Differentiation of iCas9 Human Neural Stem Cells

The iCas9 hPSCs were gifted by Dr. Danwei Huangfu at Sloan-Kettering Institute and Dr. Jie Na at Tsinghua University. iCas9 hNSCs were differentiated from iCas9 hPSCs based on previous reports.^{49 (link)} Briefly, iCas9 hPSCs were cultured on matrigel-coated dishes and fed daily with mTeSR (STEMCELL) for 7 days. On the next day, mTeSR was substituted by N2 medium (DMEM/F12 supplemented with 0.5× N2 supplement (Gibco), 1 μM dorsomorphin (Tocris), and 1 μM SB431542 (STEMCELL)) for 1–2 days. hPSC colonies were lifted off, cultured in suspension on the shaker (95× rpm at 37 °C) for 8 days to form embryoid bodies (EBs) and fed with N2 media. EBs were then mechanically dissociated, plated on a matrigel-coated dish, and fed with hNSC maintenance medium (DMEM/F12 supplemented with 1× N2 supplement, 1× B27 supplement (Gibco), 1% penicillin/streptomycin, and 20 ng/mL bFGF (Gibco)). The emerging rosettes were picked manually, dissociated completely using Accutase (Gibco), and plated on a poly-ornithine/laminin-coated plate. The resultant hNSCs were expanded and maintained in the hNSC maintenance medium. The 293T cells were purchased from the cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin (Gibco).