Authors K. ARNAUTS (1), B. VERSTOCKT (2), M. VANCAMELBEKE (2), S. VERMEIRE (2), C. VERFAILLIE (3), M. FERRANTE (2) / [1] Translational Research Center for Gastrointestinal Disorders (TARGID), KULeuven, Leuven, Belgium, Department of Chronic Diseases, Metabolism and Ageing, [2] Translational Research Center for Gastrointestinal Disorders (TARGID), KULeuven, Leuven, Belgium, Department of Chronic Diseases, Metabolism & Ageing, [3] Stem Cell Institute (SCIL),KU Leuven, Leuven, Belgium, Department of Development and Regeneration Introduction Patient-derived intestinal organoids provide an excellent tool to unravel the multifactorial 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 if organoids retain the inflammatory character of their origin. Aim To address if organoids maintain the inflammatory character, 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 in both inflamed and non-inflamed segments were obtained during endoscopy from 8 patients with active UC (endoscopic Mayo sub-score of ≥2) with an accessible border of inflammation. Crypts were isolated from fresh biopsies 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. All p-values are adjusted for multiple testing (False Discovery Rate). Results Biopsies and crypts from inflamed regions showed separate clustering on principal component analysis (PCA) 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 to biopsies and crypts of non-inflamed regions. 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 to inflamed crypts. After 4 weeks in culture, organoids derived from inflamed and non-inflamed regions were indistinguishable in PCA clustering. Expression levels of inflammatory signalling pathways were not significantly different in organoids derived from inflamed and non-inflamed biopsies after 4 weeks in culture. Conclusions We conclude that organoids lose their inflammatory transcriptional signature, present in biopsies and isolated crypts, over time 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 hypothesize 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.
