Fig. 1: Astrocytes in contact with neuronal cells have increased expression of cholesterol biosynthesis genes.

a, Outline of the experimental design. Human neural progenitor cells (NPCs) and mouse astrocytes were placed in mono-culture (left) or co-culture (right) (n = 4 biological replicates). Co-cultures were differentiated for up to 8 days. RNA-seq was conducted on mono- and co-cultures at the indicated time points post co-culture. Figure created with BioRender.com. b, A heatmap of standardized expression (z-score logCPM) of canonical astrocyte marker genes across all time points in the mono- and co-cultured astrocytes reveals changes in expression over time (n = 4 per time point). c, PCA of the astrocyte normalized read counts shows clustering of samples by time (colored, 0.5 h - day 8) and culture condition (fill, CC = co-culture, MC = mono-culture) across PC1 and PC2, explaining 36.35% and 6.42% of variation, respectively. d, A heatmap of mean factor values (sample weight mean) across time and culture condition from latent factor (LF) analysis of gene expression in astrocytes. e, A qq-plot of p-values from linear regression of LF values with time in co-culture reveals that LF2 has the strongest, significant association with time in co-culture. Expected p-value distribution shown by black line. f, Gene ratio of top enriched gene ontology (GO) terms from GSEA of the gene weights in LF2 show an enrichment for upregulated genes involved in cholesterol biosynthesis and lipid metabolism. g, Heatmap of standardized expression (z-score logCPM) of the sterol biosynthetic process genes (n = 24) show a distinct increase in expression between 6 hours and 2 days post co-culture. Colored annotations on the right of the heatmap show association of the gene with LF2 gene loading. Genes that are significantly differentially expressed between 2 days and 1 day in co-culture, 1 day and 6 hours in co-culture and between astrocytes in CC and MC at 2 days (p < 4.63908e-06 [Bonferroni cut-off] in red, FDR < 5% cut-off in pink). h, The presynaptic genes in LF2 include genes (n = 3) in the SNAP-a program in the human brain, which are highlighted in red. i, Boxplots of normalized expression (logCPM) for Nrxn1 in astrocytes (n = 4 per time point). The boxplots’ limits show the upper- and lower quartile ranges, the center lines show the median values. The whiskers show the minimum and maximum values. j, Gene expression trends from qPCR experiment of Nrxn1 at various time points after co-culture of human NPCs and mouse glia, along with the corresponding ANOVA test p-value.