Fig 1: Activation of NC genes in the adult leptomeninges following dMCAO as identified using snRNA-seq(A) Single nuclei were isolated from cortices after dMCAO on days 3 (n = 3), 7 (n = 4), 14 (n = 3), and 28 (n = 4) and compared with sham controls (n = 3). (B)–(H) derive from these datasets.(B) Unsupervised clustering of 107,020 nuclei identified 14 clusters. PC, progenitor cell.(C) Early NC signature scoring (Wnt1-associated) highlighted a high-scoring subpopulation within C11.(D) NC regulatory genes (Tfap2b, Foxd1, Sned1, and Eya2) were enriched in C11.(E) DEG analysis of C11 visualized by UMAP and force-directed layout/embedding (FLE) identified NC-related subsets.(F) GO analysis revealed SC2, SC3, and SC5 subclusters as enriched for NCC migration, development, and differentiation.(G) Subclusters refined into seven subsets: SC2 (subsets 1, 2, 4, and 6), SC3 (subsets 0 and 3), and SC5 (subset 5).(H) Upregulated genes in each cluster were consistent with NC lineage programs.(I) GSEA showed subset 1 enriched for stromal signatures and subsets 0 and 3 enriched for radial glia-like signatures.(J) Venn diagram comparing pericyte cluster C13 and subsets 2, 4, and 6.(K–P) Immunohistochemical validation of cluster- and subset-specific markers (low-power anatomical context in Figure S8).(K) Klf5+ (subsets 1 and 5) and Bmp7+ (subsets 0 and 3) cells co-localized with Foxd3.(L) CD271+ cells were present in layers I–IV ipsilateral to dMCAO; a subset co-expressed Bmp7.(M) Klf5+ cells co-localized with CD271 in cortical layer II/III.(N) Subset 4: PDGFRβ+/Col3a1+ cells; subset 6: Col3a1+/PDGFRβ− cells.(O) Subset 2: Ebf2+/Cped1+ cells adjacent to CD31+ vessels.(P) No co-expression of PDGFRβ and Cped1 (specificity control). DAPI labels nuclei.(Q) Integration of snRNA-seq with spatial transcriptomics showed subsets 2 and 5 enriched ipsilaterally on day 3 post dMCAO and SC3 subsets localized to leptomeninges on days 3 and 14.(R) Representative genes of refined SC2/SC3/SC5 plotted on UMAP.(S–U) CD271+ meningeal cells isolated 3 days post tMCAO (S), expanded in vitro (T), and confirmed to express CD271 and Foxd3 (U).(V) C11 subdivided into SC2, SC3, and SC5, each further resolved into seven subsets based on upregulated DEGs, with NC-lineage enrichment.PC, pericyte; SMC, smooth muscle cell; MG, microglia; FB, vascular fibroblast-like cell; OL, oligodendrocyte; EC, endothelial cell; AC, astrocyte; v, venous; capil, capillary; a, arterial; aa, arteriolar; 1/2/3, subtypes.