Cultivation of Induced Pluripotent Stem Cells

The piPSCs constructed by Ma et al. (2018) (link) were used in this study. The piPSCs were grown on feeder cells [mouse embryonic fibroblasts (MEFs) treated by mitomycin-C] and maintained in the standard medium as follows (termed LBCS medium): DMEM (Hyclone, United States) supplemented with 15% FBS (VISTECH, VIS59216269, New Zealand), 0.1 mM NEAA (Gibco, United States), 1 mM L-glutaMAX (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, United States), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 10 ng/ml of LIF (Sino Biological, China), 10 ng/ml of bFGF (Sino Biological), 3 μM CHIR99021 (MCE, United States), 2 μM SB431542 (Selleck, China), and 4 μg/ml of doxycycline (DOX, Sigma-Aldrich). The piPSCs were subcultured by using TrypLE^TM Select (Invitrogen, United States) at a 1:50 ratio every 5–6 days. HEK293T cells used in this study were cultured in DMEM supplemented with 10% FBS, 0.1 mM NEAA (Gibco, United States), 1 mM L-glutaMAX (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, United States), 100 U/ml of penicillin, and 100 μg/ml of streptomycin.