Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients

Abstract

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation

“…To generate human colonic organoids, fresh colonic tissues from control patients ( n = 13) were harvested and colon crypts were isolated and cultured in a three-dimensional Matrigel matrix for 10–12 days as previously described. 42 Organoids were generated in 48 wells in Matrigel plus culture medium (50% Wnt3a-conditioned medium (supernatant from L-Wnt3a cells), 50% advanced DMEM/F12, 100× GlutaMAX-CTS, 100× HEPES, serum-free B27, N2, N-acetylcysteine, nicotinamide, recombinant human epithelial growth factor, human Noggin, human R-spondin, gastrin, SB202190, LY2157299, and PGE2. The culture medium was changed every 2–3 days (without NAC and LY2157599).…”

Intestinal organoids in farm animals

“…In our hands, we observed a rapid viability loss of organoids cultured in suspension (< 3 days) which restricts the use of this culture condition to short-term experimental treatments. Alternatively, incubation of human intestinal organoids with an inflammatory cocktail composed of tumor necrosis factor-alpha, interleukin-6 and interleukin-1 induced the inversion of organoid polarity [ 35 ]. This technique has not been applied yet to reverse epithelial polarity in farm animal organoids.…”

Gastrointestinal epithelial innate immunity—regionalization and organoids as new model

“…Characterization of organoids derived from patients with IBD revealed a phenotype with decreased size and budding capacity, increased rate of cell death, luminal debris and partially inverted polarization of epithelial cells [106]. Global comparison of organoids from UC or CD patients and healthy controls showed that transcriptional and methylation differences seen in the intestinal epithelium were maintained in vitro [102,103,107].…”

“…This was especially the case when organoids were generated from sites of severe inflammation [105]. A decrease in junctional proteins could also be induced in organoids from healthy donors by application of the proinflammatory cytokine TNF-α and/or IFN-γ [106,111]. However, the fixed pattern of changes of junctional proteins in organoids from patients with IBD was observed only on the protein level but not on the mRNA level [105].…”