Figure 4. KNL1^{c.6125G > A} Differentiating Neuro-Glial Cultures Differentiate Prematurely, at the Expense of Neural Stem Cell Proliferation.

(A–C) Time-course analysis of neural progenitors marker PAX6 (A), neuronal marker MAP2 (B), and glial marker GFAP (C), using real-time qPCR in a wild-type and KNL1^{c.6125G > A} neuro-glial differentiating culture. ANOVA was performed followed by a post hoc group comparisons using a Bonferroni test.

(D–F) Time-course analysis of neural progenitors (D), neurons (E), and astrocytes (F), using quantification based on cell-surface markers of wild-type and KNL1^{c.6125G > A} neuro-glial differentiating culture. ANOVA was performed followed by a post hoc group comparisons using a Bonferroni test.

(G) Immunostaining using antibodies against S100b and Tuj1 in wild-type and KNL1^{c.6125G > A} in a neuro-glial culture, at 4 weeks of differentiation (scale bar, 50 μm).

(H) Immunostaining using antibodies against GFAP and MAP2 in wild-type and KNL1^{c.6125G > A} in a neuro-glial culture, at 6 weeks of differentiation (scale bar, 50 μm).

(I) FACS-based quantification of EdU⁺TUJ1⁺/EdU⁺ cells following 1-day post EdU pulse-chase assay in 2-week-old neuro-glial differentiating culture. Welch’s t test (two-tailed) was applied.

(J) FACS-based quantification of EdU⁺S100β⁺/EdU⁺ cells following 1-day post EdU pulse-chase assay in 2-week-old neuro-glial differentiating culture. Welch’s t test (two-tailed) was applied.

(K) Quantification of the mitosis time in wild-type and KNL1^{c.6125G > A} 1-week differentiating culture using live imaging, in the presence and absence of nocodazole. Each point represents a dividing wild-type or KNL1^{c.6125G > A} cell, from three different wild-type and KNL1^{c.6125G > A} clones. ANOVA was performed followed by a post hoc group comparisons using a Bonferroni test.

(L) Quantification of EdU⁺PAX6⁺/EdU⁺ in wild-type and KNL1^{c.6125G > A} 1-week differentiating culture following a pre-incubation with EdU for 1 hr and a 90-min nocodazole treatment. ANOVA was performed followed by a post hoc group comparisons using a Bonferroni test.

One non-targeted wild-type clone, two wild-type clones and three patient mutation clones derived from the same CRISPR-Cas9 targeting are plotted in each graph. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.