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. 2023 Jan 17;29(4):1049–1066. doi: 10.1111/cns.14079

FIGURE 4.

FIGURE 4

eIF2B mutation resulted in a delay in early neuronal development and normal superficial neuronal differentiation in later stage in cerebral organoids. (A) Immunofluorescence staining for marker of NSCs (SOX2, red), IPs (TBR2, purple), and cortical plate neurons (CTIP2, green) at Week 6. Scale bars 100 μm. n = 6–13 organoids (numbers were listed within each bar) from three iPSC lines derived from three individuals, one‐way ANOVA, and Kruskal–Wallis ANOVA analysis. Change in NSCs quantified by area of fluorescence of SOX2, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.000). Change in IPs quantified by area of fluorescence of TBR2, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.000). (B) Immunofluorescence staining for markers of NSCs (SOX2, red), immature neuron (TUJ1, green), and nuclear staining (DAPI, blue) at Week 6. Scale bars 100 μm. Change in immature neurons quantified by area of fluorescence of TUJ1, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.021). Change in cortical plate neurons quantified by area of fluorescence of CTIP2, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.000). (C) Immunofluorescence staining for cortical plate neuron (CTIP2, green), deep‐layer neurons (TBR1, red), and nuclear staining (DAPI, blue) at Week 12. Scale bars 50 μm. Change in cortical plate neurons quantified by area of fluorescence of CTIP2, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.350). Change in deep‐layer neurons quantified by area of fluorescence of TBR1, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (p = 0.220). (D) Immunofluorescence staining for cortical plate neuron (CTIP2, green), superficial neurons (SATB2, red), and nuclear staining (DAPI, blue) at Week 12. Scale bars 50 μm. (E) Immunofluorescence staining for cortical plate neuron (CTIP2, green), deep‐layer neurons (TBR1, red), and nuclear staining (DAPI, blue) at Week 20. Scale bars 50 μm. (F) Immunofluorescence staining for cortical plate neuron (CTIP2, green), superficial neurons (SATB2, red), and nuclear staining (DAPI, blue) at Week 20. Scale bars 50 μm. Changes in superficial neurons quantified by area of fluorescence of SATB2, n = 6–13 in each group (numbers were listed within each bar) with three independent experiments, one‐way ANOVA analysis (Week 12: p = 0.060; Week 20: p = 0.481). NSC: neural stem cell; IP: intermediate progenitor cells. Data were presented as the mean ± SEM values, ns: p > 0.05, **: p < 0.01, ***: p < 0.001.