Transcriptional heterogeneity of cells derived from TS microglia. a) UMAP shows the distribution of cell types from humans, macaques, TS, pigs, and mice. b) Heatmap shows the correlation of microglia across species (Spearman, *P < 0.05, **P < 0.01, ***P < 0.001, significance test for Spearman's rank correlation coefficient using t-distribution approximation). c) The violin plot demonstrates the expression of the canonical marker genes of microglia across species. d) Network plot show species-specific transcriptional regulators of microglia. e)–h) Manual tracking and Sholl analysis-based quantification of cell morphology of hippocampal IBA1-positive microglia (red). Each symbol represents one individual; three to five cells analyzed per individual. Sholl analysis: step size = 20 μm. Blue, DAPI. Scale bar: 25 μm. Similar outcomes were obtained in three repeated independent experiments. One-way ANOVA, *P < 0.05, ns, not significant. i) Quantification of IBA1-positive cells in the hippocampal DG from infancy, adult, and old TS. One-way ANOVA, ***P < 0.001. j) Violin plots showing expression levels of MEF2C in microglia in the TS hippocampus from infancy, adult, and old groups (two-sided Wilcoxon rank-sum test, **P < 0.01, ***P < 0.001). k) Representative microscopic fields of IBA1/MEF2C double-positive cells in the hippocampal DG from infancy, adult, and old TS. Blue, DAPI. Scale bar: low magnification, 50 μm; high magnification, 10 μm. n = 3. l) Density plot showing gene set scores of HuMi_Aged microglia genes in different cell types in the TS hippocampus. m) Top, boxplot showing increasing gene set scores in TS hippocampus with age. Bottom, violin plots showing increasing HuMi_Aged gene set scores in microglia in the TS hippocampus from infancy, adult, and old groups (two-sided Wilcoxon rank-sum test). n) Visualization of microglia subclusters using UMAP. o) Left, bar plot showing the proportions of microglia subclusters in the hippocampus from infancy, adult, and old groups. Right, enriched GO terms in Micro1 and Micro3. p) Visualization of Micro1 subclusters using UMAP. q) Left-top, bar plot showing the proportions of Micro1 subclusters in the hippocampus from infancy, adult, and old groups. Left-bottom, UMAP showing the distribution of cluster-related DEGs in the Micro1. Right, enriched GO terms in Micro1_1 and Micro1_2.