(A) Quantitative analysis for MKI67 and PH3 in Iso 1, Iso 2, and Mut Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells) reveals a decrease in G1-M (MKI67+) but not in mitotically active (PH3+) cells (MKI67+, replicates n=4, total cell N=3315, one-way ANOVA, post hoc Tukey’s test, p-value <0.001; PH3+, replicates n=4, total cell N=12684, one-way ANOVA, post hoc Tukey’s test, p-value >0.05). (B) Scheme of synchronization experiment in iPS-NES cells. (C) Representative confocal images and quantification of mitotic figure distribution in iPS-NES cells. The distribution of mitotic figures (quantified as % of mitotic figures at each phase) is not significantly different in unsynchronized (T0) Iso 1, Iso 2, and Mut iPS-NES cell cultures (replicates n=4, total cell N=12684, p-value <0.05, two-way ANOVA, post hoc Dunnett’s test). Quantitative analysis after 16 hr of treatment (T1) shows increased mitotic cell fraction in P-M. The percentage of cells arrested in P-M is higher in Iso 1 and Iso 2 than in Mut iPS-NES cell cultures. Fewer Mut iPS-NES cells proceed to M following nocodazole treatment and subsequent release in standard culture conditions (T2) compared to Iso 1 and Iso 2 iPS-NES cells (replicates n=4, total cell N=8764, P-value <0.0001, two-way ANOVA, post hoc Dunnett’s test and replicates n=4, total cell N=9751, p-value <0.05, two-way ANOVA, post hoc Dunnett’s test). P (prophase); P-M (prometaphase); M (metaphase); A (anaphase), and T (telophase). (D) Schematic 3D representation of inter-centrosomal distance (ICD) and spindle angle α measurements in iPS-NES cell cultures. α was measured as the angle of the spindle plane relative to the surface of the culture dish (baseline). (E) Representative confocal images of PCTN and DAPI staining in Iso 1 and Mut iPS-NES cells in metaphase and example of ICD measurements. Quantitative analysis shows no differences in ICD (replicates n=6, total cell N=81, p-value >0.05, one-way ANOVA, post hoc Sidak’s test). (F) Confocal Z-stack images of PCTN in Iso 1 and Mut iPS-NES cell lines during metaphase. Quantitative analysis shows smaller α in Mut iPS-NES cell compared with Iso 1 and Iso 2 iPS-NES cells (replicates n=6, total cell N=81, p-value <0.01, one-way ANOVA, post hoc Dunnett’s test). (G) Representative confocal images of ZO-1 in COs of Iso 2, Het, and Mut at 30 days in vitro (DIV30) showing the lumen of neural rosettes. (H) Confocal images of PCTN in Iso 2, Het, and Mut COs at DIV30. (I) Analysis of the cleavage plane angle θ in neural progenitors (% mitotic cells) showing a higher percentage of asymmetric (horizontal) divisions in Mut COs compared with Iso 2 and Het COs (COs n=6, total cell N=184, data are shown as %, p-value >0.001, Chi-square test). Data are shown as mean ± SD. Scale bar = 5 μm in (C), 10 μm in (E, F), and 20 μm (G, H).