12, the neuroepithelial sheet was mechanically broken into large aggregates and replated onto laminin (Sigma) coated MW6 plates. At DIV 13 and 15 medium was replaced with N2B27 medium supplemented with 20 ng/ml FGF2 (Stem Cell Technologies) for neural precursor cell (NPC) expansion. Neural rosettes were purified on DIV 18 using dispase (Sigma, 10 mg/ml in PBS, 1/10 diluted in medium). Large rosette clumps were maintained for another week with 1 or 2 more dispase passages depending on purity of the rosettes. Around day 25 of neural induction, cells were passaged with accutase (Thermo Fisher Scientific) to dissociate cell clumps into a single-cell suspension followed by plating on laminin-coated MW6 plates with N2B27 medium changes every other day. NPCs (DIV 27–30, passage 0) were cryopreserved, further proliferated/passaged in N2B27 medium with 10 ng/ml FGF2, or used for final plating and differentiation into cortical neurons.
