Evaluation of hNSC proliferation. (A-E) Representative image of (A) undifferentiated, (B) 1W 2D differentiated, (C) 3W 2D differentiated, (D) 1W 3D differentiated, and (E) 3W 3D differentiated hNSCs stained with Ki67, marker of cell proliferation; scale bar, 50 μm; Ki67+ hNSCs (red), nuclear Hoechst counterstain (blue). (F) Quantification of cell proliferation measured as percentage of cells positive for Ki67. Statistical analysis showed a significant reduction in proliferation in 2D and 3D cultures compared with the proliferative control and between 2D and 3D samples at the same time point; ±SDMs. *P < 0.05, **P < 0.001, ***P < 0.005, Student t test.