Fig. 3

Single-cell gene expression analysis of iMG. A. K-means clustering of ~ 2000 single-cell transcriptional profiles from iMG at day 20, identifying four transcriptionally distinct clusters. These iMG clusters are presented in a Uniform Manifold Approximation and Projection (UMAP), where each dot represents a cell. The cells are color-coded based on their cluster affiliation. B-D. The expression of microglial genes IBA1, CD45, and C1QA projected on the UMAP representation of the clusters. The colored scale represents the log2 expression. E. Stacked violin plots of ~ 2000 iMG showing cluster-wise the high expression levels of microglial genes (left panel) compared to the low or absent expression of monocytic genes (right upper panel). The expression of glial and neuronal markers (e.g., astrocytes (GFAP), oligodendrocytes (OLIG1 and OLIG2) and neurons (RBFOX3 and MAP2)) was not detected (right lower panels). F. UMAP showing the integration of our iMG-scRNA-Seq data with the post-mortem single-nucleus RNA-Seq data from aging human frontal cortex (Cain, A. et al. 2023). The integration revealed that iMG signature resembles the microglial signature but differs from the signature of other cell types in the brain, including neurons, neuroglia, and vascular cells. G. Principal component analysis of expression of microglial genes in iMG (red) human adult microglia (blue), human fetal microglia (green) and iPSC-derive iMG from other studies, showing strong resemblance of our iMG to primary microglia. H. Scatter plot showing the comparison of iMG mRNA expression with that of primary adult aged microglia (pMG) from a previous study, which were purified from the dorsolateral prefrontal cortex of deceased patients (Olah, M. et al. 2020). Each dot represents a gene. A strong correlation between the transcriptome of iMG and pMG was observed (Kendall’s tau correlation coefficient (T_b) = 0.6826, p-value = 0.0, Pearson’s r = 0.2658 and p-value = 8.771e^− 288). I. Bar graph comparing CX3CR1 and P2RY12 expression in iMG differentiated for 20 days and iMG cultured in homeostatic conditions for additional three days (total 23 days). Fold changes were identified by quantitative RT-PCR (n = 3, C1 iPSCs). Error bars represent the standard error of three independent experiments. J-K. Gene enrichment analysis of the top 25 upregulated genes in each cluster. Lollipop charts provide information about Gene Ontology (GO) fold enrichment, significance (FDR log10), and the number of genes in each enriched term in iMG cluster 1 (J) and cluster 2 (K). scRNA-Seq was performed on iMG derived from C1-iPSC line. Abbreviations: Uniform Manifold Approximation and Projection (UMAP); PCA principal component analysis; VLMC vascular leptomeningeal cell; OPC oligodendrocyte precursor cells; FDR false discovery rate