Generation of Human Cortical Spheroids from iPSCs

Human cortical spheroids were generated from iPSC as previously described^{31 (link)}. Briefly, iPSC lines derived from two healthy control individuals were grown on inactivated mouse embryonic fibroblast feeders in the following medium: DMEM/F12, Knockout Serum 20%, 1 mM non-essential amino acids (1:100), GlutaMax (1:200), β-mercaptoethanol (0.1 mM), penicillin and streptomycin (1:100) (Life Technologies). Cultures were regularly tested and maintained mycoplasma free. Colonies of iPSCs were detached intact with dispase (0.35 mg/ml, Invitrogen) and transferred into low-attachment plates in iPSC medium supplemented with dorsomorphin (5 μM, Sigma) and SB-431542 (10 μM, Tocris), and the medium was changed daily. On day six of in vitro differentiation, neural spheroids were transferred to NPC medium (Neurobasal A, B27 without vitamin A, GlutaMax (1:100), penicillin and streptomycin; Life Technologies), which was supplemented with EGF (20 ng/ml) and FGF2 (20 ng/ml) until day 24, and then supplemented with BDNF (20 ng/ml) and NT3 (20 ng/ml) from day 25 to 42. From day 43 onwards, cortical spheroids were maintained in NPC medium, which was changed every 4 days.