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Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-β. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-β. Surprisingly, addition of TGF-β induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-β significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.

Background Intestinal stem cells (ISCs) play indispensable roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn’s disease (CD) affects properties of resident small intestinal stem cells remain uncertain. Methods CD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay. Results ISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCD-SIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISC-cluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs. Conclusions aCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD. Electronic supplementary material The online version of this article (10.1007/s00535-018-1437-3) contains supplementary material, which is available to authorized users.