Figure 4A Schematic of PSC aggregation and development into cortical organoids. B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm. C Ki67+ proliferative area (Student's t-test, t10 = 0.36, P = 0.73, N = 6 each). Left: representative immunofluorescence images of progenitor, proliferative, and neuronal markers in cortical organoids; Right: quantification of Ki67+ proliferation in cortical organoids. Scale bar = 50 µm. RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways. E, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values. RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways. G, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values. H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm. MECP2-KO and control organoids had proportions of cells positive for the neuronal marker NeuN (I; Student's t-test, t14 = 0.26, P = 0.80, N = 8 each) J MECP2-KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 (J; one-way ANOVA, F3,40 = 2.489, P = 0.074 and Z < 1, WT83/Q83X and WT82/K82fs cell lines were used; N = 8-14 organoids each). J, left: representative image of CTIP2+ immunofluorescent staining in cortical organoids; J, right: quantification of CTIP2+ neurons. Scale bar = 50 µm. MECP2-KO cortical organoids plated on MEA (K; left: schematic depicts process of plating organoids on MEA; right: top-down view of organoid on MEA; scale bar = 200 µm) showed decreased population spiking L; left and center: representative population spiking traces of control and MECP2-KO organoids; right: quantification of organoid population spiking activity, N = 6 replicates each). Drug treatment increased spiking network activity of MECP2-KO cortical organoids to a level not significantly different from that of control organoids (M; one-way ANOVA, F3,31 = 3.775, Dunnett's multiple comparisons test vs control: untreated KO, **P < 0.01 and Z = 3.192; Nefiracetam, P = 0.98 and Z = 2.117; PHA 543613, P = 0.12 and Z = 1.101, WT83/Q83X cell lines were used; N = 7-10 MEA wells per condition).