Figure 3. Reduced neuronal growth and impaired mitochondrial health in forebrain neurons differentiated from MECP2 mutant hESC and iPSC lines. A, Representative images of neuronal morphology in Synapsin-GFP-labeled H9, MECP2T158M/T158M, and MECP2-KO neurons. Scale bar, 20 μm. B, Sholl analysis of dendritic complexity in Synapsin-GFP-labeled H9, MECP2T158M/T158M, and MECP2-KO neurons. Reduced number of neurite intersections indicates reduced complexity. Data points are represented as mean ± SEM. A total of 47–55 neurons per line from three independent differentiation experiments were included in the analysis. Two-way ANOVA analysis was followed by Dunnett's post hoc test. ****p < 0.0001, H9 versus MECP2T158M/T158M. ***p < 0.001, H9 versus MECP2T158M/T158M. *p < 0.05, H9 versus MECP2T158M/T158M. ####p < 0.0001, H9 versus MECP2-KO. ###p < 0.001, H9 versus MECP2-KO. ##p < 0.01, H9 versus MECP2-KO. #p < 0.05, H9 versus MECP2-KO. C, Quantification of total neurite length in neurons analyzed in B. ****p < 0.0001. **p < 0.01. D, Representative images of neuronal morphology in Synapsin-GFP-labeled V247fs-WT, V247fs-MT, and V247fs-MT-correction neurons. Scale bar, 20 μm. E, Sholl analysis of dendritic complexity in Synapsin-GFP-labeled V247fs-WT, V247fs-MT, and V247fs-MT-correction neurons had restored the number of neurite intersections compared with V247fs-MT neurons. Reduced number of neurite intersections indicates reduced complexity. Data points are represented as mean ± SEM. A total of 47–54 neurons per line from three independent differentiation experiments were included in the analysis. Two-way ANOVA analysis was followed by Dunnett's post hoc test. ****p < 0.0001, V247fs-MT versus V247fs-WT. ***p < 0.001, V247fs-MT versus V247fs-WT. *p < 0.05, V247fs-MT versus V247fs-WT. ####p < 0.0001, V247fs-MT versus V247fs-MT-correction. ###p < 0.001, V247fs-MT versus V247fs-MT-correction. ##p < 0.01, V247fs-MT versus V247fs-MT-correction. #p < 0.05, V247fs-MT versus V247fs-MT-correction. F, Quantification of total neurite length in neurons analyzed in E. ****p < 0.0001. **p < 0.01. G, Representative images of H9, MECP2T158M/T158M, and MECP2-KO neurons expressing Synapsin-mitoDsRed2. Enlarged view of the boxed area is shown at the bottom of each image. Scale bars, 20 μm. H, Quantification of mitochondrial aspect ratio in Synapsin-mitoDsRed2-labeled H9, MECP2T158M/T158M, and MECP2-KO neurons. A total of 56–60 neurons per line from three independent differentiation experiments were included in the analysis. ****p < 0.0001. ***p < 0.001. I, Representative images of V247fs-WT, V247fs-MT, and V247fs-MT-correction neurons expressing Synapsin-mitoDsRed2. Enlarged view of the boxed area is shown at the bottom of each image. Scale bars, 20 μm. J, Quantification of mitochondrial aspect ratio in Synapsin-mitoDsRed2-labeled V247fs-WT, V247fs-MT, and V247fs-MT-correction neurons. A total of 25–33 neurons per line from three independent differentiation experiments were included in the analysis. ****p < 0.0001. **p < 0.01. K, Representative images of JC-10 dye-stained H9, MECP2T158M/T158M, and MECP2-KO neurons. Scale bar, 20 μm. L, Quantification of the ratio of red (F590) and green (F520) fluorescence in H9, MECP2T158M/T158M, and MECP2-KO neurons. A total of 128–192 neurons per line from three independent differentiation experiments were included in the analysis ****p < 0.0001. Bar graphs, Data are mean ± SEM.