Differentiation of iPSC-derived Neurospheres

Control NPCs were derived from human iPSCs (WT83 c9 and c6), as previously described^{4 (link)}. All iPSC lines were negative for mycoplasma contamination. Briefly, high-passage iPSC/hESC colonies on feeder-free plates were grown to 80% confluency. The medium was changed to N2 (DMEM/F12 medium containing 1 × N2 supplement [Invitrogen], 1 μM dorsomorphin [Tocris], and 1 μM SB431542 [Stemgent]) for 48 h. Colonies were then detached from the plate and cultured in suspension as embryoid bodies for 5 days at 90 rpm in N2 medium. Embryoid bodies were plated on Matrigel (Corning) in NGF medium (DMEM/F12 medium supplemented with 0.5 × N2, 0.5 × B27 supplement [Gibco], 20 ng/ml fibroblast growth factor, and 1% penicillin/streptomycin) for 1 week. Rosettes were manually picked, dissociated, and added to plates double-coated with polyornithine (10 μg/mL, Sigma-Aldrich) and laminin (2.5 μg/mL, Gibco) in NGF medium. To generate neurospheres, NPCs were scraped from the plates and continuously shaken for 2–5 days at 90 rpm in NGF medium. Neurospheres were then treated with NG medium supplemented with 10 μM ROCK inhibitor (Y-27632, Calbiochem) for 48 h. Neurospheres were maintained in NG medium for 4 weeks to allow neuronal maturation.