Abstract
The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms—the blood–brain barrier—impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer’s disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer’s disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer’s disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer’s disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.
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Data availability
Raw sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession code GSE163577. Data are also available to explore via an interactive web browser: https://twc-stanford.shinyapps.io/human_bbb.
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Acknowledgements
We thank T. Iram, E. Tapp, N. Lu, M. Haney, O. Hahn, M. J. Estrada, S. M. Shi and other members of the Wyss-Coray laboratory for feedback and support; H. Mathys, D. A. Bennett and participants in the CSHL BBB 2021 meeting for advice; and H. Zhang and K. Dickey for laboratory management. This work was funded by the NOMIS Foundation (T.W.-C.), the National Institute on Aging (T32-AG0047126 to A.C.Y. and 1RF1AG059694 to T.W.-C), Nan Fung Life Sciences (T.W.-C.), the Bertarelli Brain Rejuvenation Sequencing Cluster (an initiative of the Stanford Wu Tsai Neurosciences Institute) and the Stanford Alzheimer’s Disease Research Center (P30 AG066515). This work was supported by a grant from the Simons Foundation Award (811253TWC). A.C.Y. was supported by a Siebel Scholarship. F.K. and A.K. are part of the CORSAAR study supported by the State of Saarland, the Saarland University and the Rolf M. Schwiete Stiftung. This study was supported by the AHA–Allen Initiative in Brain Health and Cognitive Impairment (19PABHI34580007). The statements in this work are solely the responsibility of the authors and do not necessarily represent the views of the American Heart Association (AHA) or the Paul G. Allen Frontiers Group. Graphics were created with BioRender.com.
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A.C.Y. and T.W.-C. conceptualized the study. A.C.Y. devised the isolation method. M.W.M. and W.J.S.-S. provided and A.C.Y. organized tissue samples. D.P.L. and A.C.Y. performed tissue dissociations. N.S., R.T.V., D.G., K.C., H.S. and A.C.Y. prepared libraries for sequencing. R.T.V., F.K., A.K., C.A.M., M.B.C., R.P., A.S., N.K., J.A.S. and A.C.Y. performed computational analysis. D.P.L., C.A.M., M.A., D.G., E.Y.W., J.L. A.T., P.C. and A.C.Y. performed immunohistochemical stains. P.M.L. developed the searchable web interface (Shiny app). C.A.M. and A.C.Y. drew diagrams. A.C.Y. wrote the manuscript with input from all authors. A.C.Y. and T.W.-C. supervised the study.
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T.W.-C. is a co-founder and scientific advisor of Alkahest. A.C.Y., R.T.V. and T.W.-C. are co-founders and scientific advisors of Qinotto.
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Extended data figures and tables
Extended Data Fig. 1 Enhanced capture and characterization of human brain vascular nuclei.
a, Detailed schematic of the VINE-seq method to capture human brain vascular and immune cell types for single-nucleus sequencing. b, Total number of nuclei, median number of unique molecular identifiers (UMI), and median number of genes for each human sample sequenced from hippocampus and superior frontal cortex. c, Quantification of the median number of genes detected per nuclei across groups (n = 17 hippocampus and n = 8 cortex; n = 8 NCI and n = 9 AD, two-sided t-test; mean +/− s.e.m.). d, e, Quantification of the number (d) and proportion (e) of cerebrovascular cell types captured via the VINE-seq method introduced here compared to recent snRNA-seq studies16,17. f, Summary quantification of the proportion of captured cell types, by individuals with NCI and individuals with AD. g, Quantification of the proportion of captured cell types across individuals. h, Summary (left) and quantification (right) of the proportion of captured cell types by brain region (n = 17 hippocampus and n = 8 cortex; n = 8 NCI and n = 9 AD, two-sided t-test; mean +/− s.e.m.).
Extended Data Fig. 2 Diversity and heterogeneity of human brain vascular cell types.
a, Representative H&E images used by two neuropathologists to evaluate NCI cortical and hippocampal tissue for vascular pathology. No significant vascular pathology was observed. Scale bars, 200 μm. b, Discovery of the top cell-type-specific marker genes across the major classes of cells captured. The colour bar indicates gene expression from low (blue) to high (yellow). c, Validation of cell-type annotations and confirmation of minimal doublet contamination using established cell-type markers. d, UMAP projection of captured myeloid cells, forming two distinct clusters corresponding to parenchymal microglia and brain barrier macrophages. Example marker genes listed. e, Immunohistochemical validation of microglial and perivascular macrophage markers. Scale bars, 50 μm. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org). f, Global view of DEGs comparing human brain macrophages and microglia (left, MAST, Benjamini–Hochberg correction; FDR < 0.01 and logFC>0.5 [log2FC>0.72] to be coloured significant). Pathways enriched in microglia versus macrophages (right), recapitulating interesting biology such as the unique TGF-β molecular signature in microglia75. g, Expression of top gene markers for various T cell subtypes (top), and quantification of their expression as a module (bottom)76. Brain T cells exhibit highest expression of markers corresponding to CD8 cytotoxic and CD4 Naive/Central memory (NV/CM) T cells. h, UMAP projection of captured astrocytes, forming two distinct clusters, and split by brain region. Example marker genes listed. i–j, Quantification of astrocyte cluster 0 (b) and 1 (c) frequency in the cortex and hippocampus (n = 8 cortex and n = 17 hippocampus, Mann-Whitney t-test; mean +/− s.e.m.). k, Immunohistochemical validation of the brain region-specific astrocyte marker TENM4. Scale bars, 50 μm.
Extended Data Fig. 3 Species-specific gene expression across brain cell types and their pharmacological relevance.
a–f, Identification of species-specific genes. Both mouse and human transcriptomes were generated and analysed similarly via single-nucleus RNA-sequencing. Mice were 19 months of age to match the average age of our human cohort. Species-specific/enriched are coloured. g, Immunohistochemical confirmation of genes predicted to be enriched or specific to human cerebrovascular cells compared to mouse (isolated mouse nuclei and per Vanlandewijck, et al., 2018)12, in terms of overall expression or zonation. In parenthesis is the cell type predicted to be uniquely or exhibiting enriched expressed in human over mouse. Scale bars, 50 μm. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org). h–i, Mouse and human BEC expression of genes mediating protein transcytosis (h) and small molecule influx and efflux (i).
Extended Data Fig. 4 Human brain vascular expression of genes relevant to disease.
a, Brain vascular expression of genes relevant to SARS-CoV-2 brain entry, as summarized in Iadecola, et al. 202077. b, Expression of the mouse perivascular fibroblast-like gene Spp1 is instead specifically expressed in human myeloid cells and oligodendrocytes (SPP1, top)78. c, No expression of the immuno-oncology target CD19 and its chaperone CD81 across human adult brain pericytes and SMCs79. Note: cells with any finite expression are ordered to the front to ensure all expression is visible, but this carries the potential to visually overestimate average expression.
Extended Data Fig. 5 Brain endothelial and mural cell zonation and subpopulations.
a, UMAP projection of captured BECs, organizing by arteriovenous zonation. Bottom, tip cell markers expressed in the tip-like/ proteostatic EC cluster. b, Validation of BEC zonation clusters using established zonation markers12. Violin plots are centred around the median, with their shape representing cell distribution. c, d, As in a, b but for pericytes and SMCs. Note that the anatomical locations of pericyte 0 and 1 have not yet been determined. e, Immunohistochemical validation of ACTA2 (α-SMA) expression in human SMCs and less so in capillary pericytes. A denotes arterial and C denotes capillary. Arrowheads specify capillary pericytes expressing ACTA2. Scale bars, 50 μm. f, g, As in a, b but for perivascular fibroblast-like cells, as recently discovered in mice12.
Extended Data Fig. 6 Brain endothelial zonation and mural cell subtype markers.
a, Immunohistochemical validation of zonation and cell subtype markers in BECs. Scale bars, 50 μm. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org). b, Comparison of the zonal specificity of genes in arterial, capillary, and venous cells. Axis plot a specificity score, as defined in the Methods. For example, specificity score for capillaries = avg(logFC(cap/ven), logFC(cap/art)). c, Immunohistochemical validation of capillary expression in human brains of the mouse venous-specific marker VWF and CA4, with similar patterns observed across multiple primary antibody clones. Scale bars, 100 μm. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org). d, Immunohistochemical validation of zonation and cell subtype markers in brain SMCs and pericytes. Scale bars, 50 μm. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org).
Extended Data Fig. 7 Specialization and functions of human brain fibroblasts.
a, Expression of example markers demarcating perivascular from meningeal fibroblasts. b, UMAP of 428 meningeal fibroblast nuclei, subclustering into anatomically segregated dural and arachnoid space fibroblasts. c, Expression of the genes constituting the major fibrotic scar component collagen I in pericytes and fibroblasts. Collagen I is composed of two components, COL1A1 and COL1A2. Column annotations: T-PC = solute transport pericyte and M-PC = Extracellular matrix regulating pericyte, P. FB = Perivascular fibroblast, and M. FB = Meningeal fibroblast. d, e, Protein immunostaining validation of polarized expression of human brain meningeal and perivascular fibroblast pumps: the common marker CYP1B1 (d, serves as a control) and the meningeal fibroblast-specific influx pump SLC47A1 (e). Scale bars, 50 μm. f, Overlap between the top 100 perivascular fibroblast-like cell markers and those identified in mice. A more lenient set of 500 (instead of 100) mouse markers80 were used for comparison to ensure claims of species-specificity were robust. Note: the species-conservation of a cell-type marker depends on species-specific changes in the given cell type and changes amongst the remaining background cell types.
Extended Data Fig. 8 Vascular cell-type-specific perturbations in patients with AD and ApoE4 carriers.
a, Immunohistochemistry with anti-β-amyloid antibody (D54D2, white), Thioflavin S (green), and Hoechst (blue) in the hippocampus of individuals with NCI and individuals with AD. Scale bars, 40 μm. b, Quantification of β-amyloid immunostaining in a for overall β-amyloid (n = 4 NCI and AD, two-sided t-test; mean +/− s.e.m.). c, As in b but for cored and neuritic β-amyloid plaques (n = 3 NCI and AD, two-sided t-test; mean +/− s.e.m.). d, UMAP of 143,793 nuclei captured from 17 human hippocampus and superior frontal cortex samples, coloured by AD diagnosis. e, Quantification controls for Fig. 5b. Quantification of Collagen IV+ vasculature (left) and number of total (regardless of Collagen IV+ overlap) Hoechst+ nuclei (n = 5 NCI and AD, nested two-sided t-test; mean +/− s.e.m.). f, Matrix layout for intersections of AD DEGs shared across and specific to each cell type. Circles in the matrix indicate sets that are part of the intersection, showing that most DEGs are cell-type-specific. g, Example DEGs in AD: arterial (Art), capillary (Cap), venous (Vein), pericyte (Peri), perivascular fibro blast-like cell (P. fibro), and SMC. Blue arrow indicates upregulated and grey arrow downregulated genes. h, Summary of the number of AD DEGs by pericyte class: T-, M-, and all pericytes combined to evaluate DEGs that could arise due to a disproportionate loss of M-pericytes in AD. i, DEG counts for each cell type in ApoE4 carriers (n = 5 ApoE3/3, n = 11 ApoE3/4 or ApoE4/4): arterial (Art), capillary (Cap), venous (Vein), pericyte (Peri), perivascular fibro blast-like cell (P. fibro), and SMC. The intensity of the blue colour and the size of the squares are proportional to entry values. j, Matrix layout for intersections of ApoE4 DEGs shared across and specific to each cell type. Circles in the matrix indicate sets that are part of the intersection, showing that most DEGs are cell-type-specific. k, Immunohistochemical validation of the predicted upregulated anti-inflammatory DEG SLC39A10 in venous BECs of ApoE4 carriers. Scale bars, 50 μm (n = 4 ApoE3/3 and ApoE4 carriers, nested two-sided t-test; mean +/− s.e.m.). l, Among patients with both hippocampus and superior frontal cortex profiled (n = 4 NCI and n = 4 AD), quantification of the relative abundance of major vascular cell types (NCI hippocampus set as reference, unpaired two-sided t-test; mean +/− s.e.m.). *BEC P = 0.0260, **BEC P = 0.0023, *Pericyte P (left) = 0.0357, *Pericyte P (mid) = 0.0237, **Pericyte P = 0.0077, **SMC P = 0.0075, *Fibroblast P = 0.0109, *Astrocyte P = 0.0357. m, As in (l), but comparison of the number of DEGs between brain regions for each cerebrovascular cell type. Analysis done separately for NCI and AD samples (n = 7 cell types, unpaired two-sided t-test; mean +/− s.e.m.).
Extended Data Fig. 9 Re-evaluation and characterization of top AD GWAS genes expressed in the human brain vasculature.
a, Heterogeneous expression of AD GWAS genes across T- and M-pericyte subtypes. b, RNA-seq data of the predicted T cell-specific AD GWAS genes EPHA1 and ABCA7 in an independent dataset81, corroborating minimal expression across resident/ parenchymal brain cells. c, Immunohistochemical confirmation of vascular localization of proteins encoded by 12 top AD GWAS genes from a. Scale bars, 25 μm. Arrowheads in APOE point to signal around larger-diameter vessels, consistent with predicted SMC expression. Image credit: Human Protein Atlas25,74 (http://www.proteinatlas.org). d, Heat map comparing expression patterns of top AD GWAS genes in the hippocampus and superior frontal cortex: e.g., several microglia-expressed GWAS genes like APOE, MS4A4A, and TREM2 are more highly expressed in hippocampal compared to cortical microglia/ macrophages. e, GWAS genes found to be expressed specifically in microglia among cells captured using the conventional nuclei isolation process (from Grubman et al. 2019)17 are also expressed in vascular cells (asterisks). f, Summary of AD GWAS genes enriched in microglia and vascular cells mediating common pathways in protein clearance and inflammation. Mouse and human superscripts denote whether expression has been confirmed in that species for a given gene. Proposed model is described in Discussion.
Extended Data Fig. 10 Brain vascular and perivascular expression of AD and AD-related GWAS genes.
a, Expression of AD and AD-related GWAS risk genes (from Grubman et al. 2019)17 across human vascular cells. b, Enriched biological pathways amongst AD and AD-related trait GWAS genes expressed in each cell type. c, For each cell type, the top 10 most specifically expressed AD and AD-related trait GWAS genes.
Supplementary information
Supplementary Table 1
Patient samples.
Supplementary Table 2
Cell type markers.
Supplementary Table 3
Brain region enriched genes.
Supplementary Table 4
Mouse versus human gene expression.
Supplementary Table 5
Vascular cell subtype markers.
Supplementary Table 6
Vascular Alzheimer’s disease differentially expressed genes.
Supplementary Table 7
Vascular ApoE4 carrier disease differentially expressed genes.
Supplementary Table 8
Expression of top Alzheimer’s disease and related GWAS genes.
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Yang, A.C., Vest, R.T., Kern, F. et al. A human brain vascular atlas reveals diverse mediators of Alzheimer’s risk. Nature 603, 885–892 (2022). https://doi.org/10.1038/s41586-021-04369-3
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DOI: https://doi.org/10.1038/s41586-021-04369-3
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Molecular Neurodegeneration (2025)
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Cell-specific transcriptional signatures of vascular cells in Alzheimer’s disease: perspectives, pathways, and therapeutic directions
Molecular Neurodegeneration (2025)
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Eppur si muove: the dynamic brain pericyte
Fluids and Barriers of the CNS (2025)
Fran Krstanovic
Could this novel VINE-seq protocol be applicable to mice? As I understood correctly, I the paper you did not use VINE-seq for mice-sc, but rather whole cell isolation.
I understand the size limitations between human and mice vessels. Could this potentially be overcome with grouping more than one brain per sample to get more vascular nuclei?