Embryoid Body Formation and Spontaneous Differentiation

The piPSCs were digested by TryPLE select^TM, cultured in low-cell-binding dishes with LBCS medium for 1 day, and then transferred to the DMEM medium including 15% FBS (Gibco), 0.1 mM NEAA, 1 mM L-glutaMAX, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 0.1 mM β-mercaptoethanol for 6 days. Embryoid bodies (EBs) were transferred to 0.1% (w/v) gelatin-coated dishes and cultured in DMEM medium for 7 days. The medium was changed every day. Morphology observation and immunofluorescence were used to evaluate the differentiation of the three germ layers as reported previously (Shen et al., 2019).