Fig. 2: Cortical-cell-type diversity and maturity are recapitulated in C9 and control ALI-COs.

a, Violin plots (upper) and UMAP plots (lower) illustrating AQP4 (astrocyte), CTIP2 (DLN) and GAD2 (IN) marker gene expression identified by scRNA-seq in 20 ALI-COs at 150 DIV. Cluster numbers (x axis) correspond to color-coded cell-type identities as indicated above the plots. b, Representative images demonstrating the immunoreactivity of cell-type marker proteins in ALI-COs (three biological repeats per group). c, Pseudotime analysis of scRNA-seq data with branches representing the color-coded trajectory of cell-subtype specification (upper) or cluster identities (lower). n = 75,497 cells for a and c. d, Projected transcriptomic data of four control and four C9 ALI-CO slice-pairs onto two fetal brain reference datasets^16,17 using scmap¹⁸. Bars indicate the proportion of cells assigned to different fetal ages and expressed as human gestational weeks. e, Representative original confocal microscopy images (upper) illustrate pre- and post-synaptic proteins, synaptotagmin-1 (SYT1; red) and HOMER1 (HOMER; green) and their colocalization over MAP2⁺ dendrites (gray) in deconvoluted digital images (lower) in ALI-COs at 150 DIV and quantification (right). Data represent the mean ± s.e.m. synapse densities; n = 4, 6, 6, 5 independent ALI-CO slices for H-L1, H-L2, C9-L1, C9-L2 organoid lines, respectively; one-way ANOVA with Tukey’s post hoc test (overall ANOVA P value is indicated in the graph). f, Neuronal network analysis using MEAs (left) reveals rich spontaneous network activity in all ALI-COs. Highly connected nodes (circles) represent individual electrodes for the degree of connectivity (node size), the spike rates (node color) and the connection strength (line thickness). The graph (right) shows the firing rates (spike number per second) across all electrodes for each recording. Data are expressed as individual recordings and the mean ± s.e.m.; n = 9, 9, 11, 6 ALI-CO slices for H-L1, H-L2, C9-L1, C9-L2 organoid lines, respectively; one-way ANOVA with Tukey’s post hoc test (overall ANOVA P value is indicated in the graph). Scale bar, 10 μm (b,e). See Supplementary Table 3 for detailed statistics.