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. 2018 Oct 4;175(2):372–386.e17. doi: 10.1016/j.cell.2018.08.067

Figure 7.

Figure 7

Functional Attributes of Crypt Niche and IBD-Associated Mesenchymal Cells

(A) Epithelial characterization after in vitro co-culture with and without S2. S2 was isolated by fluorescence-activated cell sorting (FACS) for F3 (CD142). Crypts with (ii) and without (iii) F3+ stromal cells grown in culture containing Rspo1 and assessed for up to 10 days of culture. Representative images from day 4 and day 10 are shown. (i) Normal growth of human colon organoids without any stromal cells. Bar graph shows quantification of organoid complexity during the course of co-culture.

(B) (i) Violin plots from the scRNA-seq data showing IL-6 and TNFSF14 (LIGHT) upregulated by S4. (ii) Human colon organoids were treated with 100 ng/mL of either IL-6 or LIGHT. Confocal immunofluorescence images show EdU-labeled nuclei (red) and total nuclei stained with DAPI (blue). Epithelial proliferative capacity was assessed by quantification of the total numbers of EdU positive nuclei and DAPI-stained nuclei to calculate the fraction of proliferating cells in a section of interest. For each experiment, 15 random fields were quantified for each treatment. n = 3 independent experiments. ∗∗p < 0.0001, ∗p < 0.001 Mann-Whitney U test.

(C) Real-time qPCR measured stem cell markers (LGR5, OLFM4, AXIN2, NOTCH1, and ALDH1A1) and CDX2 gene expression after treatment of human colon organoids with IL-6 or LIGHT for 4 days in the presence of Wnt containing medium.

(D) Real-time qPCR measured stem cell marker (LGR5, OLFM4, AXIN2, ALDH1A1, MSI1, and SOX9) and differentiation marker (KRT20, MUC2, and CDX2) gene expression after treatment of human colon organoids with IL-6 or LIGHT for 4 days in the presence of Wnt containing medium, with subsequent Wnt withdrawal and treatment with IL-6 and LIGHT for another 4 days.

(E) OLFM4 gene expression from scRNA-seq of over 11,175 single cells isolated from healthy, non-inflamed and inflamed colonic biopsies (i), and gene expression from bulk RNA of inflamed and non-inflamed mucosa of IBD patients compared to healthy control samples.

(F) (i) Violin plots of relative gene expression of Lox and Loxl1 in DSS-induced colitis. (ii) Cumulative diarrhea score, blood score, and large bowel weight to length ratio of vehicle-only Ctrls versus BAPN-treated animals.

(G) Lipid peroxidation measured by malondialdehyde (MDA) plasma levels of vehicle-only and BAPN-treated animals.

Error bars represent the SEM.