Fig. 2: Composition of intestinal stroma based on sc-RNAseq validated across colon segments and intestinal layers using 25-color flow cytometry.

a scRNA-seq design and validation analysis. b Clustering of scRNA-seq data showing clusters 0–10 in UMAP space. c Heatmap of top 50 differentially expressed genes among clusters 0–10. d Violin plots showing differential PDPN expression in clusters 0–10 from non-inflamed and inflamed samples. e Heatmap illustrating pathway significance, calculated as −log10(p-value) using right-tailed Fisher’s Exact test, from IPA analysis on genes upregulated in inflamed compared to non-inflamed samples, assessed in each cluster independently; ^#Hepatic Fibrosis/Hepatic Stellate Cell Activation, ^##Tumor Microenvironment Pathway. f Dot plot of gene expression, selected for validation on protein level, non-inflamed; for inflamed, see Supplementary Fig. 3a. g Gating strategy of stoma populations using FACS; gated populations shown in UMAP space see Supplementary Fig. 3b for more details. h Percentage of total stromal cells in 81–97 biologically independent non-inflamed and inflamed biopsies from pediatric patients undergoing first diagnostic colonoscopy. i Heatmap illustrating percentage of total stromal cells across the segments of the colon in matched samples from treatment naïve pediatric patients with no inflammation in the colon (Non-inf) or total colitis (Inf). j Percentage of total stromal cells across dissected intestinal layers from three adult IBD patients undergoing gut resection. k Representative flow cytometry plot of Fibro_4 subset across intestinal layers. l Levels of stromal activation markers across stromal subsets in non-inflamed and inflamed colon biopsies from children undergoing first diagnostic colonoscopy. m MFI levels of stroma activation markers in non-inflamed and inflamed colon biopsies from children undergoing first diagnostic colonoscopy, comparing levels across non-immune (epithelial, stromal) and immune cells (upper panels); and PDPN levels across immune cells only (lower panel), for gating strategy see Supplementary Fig. 3e. n Significant (p < 0.05) Spearman correlations between PDPN levels (MFI) in Fibro_0_2 and immune cell frequencies (two-tailed p-value). Mean with SE in (h) and (j); Mann–Whitney test was used to compare differences between two groups (two-tailed p value) (h, l), significance level for (l): ns > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, for p values see Supplementary Fig. 3c.