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OP11 Organoids derived from inflamed intestinal biopsies of patients with ulcerative colitis lose their inflammatory phenotype during ex vivo culture

K. Arnauts*1,2, B. Verstockt1,3, M. Vancamelbeke1, S. Vermeire1,3, C. Verfaillie2, M. Ferrante1,3

1KU Leuven, Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), Leuven, Belgium, 2KU Leuven, Department of Development and Regeneration, Leuven, Belgium, 3KU Leuven, Department of Gastroenterology and Hepatology, Leuven, Belgium

Background

Patient-derived intestinal organoids provide an excellent tool to unravel the multi-factorial mechanisms underlying ulcerative colitis (UC). Organoids develop from stem cell-containing intestinal crypts and recapitulate many features of the source tissue. However, it remains unclear whether ex vivo organoids retain the inflammatory character of their origin. To address this, we isolated crypts from both inflamed and non-inflamed regions of the colon, created organoids, and compared the transcriptome of whole biopsies, crypts and ex vivo cultured organoids.

Methods

Fresh biopsies from both inflamed and non-inflamed segments were obtained during endoscopy from eight patients with active UC (endoscopic Mayo sub-score of ≥2) and an accessible border of inflammation. Crypts were isolated and cultured as organoids for 4 weeks with weekly mechanical splitting. RNA was extracted from biopsies, crypts, and 1- and 4-week-old organoids. RNA sequencing was performed by Lexogen QuantSeq for Illumina. Differential gene expression and pathways were studied through DESeq2 and Ingenuity Pathway Analysis (FDR < 0.05).

Results

Biopsies and crypts from inflamed regions showed separate clustering on principal component analysis (PCA, Figure) and significantly higher activation of inflammatory pathways, including antigen presentation (p < 0.01 and p < 0.001), interferon signalling (p < 0.05 and p < 0.001) and granulocyte adhesion (both p < 0.001) compared with non-inflamed biopsies and crypts. However, organoids derived from inflamed crypts lost part of their inflammatory character after 1 week in culture. Several inflammatory markers (IFN-γ [p = 0.01], IL-1β [p < 0.001], JAK1 [p < 0.001]), and pathways involved in antigen presentation (p < 0.005) and interferon signalling (p < 0.001) were significantly decreased after 1 week ex vivo culture compared with inflamed crypts. After 4 weeks in culture, organoids derived from inflamed and non-inflamed regions were indistinguishable in PCA clustering, and expression levels of inflammatory signalling pathways were not significant.

Conclusion

We conclude that ex vivo organoids lose their inflammatory transcriptional signature in culture. After 4 weeks in culture, organoids derived from inflamed and non-inflamed biopsies were no longer distinguishable. Therefore, it is not essential to obtain biopsies from inflamed regions to culture organoids from UC patients. We hypothesise that to mimic the inflammatory phenotype and create a physiological representative model, inflammatory components, and/or immune cells should be added to the ex vivo culture system.

Principal component analysis (PCA) shows separate clustering of biopsies and crypts from inflamed regions vs. non-inflamed regions. After 1 and 4 weeks, organoids of inflamed and non-inflamed origin cluster together and are no longer distinguishable.