Fig. 1: uPAR⁺ cells accumulate in aging in murine and human intestines.

a, Surface uPAR expression as determined by flow cytometry on isolated intestinal crypts from young (3 months) and old (20 months) mice (n = 3 per group). b, Percentage of uPAR⁺ cells that are either EpCAM⁺ CD45⁻ or EpCAM⁻ CD45⁺ as determined by flow cytometry on isolated intestinal crypts from old (20 months) mice (n = 3 per group). c, Surface uPAR expression in SPiDER-β-gal⁺ cells as determined by flow cytometry on isolated intestinal crypts from old (20 months) mice (n = 3 per group). d, Representative co-immunofluorescence of uPAR (red) and EdU (green) in the proximal jejunum of aged (20 months old mice) (n = 3 mice). White arrows signal uPAR⁺ EdU⁻ cells. e, Percentage of uPAR⁺ cells that are EdU⁺ or EdU⁻ in the proximal jejunum of aged (20 months) mice (n = 3 mice). f, Representative co-immunofluorescence of uPAR (red) and p21 (green) in the proximal jejunum of aged (20 months) mice (n = 3 mice). White arrows signal uPAR⁺ cells. g, Percentage of uPAR⁺ cells that are p21⁺ or p21⁻ in the proximal jejunum of aged (20 months) mice (n = 3 mice). h–k, uPAR⁺ and uPAR⁻ cells from isolated intestinal crypts of duodenum, jejunum and ileum (whole small intestine) from old (20 months) mice were FACS sorted and subjected to scRNA-seq (n = 4 mice per group pooled into two replicates per group). i, Uniform Manifold Approximation and Projection (UMAP) visualization of small intestinal cell types generated by 10X chromium protocol. Color scale indicates differences in density of cellular populations between uPAR⁺ and uPAR⁻ cells. j, Pathway analysis using enrichR comparing differentially expressed genes between uPAR⁺ versus uPAR⁻ cells in scRNA-seq data. Size scale represents number of genes in each ontology, and color scale represents degree of significance. k, UMAP visualization of small intestinal cell types generated by 10X chromium protocol. Color scale indicates log₂ fold change (log2FC) in senescence signature³⁴ between uPAR⁺ and uPAR⁻ cells. Right: quantification of the proportion of uPAR⁺ and uPAR⁻ cells contributing to the senescence signature. l–n, scRNA-seq of small intestinal non-immune cell types in the whole small intestine of young (25–30 years old) and old (65–70 years old) subjects generated by 10X chromium protocol³⁵ (n = 1 per group). m, Split-violin plot indicates the expression level of PLAUR in the ISC and epithelial lineage. Boxplots display median (center line) and interquartile range (box). n, Pathway analysis using enrichR comparing differentially expressed genes between non-immune PLAUR⁺ vs PLAUR⁻ cells in scRNA-seq data. Size scale represents number of genes in each ontology, and color scale represents degree of significance. o–q, scRNA-seq of small intestinal immune cell types in the whole small intestine of young (25–30 years old) and old (65–70 years old) subjects generated by 10X chromium protocol³⁵ (n = 1 per group). p, Split-violin plot indicates the expression level of PLAUR in B cells, myeloid cells and T cells. Boxplots display median (center line) and interquartile range (box). q, Pathway analysis using enrichR comparing differentially expressed genes between immune PLAUR⁺ versus PLAUR⁻ cells in scRNA-seq data. Size scale represents number of genes in each ontology, and color scale represents degree of significance. r, Multiplex immunofluorescence of uPAR, ki-67, p21, γH2A.X, cleaved caspase-3, CD45, CD31, E-cadherin and DAPI in human intestinal samples from subjects aged 51–91 years (n = 3 subjects). Green arrows highlight uPAR⁺ p21⁺ cells, pink arrows highlight uPAR⁺ γH2A.X⁺ cells. s, Percentage of cells in the tissues from t that are uPAR⁺ CD45⁺ uPAR⁺, CD68⁺uPAR⁺ or E-Cadherin⁺ uPAR⁺ (n = 3 subjects). t, Percentage of E-Cadherin⁺ uPAR⁺ from t that are Ki-67⁻, p21⁺, Ki-67⁻ and p21⁺, γH2A.X⁺, or Ki-67⁻, p21⁺ and γH2A.X⁺ (n = 3 subjects). Shown are results of one independent experiment (a–t). Data are mean ± standard error of the mean (s.e.m.) (a–c,e,g,s–t). Significance was determined using a two-tailed unpaired Student’s t-test (a–c,e,g), two-tailed Fischer’s exact test (j,n,q) or two-tailed Wilcoxon rank-sum test (*P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001 (m,p). Illustration was created with Biorender.com (h).

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