iPSC colonies (>10 passages in defined medium with a normal karyotype) were harvested using a scraper and cultured in suspension as embryoid bodies (EBs) for 8 days in StemPro defined medium minus FGF2. EBs were then cultured for additional 2–3 days in suspension in neural induction medium containing Dulbecco's Modified Eagle Medium (DMEM/F12) with Glutamax, NEAA, N2, and FGF2 (20 ng/ml) prior to attachment on cell culture plates coated with CellStart. Neural rosettes formed 2–3 days after adherent culture were isolated manually using stretched glass Pasteur pipette and placed in fresh culture dishes. Then, the rosettes were dissociated into single cells using accutase and replated onto culture dishes to obtain a homogeneous population of NSCs. The NSCs population was expanded in Neurobasal media containing NEAA, 2 mM glutamine, B27, and 20 ng/ml FGF2.
