Fig. 2: Single-cell RNA sequencing analysis reveals progression of neuronal maturity in cortical organoids but no difference in cell type composition between genotypes.

a Uniform manifold approximation and projection (UMAP) dimensionality reduction of all sequenced cells reveals distinct populations, including: cycling radial glia in G2/M and S phases (cRG (G2/M), cRG (S)); non-cycling radial glia (RG); outer and truncated radial glia (oRG, tRG); vascular leptomeningeal cells (VLMC); choroid plexus (CP); intermediate progenitors (IPC); newborn glutamatergic neurons (nGluN, EOMES⁺ nGluN); immature neurons of a mixed population (imN), GABAergic immature neurons (imGABA N), glutamatergic immature neurons (imGluN); and mature glutamatergic neuron groups with high expression of layer II-III marker CUX2 (CUX2⁺ mGluN), subcortical projection marker FEZF2 (FEZF2⁺ mGluN), or upper layer neuron marker SATB2 (SATB2⁺ mGluN). Data in (a–f) is shown from 9 organoids total across four independent differentiations (Day 40: one C1, one C3, one CRISPR R203W, one A1, and one A2 organoid; Day 88: two C2, one CRISPR R203W, and one A1 organoid. See Table S1). b UMAP reductions divided by genotype and time point. Individual samples are shown in Fig. S8a. c Bar chart showing the cell type composition of samples in each genotype and time point. Individual samples are shown in Fig. S8b. Data from PACS1^(+/R203W) lines is indicated by the dot pattern. d Hierarchical clustering of Pearson’s correlation of variable feature expression across sample and cell type. e Violin plots of feature expression. G2/M phase marker AURKA is restricted to cRG (G2/M) group and S phase marker CLSPN is strongly enriched in cRG (S). Radial glia marker FABP7 is enriched in proliferative groups. HES6 is enriched in IPCs. DLX6 expression is restricted to the imGABA N group. GAP43 is expressed across young and mature neurons while BCL11B and SLC17A7 are enriched in mature glutamatergic neurons. Enrichment of CUX2, FEZF2, and SATB2 define mGluN subpopulations. VLMC-like cells express collagen marker COL1A2 and CP exclusively express FOLR1. f Feature plots of all cells from both genotypes in UMAP space show expression of PACS1, proliferative marker MKI67, RG marker FGFR3, oRG marker HOPX, tRG marker CRYAB, IPC/newborn neuron marker EOMES, immature neuronal vesicular glutamate transporter SLC17A6 (vGLUT2), more mature neuronal transcripts BCL11B and SLC17A7 (vGLUT1), and upper cortical layer marker SATB2. Scale is in units of log₁₀(value + 0.1).