Cortical Spheroid Differentiation from hESCs/hiPSCs

Three-dimensional differentiation of hESCs and hiPSCs into cortical spheroids was performed as described previously^{43 (link)}. Briefly, confluent, undifferentiated colonies of hESCs were removed from MEFs using collagenase. Colonies were washed once with media and suspended in hESC media without FGF2, supplemented with 10 μm Y-27632 and plated into 6-well low attachment plates (Corning: 3471). On days 1-5, media was changed to hESC-FGF2 media, supplemented with 10 μm Dorsomorphin (Abcam: ab146597) and 10 μm SB431542. On day 6, developing spheroids were put into neural induction media composed of Neurobasal-A (ThermoFisher: 10888022), B-27 Supplement–A (ThermoFisher: 12587-010), PenStrep, and Glutamax, supplemented with 20 ng/ml FGF and 20 ng/ml EGF. Media was changed in this manner every day from days 6-15 and then every other day until day 25. From days 25-43, the developing spheroids were grown in neural induction media supplemented with 20 ng/ml BDNF and 20 ng/ml NT-3, with media changes every 4 days. From day 43 on, spheroids were maintained in neural induction media without BDNF or NT-3, with media changes every 4 days until harvest.