The ESCs were cultured under feeder-free conditions with DMEM supplemented with 15% fetal bovine serum, 2 mM Glutamax-1,0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 450 lM 1-thioglycerol, 0.5 l M mitogen/extracellular signal-regulated kinase inhibitor PD0325901, 3 lM glycogen synthase kinase 3 inhibitor CHIR99021, and leukemia inhibitory factor.