2.5 |. EB formation and neural differentiation

hESC colonies were treated with collagenase and subsequently split using accutase for 5 min at 37°C. The cells were replated into a round-bottomed 96 well ultra-low attachment plate with EB medium (80% DMEM/F12, 20% HI-FBS, 1% NEAA, and 1% GLUTAMAX) and centrifuged for 2 min at 3,000 rpm. After 2–3 days, EBs were individualized by applying protocol from Vichier-Guerre, Parker, Pomerantz, Finnell, and Cabrera (2017), and replated onto matrigel (9–12 mg/ml; cat# 354230; BD bioscience) coated wells in neural differentiation media (DMEM/F12, Neural Basal [Gibco] 1:1; 1:100 N2 [Invitrogen]; 300 mg/ml bovine serum albumin fraction V; 1: 50 B27 [Invitrogen]; bFGF 1:1000, EGF 1:1000 [Invitrogen growth factor], insulin 20 mg/ml and Anti-anti 1:100). After 21 days, the cells were lifted with accutase and replated on matrigel for neural-organoid formation.