Generation of 3D spheroids from iPSCs was accomplished using a modified protocol [23]. Briefly, the iPSCs in E8 medium with a ROCK inhibitor (Thiazovivin, 1 uM) were transferred into 100 mm ultra-low-attachment plastic plates (Corning, Tewksbury, MA). On the day following formation of the spheroid, the medium was replaced with neural induction medium (Invitrogen) for 6 days. Then the floating spheroids were moved to neural medium (NM) containing Neurobasal, B-27 serum substitute without vitamin A, GlutaMax, penicillin and streptomycin (Invitrogen). The NM was supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF (R&D Systems, Minneapolis, MN). Cells were grown in this medium for 21 days with daily replacement during the first 10 days, and every other day for the subsequent 11 days. To promote differentiation of the neural progenitors into neurons, FGF2 and EGF were replaced with 20 ng/ml BDNF and 20 ng/ml NT3 (Peprotech, Rocky Hill, NJ) starting at day 27. From day 48 onwards, NM without growth factors was used and replaced every four days.
