*2.6. Immunofluorescence Staining*

First, the cells were rinsed in PBS and fixed with 1% (*v*/*v*) paraformaldehyde for 10 min followed by treatment with 70% ethanol (*v*/*v*) for 10 min at room temperature. The cells were permeabilized with 0.1% NP-40 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, then washed twice with PBS. To block cells, blocking solution (0.3% BSA and 5% serum in PBS) was applied for 30 min. Cells were incubated with primary antibodies in the blocking solution overnight at 4 ◦C, washed three times in PBS, then stained with secondary antibodies at 1:200 in PBS for 1 h at room temperature. Cells were washed three times in PBS, and nuclei were stained with Hoechst 33,342 (Life Technologies, Thermo Fisher Scientific) at 1:5000 in PBS for 5 min at room temperature. Prior to imaging, cells mounted with SlowFade Gold Antifade Mountant (Millipore, Sigma, Burlington, MA, USA). Images were obtained using fluorescent microscopy and a digital camera. Antibody for characterization of pluripotency is listed in Supplementary Table S3.
