Immunofluorescence staining

The cells were cultured on poly-d-lysine/Matrigel-coated (growth factor-reduced) coverslips. The immunofluorescence staining of hPSC-derived NSCs, OPCs, and oligodendrocytes was performed using the following primary antibodies: anti-NESTIN (Santa Cruz Biotechnology, USA), anti-sex determining region Y-box 2 (SOX2) (Millipore, USA), anti-NG2 (Santa Cruz Biotechnology, USA), anti-GFP (Millipore, USA), anti-PDGFR-α (Santa Cruz Biotechnology, USA), anti-galactosylceramidase (GALC) (Santa Cruz Biotechnology, USA), anti-O4 (R&D Systems, USA), anti-MBP (R&D Systems, USA), anti-myelin oligodendrocyte glycoprotein (MOG) (Santa Cruz Biotechnology, USA), anti-proteolipid peptide (PLP1) (Santa Cruz Biotechnology, USA), and anti-Glial fibrillary acidic protein (GFAP) (Santa Cruz Biotechnology, USA) as per manufacturer’s recommendations. Alexa Fluor 488, 545, 595, or 647 conjugated secondary antibodies (Thermo Fisher Scientific, USA, 1:1000) were used. In brief, coverslips were washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. The cells were permeated by 0.25% Triton X-100 for 10 min at room temperature and blocked with 1% bovine serum albumin (BSA) in DPBS. The primary antibodies were stained overnight at 4 °C and washed three times prior to secondary antibody staining for 30 min at room temperature. The coverslips were mounted with Fluoromount-G containing the nuclear stain DAPI (Southern Biotech, USA). The images were taken by a Nikon C1 confocal microscope.