Fig 2: Human Colonic Mesenchymal Heterogeneity in Health(A) Flow cytometry analysis of the indicated surface markers on colonic single-cell suspensions following removal of epithelial and hematopoietic cells by MACS. Column flow-through is shown in red, and column-retained fraction is in blue.(B) t-SNE plot of the healthy human colonic mesenchyme dataset. Single cells colored by cluster annotation.(C) Violin plots for pan-fibroblast marker genes vimentin (VIM) and collagen types 1 and 3 (COL1A2, COL3A1) across clusters.(D) Violin plots for high-ranked transcriptional regulators and marker genes sharing GO annotation for significantly enriched terms for (i) S1 subset, (ii) S2 subset, (iii) S3 subset, (iv) S4 subset, and (v) myofibroblasts. Crossbars indicate median expression.(E) Single-molecule ISH staining of healthy human colonic tissue showing distribution of S1 markers (ADAMDEC1, DCN, SLIT2, and CXCL12) (left) and S2 markers (F3 (CD142), WNT5A, HSD17B2, WNT5B, POSTN, BMP2, FRZB, BMP5) (right).(F) Identification of SOX6-ZEB2+/ZEB1-ZEB2+ S1 and SOX6+ZEB2-/ZEB1+ZEB2- S2 subsets in healthy human colon.(G) Single (left) and co-staining with CD45 (right) and F3/CD142 (S2), ZEB2 (S1), and SMAD7 (S3) by IHC in colonic sections. The lower far-right panel is a quadruple stain of all 4 markers.(H) Differential expression analysis between S2a and S2b reveals 302 differentially expressed genes.(I) t-SNE plots showing examples of genes differentially expressed between S2a and S2b.(J) GO enrichment terms for S2a and S2b.See also Figures S1–S3 and Tables S1–S4.

Fig 3: Predicted separation between EPDCs and Mesothelial epicardium with UPK3B, TM4SF1, and POSTN using a, pseudostain on integrated uniform manifold approximation and projection embeddings followed by immunocytochemical staining using antibodies.The UPK3B antibody did not label the epicardium clearly as a result of undefined technical difficulties. Lower-magnification imaging of b, immunofluorescence using antibodies for KRT19 and POSTN as one strategy for distinguishing between EPDCs and Mesothelial epicardium in fetal cardiac tissue. Images shown are representative of stains carried out on three sections with each combination on a single heart (a, BRC2281; b, BRC2375).

Fig 4: CYTOF Analysis of Key Mesenchymal Subset Markers Reveals Colitis-Associated Stromal Remodeling(A) CyTOF panel detected colonic mesenchymal populations. Stromal subsets are represented by indicated markers.(B) Heatmaps of selected markers on concatenated healthy and inflamed t-SNE plots representing key stromal subsets. Color maps by F3 (CD142), POSTN, IL-33, CCL19, BCL6, and PTGS2 shown.(C) Expansion of S4 in UC detected by scRNA-seq.(D) Histogram comparisons of CCL19 and TNFSF14 (LIGHT) levels in healthy versus inflamed colonic mesenchyme marks the emergence of S4.(E) t-SNE comparisons of healthy versus inflamed colonic mesenchyme. Clustering used the following parameters: F3/CD142, POSTN, PDGFRA, PDPN, BCL6, PTGS2, CD55, CCL19, CCL21, IL-33, LIGHT, CLU, FDCSP, and aSMA. Select markers representing S2 and S4 in healthy versus inflamed tissues shown.(F) Graphical summary of the most significantly changed markers in UC. Each dot represents one independent pair of healthy donor and patient samples.See also Figure S5.

Fig 5: EPDCs were found in sub-epicardium and myocardium.a, Spatial validation of this cluster as EPDCs was carried out using a pseudostain targeting strategy from UMAP of scRNA-seq data, with gene expression displayed in RGB color space and a constant blue channel value of 0.2 for heuristic DAPI simulation. b, Staining of fetal hearts using double stains for TM4SF1 and POSTN (bi), PRG4 and DCN (bii), KRT19 and POSTN (biii) and MSLN and POSTN (biv). Images shown are representative of stains carried out on three sections, with each combination on a single heart: BRC2281 (bi and bii) and BRC2375 (biii and biv). c, Epicardial clusters were mapped onto spatial transcriptomics data from a single fetal heart using the Cell2Location algorithm. 9w4d, 9 weeks, 4 days.