Embryoid bodies were generated from hiPSCs and then transferred to nonadherent plates (Corning). Colonies were maintained in suspension in N2 media (DMEM/F12 (Invitrogen), 1x N2 (Invitrogen)) for 7 days and then plated onto polyornithine (PORN)/Laminin-coated plates. Visible rosettes formed within 1 week and were manually dissected and cultured in NPC media (DMEM/F12, 1x N2, 1x B27-RA (Invitrogen), 1 µg/ml Laminin (Invitrogen) and 20 ng/ml FGF2 (Invitrogen). NPCs are maintained at high density, grown on PORN/Laminin-coated plates in NPC media and split approximately 1:4 every week with Accutase (Millipore). For neural differentiations, NPCs were dissociated with Accutase and plated at low density in neural differentiation media (DMEM/F12-Glutamax, 1x N2, 1x B27-RA, 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mm dibutyrl-cyclicAMP (Sigma), 200 nM ascorbic acid (Sigma) onto PORN/Laminin-coated plates.
