Fig. 6

Decreased proliferation and premature differentiation of human NPCs. a, c, e Representative imaging of 2D human NPCs stained with antibodies against PAX6, BrdU (after 2-h BrdU pulse), or Ki67. Hoechst stains nuclei. Scale bars: 10 μm in a, 25 μm in c, and 50 μm in e. b, d, f Quantification of the percentage of PAX6-positive, BrdU-positive, or Ki67-positive cells out of total NPCs (~200 cells counted in each experiment). g Confocal imaging of week 5 organoid sections stained with antibodies against BrdU (green) and Ki67 (red). Hoechst stains nuclei (blue). Scale bars: 20 μm. h, i Quantification of the percentage of BrdU-positive or Ki67-positive cells out of total cells in VZ-like regions in cerebral organoids. j Western blot analysis of protein levels of Β3-Tubulin and Neurofilament (NF) in 2D human NPCs. k Quantification of western blot data from three independent experiments. l, m Representative imaging of week 6 organoid sections stained with antibodies against SOX2 (green) and DCX (red). Hoechst stains nuclei. Areas above upper white dotted lines represent DCX-positive neuron regions for quantification. Scale bars: 20 μm. n Quantification of relative thickness of DCX-positive neurons. Error bars represent SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. represents no significant difference detected (Student’s t-test)