Differentiation of human neurons from hNSCs

We generated human neurons through direct differentiation of hNSCs using an adapted protocol⁸⁰. Briefly, hNSCs were dissociated by Accutase and seeded into poly-D-lysine and laminin-coated 6-well plate (2 × 10⁵ cells /well) or 24-well plate (1 × 10⁵ cells/well), and cultured in hNSC culture medium (day 1). On day 2, bFGF was withdrawn and 5 μM Rock inhibitor (Y-27632, STEMCELL Technologies) was added to the medium for 24 h. After that hNSC culture medium minus bFGF was changed every other day until day 40. We performed immunocytochemistry to confirm the neuronal identity of differentiated cells. Cells were fixed with 4% PFA at RT for 15 minutes, washed with DPBS for three times, and blocked with 5% goat serum in DPBS for 1 h at RT. Cells were then incubated with anti-MAP2 (1:5000, Chicken, Novus, NB300-213) and anti-TUJ1 (1:5000, Rabbit, Sigma, 801201) overnight at 4 °C. Primary antibodies were visualized by anti-chicken or anti-rabbit goat secondary antibodies conjugated to Alexa 555 (1:1000, Invitrogen, A-21437) or Alexa 488 (1:1000, Invitrogen, A-11008), respectively, and the nuclei were stained with DAPI (1 μg/mL).