NPCs were generated from iPSCs by RA treatment and neurosphere formation as previously described [25]. Briefly, iPSCs were cultured in the mTeSR1 medium containing 10 μM RA and 0.1 μM sodium butyrate (NaB) (Sigma, St. Louis, MO, USA) for 7 days. Afterwards, cells were digested with dispase (1 mg/ml, Millipore, Billerica, MA, USA) into small clumps containing 200 to 500 cells and cultured in suspension with the human ESC medium for 4 days to initiate formation of neurospheres, which were then cultured in the neurosphere (NSP) medium for 7 days. On day 18 of differentiation, neurospheres were digested into single NPCs, which could be either maintained as NPCs or further induced to neuronal differentiation.
