FIGURE 5.

STAMBP^WT but not STAMBP^G307E and STAMBP^T313I overexpression rescues the phenotypes of reduced cortical size and impaired proliferation of NSCs. (A) Schematic representation of the PiggyBac-mediated STAMBP expression system. (B) Western blotting confirmed the presence of STAMBP^WT, STAMBP^G307E, and STAMBP^T313I protein generated from the STAMBP KO hESCs. (C) Representative images of control, STAMBP KO cortical organoids, and organoids generated from STAMBP^WT, STAMBP^G307E, and STAMBP^T313I overexpression on day 28. Scale bars, 200 μm. (D) Quantification of surface areas of cortical organoids. Each plot represented an individual organoid. Experiments were repeated three times. Groups were compared using a Kruskal–Wallis ANOVA with Dunn’s multiple comparisons tests. Data are presented as median ± 95% confidence interval, *p < 0.05, **p < 0.01, ***p < 0.001. (E) Immunofluorescent staining was performed on organoids on day 28. Sections were stained with Ki67 (the first row), PH3 (the second row), and CC3 (the third row). Scale bar: 100 μm. (F–H) Quantification of cell cycling marker Ki67-positive cells (F), PH3-positive cells (G), and cell apoptosis marker CC3 (H). Each plot represented an individual section. Experiments were repeated three times. Data of Ki67⁺/DAPI and PH3⁺/DAPI were compared using a one-way ANOVA followed by a Dunnett’s multiple comparisons test. Data are shown as mean ± SEM. Data of CC3⁺/DAPI were using a Kruskal–Wallis ANOVA with Dunn’s multiple comparisons tests. Data are presented as a median ± 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001.