At D15 of E6-CSFD treatment, cells were dissociated using Accutase and labeled with NCSC microbeads (20 μl per 107 cells; Miltenyi), FcR blocking reagent (20 μl per 107 cells), and MACS buffer [60 μl per 107 cells; 0.5% bovine serum albumin + 2 mM EDTA in sterile phosphate-buffered saline (PBS) without Ca2+/Mg2+] at 4°C for 15 min. Cells were washed in MACS buffer and resuspended in 500 μl of MACS buffer per 2 × 107 cells. Cells were sorted through two LS columns (Miltenyi Biotec) according to manufacturer instructions and resuspended in E6-CSFD + 10 μM Y27632 to appropriate density for specific NCSC lineage differentiations as described below.

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