To create 3D cultures, or neural organoids, we followed a published protocol [64] with some modifications. Embryoid bodies (EBs) were formed by loading 12,000 iPSCs per well into 96-well plates with cone-shaped wells (Nunc® 96-well Conical Bottom plates, VWR International, Bridgeport, NJ) pre-coated with Pluronic acid (F-127, 1%, Sigma-Aldrich, Natick, MA). The 96 well plates were transferred to an incubator at 37 C° with 95% relative humidity and 5% CO2. Media used in EB culture consisted of Glasgow-MEM supplement with KSR (20% v/v), Sodium Pyruvate (1X), NEAA (1x), 2-mercaptoethanol (0.1mM), Rock inhibitor (20 μM), TGFβ-inhibitor (SB431532 compound, Tocris Biosciences, Minneapolis, MN; 5 μM), Wnt-inhibitor (IWRe1 compound, Tocris Biosciences, Minneapolis, MN; 3 μM). The EBs maintained in this medium for 18–20 days, with Dorsomorphin (BMP signal inhibitor, Tocris Biosciences, Minneapolis, MN; 2 μM) added to the culture for the first three days to promote a neuronal lineage. Media was replaced every other day. The edges of the EBs began to appear translucent around day 10, and the tissue grew to be large than 0.6 mm in diameter by day 18. The
