Search in the Abstract Database

Search Abstracts 2016

* = Presenting author

P084 Intestinal organoids derived from patients with inflammatory bowel disease show unaltered transcriptional profiles when compared with healthy controls

M. Noben*1, 2, N. Hendriks1, 2, G. Van Assche1, S. Vermeire1, C. Verfaillie2, M. Ferrante1

1KU Leuven, Translational Research Centre for Gastrointestinal Disorders (Targid), Leuven, Belgium, 2KU Leuven, Stem Cell Institute Leuven, Leuven, Belgium

Background

Various mechanisms contribute to the pathogenesis of inflammatory bowel diseases (IBD), including microbial dysbiosis and defects in epithelial barrier, Paneth cell, or goblet cell function. The epithelium is constantly renewed by intestinal stem cells (ISCs) located at the bottom of the crypts. The ex-vivo ISC-containing organoids may be a suitable model to investigate IBD pathogenesis.

Methods

We evaluated if the organoid forming capacity of colonic crypts, as well as organoid transcriptional profiles, from IBD patients differ from that of healthy controls (HC). Colonic mucosal biopsies from 10 HC, 11 ulcerative colitis (UC) patients, and 11 Crohn’s disease (CD) patients were used to culture organoids as previously described. After 2 passages, organoids were differentiated for 4 days by withdrawal of Wnt3a, nicotinamide, and p38-inhibitor from the medium. RNA and histology samples were processed and analysed using qPCR and immunohistochemistry (IHC) for ISCs and differentiated cell types, as well as proliferation and apoptosis (Ki-67 and cleaved caspase 3 staining).

We also investigated how inflammation (IL8, TNFα , and CXCL3, amongst others) evolved when organoids were established from macroscopic inflamed areas. We included 3 patients per group (HC, UC, and CD) and cultured organoids from crypts derived from both macroscopic inflamed and non-inflamed tissue. RNA was isolated from fresh biopsies and 1-week-old organoids derived from the same tissue.

Results

There was no difference in initial organoid forming capacity between crypts of controls and IBD patients (HC 77%, UC 79%, CD 80% p = 0.51). Additionally, no changes in expression of the ISC-marker Lgr5 were found. The expression of Hath1, a positive regulator of goblet cell differentiation, was decreased in UC and CD compared with HC, both in differentiated and undifferentiated conditions. In addition, chromogranin A expression in UC patients was decreased under differentiation, whereas in CD, Muc2 expression was decreased.

Table 1 P-values from organoid differentiation experiment

Comparison colonic organoidsGeneP-valueAverage ΔCt value
UC vs HC undifferentiatedHath10.01479.48 vs 5.46
CD vs HC undifferentiatedHath10.01209.57 vs 5.46
UC vs CD undifferentiatedHath10.53629.48 vs 9.57
UC vs HC differentiatedHath10.02428.56 vs 3.53
CD vs HC differentiatedHath10.02407.98 vs 3.53
UC vs CD differentiatedHath10.96238.56 vs 7.98
UC vs HC differentiatedCHGA0.04029.49 vs 6.33
CD vs HC differentiatedMuc20.04824.84 vs 3.87

Moreover, preliminary data showed an unexpected increase in IL8 expression when both macroscopic and non-inflamed tissue was cultured as organoids, whereas expression of CXCL3 was down regulated.

Conclusion

Our data show that intestinal crypts isolated from IBD patients form organoids as efficient as crypts from healthy controls. Gene expression of markers of stemness and differentiation showed only subtle differences, and the biological implications remain to be clarified via immunohistochemistry. We are validating these results by expanding the cohort and testing more 
genes.

References

[1] Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterol 2011;141(5):1762–72.

[2] Sat T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459(7244):262–65.