Fig. 4

Transcriptional Profile of COs and CO-iMs. RNA-seq analysis of  ~ 65-day old iPSC-Ru line derived COs (n = 3) and CO-iMs (n = 3) from respective single batches. A Dot plot showing the enriched GO terms of biological processes and molecular functions amongst activated and suppressed genes identified using absolute log2 fold change > 1 and a false discovery rate (FDR) < 0.05 using the Benjamin-Hochberg procedure in CO-iMs when compared to COs. B Dot plot showing the enriched REACTOME pathways (FDR < 0.1) in activated as well as suppressed genes in CO-iMs when compared to COs. For (A, B) the x-axis indicates the ratio of genes identified by our analysis and the total number of genes that constitute the pathway, the size of the dot is based on gene count in the pathway, and the color of the dot shows the pathway enrichment significance. C Volcano plot comparison of CO-iMs vs COs indicating average log2 (fold change) versus –log10 (FDR) for all genes. Genes upregulated and downregulated by 2-fold change and FDR < 0.05 are labeled with red dots. D A heatmap showing expression of microglia sensome markers in individual replicates of CO-iMs and COs, (n = 3 for CO-iMs and COs). Color indicates the expression level scaled for each gene by centering and scaling. E RT-qPCR quantification to determine mRNA levels of microglia immune sensome markers: CX3CR1, SLCO2B1, TLR4, CSF1-R and P2RY12. F RT-qPCR quantification to determine mRNA levels of IL34, an essential cytokine for microglia maturation and survival. G RT-qPCR quantification to determine mRNA levels of C3 and C1Q, components of complement pathway. H Heatmap showing expression of microglia complement pathway genes in individual replicates of CO-iMs and COs, (n = 3 for CO-iMs and COs). Color indicates the expression level scaled for each gene. I Uniform Manifold Approximation and Projection (UMAP) plot for dimension reduction of 11,440 barcoded single cells derived from 4 individual CO-iMs (~ 120 day old) subjected to scRNA seq analysis exhibiting 12 distinct clusters as identified by graph-based clustering of cell-specific gene expression. Subsequent cell annotation allowed us to define the most likely cell type corresponding to each cluster. The bubble plot shows all the cell types that were considered by ScType for cluster annotation that were assigned to at least 100 cells with the exception of cluster 10 where no assignment reached that cut-off, and the only assignment obtained is shown. The outer bubbles correspond to each cluster with size reflecting the number of cells in the cluster, while the inner bubbles correspond to considered cell types for each cluster, with the biggest bubble corresponding to assigned cell type, except for cluster 3 and 10 where we did not obtain high quality assignments