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 cell aggregates, or organoids, were transferred to non-adherent petri-dishes (EZsphere dish) to prevent fusion of separate organoids, and cultured in a medium designed to promote neuroepithelial formation, which consisted of DMEM/F12 supplemented with Chemically Defined Lipid Concentrate (1X) and N2-supplement (1X) and maintained in an incubator with 5% CO2 and 40% Oxygen. The aggregates were kept in this media for 15 to 20 days, at which time heparin (5 μM, Sigma-Aldrich, Natick, MA), FBS (10% v/v, Gemini Bio-Products, West Sacramento CA), and Matrigel (final 1% v/v, Corning Incorporated—Life Sciences, Oneonta, NY) were added to the medium. On day 70 the amount of Matrigel was increased to 2% and B27 supplement (1X) was added to the medium. The aggregates were maintained in this final medium for the remained of the culture period. Medium in the culture dishes was replaced every 4 to 5 days.
