iNPCs were maintained on Matrigel in NSMM and passaged once 80–100% confluent using Accumax. Medium was changed every day. For the initial six passages, iNPCs were treated with 10 μM Y-27632 (Biomol Inc.) during splitting. For neuronal differentiation, ∼1 × 105 human iNPCs cells were plated onto poly-ornithine (Sigma) and laminin coated 35-mm dishes in NSMM medium. After 2 days, medium was switched to Neurobasal media (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen) and FGF2 (10 ng ml−1; Peprotech). Four days later, FGF2 was withdrawn from the medium, and after another 4 days, medium was switched to Neurobasal media supplemented with B27 and brain derived neurotrophic factor (BDNF, 20 ng ml−1, R&D Systems). Differentiated cells were maintained up to 4 weeks after FGF2 withdrawal. For astrocyte generation, cells were treated with 5% serum for 2–3 weeks.
