Colo205 cells were maintained following recommendation of ATCC.