Embryoid Body (EB) Formation Assay

Embryoid body (EB) formation assay was performed according to the previous study (Zhang et al., 2018). hiPSCs in the logarithmic growth phase were rinsed once with D-PBS, and then digested into large clones with 1 mg/mL dispase II (D4693, Sigma) for 15 min in a 37°C incubator. Cells were collected and centrifuged by low-speed centrifugation at 500 rpm for 3 min. After rinsing with DF12 and centrifugation, the supernatant was removed and the clone masses were resuspended in EB medium supplemented with 10 μM Y27632 by a 5 ml pipette, and inoculated into low-adherent cell culture dishes. Y27632 was added into the EB medium for the first 24 h to ensure a high survival rate of the cells. EB medium was changed every other day by natural precipitation in a 37°C incubator, and EBs samples were collected on day 2, 4, and 6.