Quantification of hNSC differentiation. (A-E) Representative image of (A) undifferentiated, (B) 1 week (1W) 2D differentiated, (C) 3-week (3W) 2D differentiated, (D) 1W 3D differentiated, and (E) 3W 3D differentiated hNSCs; scale bar, 50 μm. (F) Quantification of axon length on 1W and 3W differentiated hNSCs cultured on 2D and 3D substrates. (G) QRT-PCR molecular analysis for neuronal (TUBB3, DCX, and MAP2) and glial (GALC, GFAP, and S100B) markers performed on hNSCs differentiated on 2D and 3D for 1W, and 3W, and expressed as fold change compared with undifferentiated control. Statistical analysis showed significant differences between 2D versus 3D samples at the same time point; ± SDMs. *P < 0.05; **P < 0.001, ***P < 0.005, Student t test.