Through three independent assays presented in Figure 3—neurosphere migration, microfluidic device migration and laminin spot ‘chaining'—we observed aberrant migration in SZ hiPSC NPCs. Neurosphere outgrowth is an established assay whereby the distance that NPCs migrate from a neurosphere is measured. We compared outgrowth from 241 control and 324 SZ neurospheres, generated by culturing NPCs in non-adherent conditions for 72 h and then plating them in three-dimensional Matrigel conditions, in a method similar to one previously developed for embryonic stem cell-derived neurospheres.15 There was significantly reduced outgrowth from SZ neurospheres (269±6 μm) relative to controls (368±5 μm; P<0.00001), in conditions supporting either proliferation or neural differentiation (Figures 3a and b; Supplementary Figures 6A and B). Culture of SZ neurospheres with media conditioned by control hiPSC NPCs, and culture of control neurospheres with media conditioned by SZ hiPSC NPCs did not affect migration (672 neurospheres analyzed; Figure 3c). Neither mixing control and SZ hiPSC NPCs (marked through reciprocal labeling of lentiviral green fluorescent protein or red fluorescent protein expression) before neurosphere migration (288 neurospheres analyzed; Figures 3d and e) nor culturing lentiviral-green
