Alkaline Phosphatase Staining

The pluripotency of piPSCs were detected by alkaline phosphatase (AP) staining reagent according to the instructions of the manufacturer (Wu et al., 2021; Zhu et al., 2021a). In brief, the cells were fixed with 4% paraformaldehyde (Sangon Biotech) in PBS for 15 min at room temperature and washed twice with PBS. Then the cells were incubated with AP staining reagent [consisting of 1.0 mg/ml of Fast Red TR and 0.4 mg/ml of Naphthol AS-MX (Sigma-Aldrich)] in 0.1 M Tris Buffer at room temperature. The AP-positive colonies were shown in red color after 20 min of incubation. Finally, the images were captured by a fluorescent phase-contrast microscope (Nikon, Japan).