Fig. 2: Defects in neuronal differentiation of iPSCs due to mutation in TIMM8A.

A Schematic diagram of the neuronal differentiation strategy: iPSCs were differentiated sequentially into neuroepithelial cells, neural progenitor cells (NPCs), and finally mature neurons over a period of 53 days. B Morphology of CTRL-, MUT-, and MTS-derived cells at day 8 (neuroepithelial cells), day 16 (NPCs), and day 53 (mature neurons) during differentiation. Scale bar: 50 μm. C Generations of CTRL-, MUT- and MTS-NPCs was confirmed by immunostaining with NPC markers, including PAX6 (red), SOX1 (green), SOX2 (red), and Nestin (green) in rosette-like cells at day 16. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. D, E Expression levels of Nestin, PAX6, and SOX2 in CTRL-, MUT-, and MTS-NPCs were analyzed by Western blot. Relative protein levels were quantified and normalized to β-Tubulin as a loading control. n = 3 independent biological replicates. Data are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s post hoc test: ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001. F Generation of early neurons at day 30 and mature neurons at day 53 was confirmed by immunostaining with the early neuronal marker TUJ1 (green) and the mature neuronal marker MAP2 (green), respectively. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. G Quantification of branch numbers, soma size, and neurite length in early neurons at day 30. n = 3 independent biological replicates (42 neurons for CTRL, 53 for MUT, and 46 for MTS). Data are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s post hoc test: *p < 0.05; ***p < 0.001; ****p < 0.0001.