On day 0 of organoid culture, any pre-differentiated cells in the hPSCs culture were removed by scraping under microscope and ES or iPSC colonies were dissociated into single cell suspension with Accutase. 9,000 cells were then plated into each well of a U-bottom ultra-low-attachment 96-well plate in neural induction media supplemented with 50 μM Y-27632 compound and 5% (v/v) heat-inactivated FBS. Neural induction media contained DMEM-F12, 15% (v/v) knockout serum replacement, 1% (v/v) MEM-NEAA, 1% (v/v) Glutamax, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, and 2 μM XAV939. Media was replenishced every other day until day 10. On day 2, neural induction media was supplemented with 50 μM Y-27632 compound without heat-inactivated FBS. From day 4, Y-27632 compound was no longer supplemented. From day 0 to day 10, cell aggregates in the ultra-low-attachment 96-well plate were cultured statically in 37° incubator with 5% CO2.
