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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.

Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture

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.

Inflammatory bowel disease (IBD) consists of two major idiopathic gastrointestinal diseases: ulcerative colitis and Crohn's disease. Although a significant advance has been achieved in the treatment of IBD, there remains a particular population of patients that are refractory to the conventional treatments, including the biologic agents. Studies have revealed the importance of “mucosal healing” in improving the prognosis of those difficult-to-treat patients, which indicates the proper and complete regeneration of the damaged intestinal tissue. In this regard, organoid-based regenerative medicine may have the potential to dramatically promote the achievement of mucosal healing in refractory IBD patients, and thereby improve their long-term prognosis as well. So far, studies have shown that hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) may have some beneficial effect on IBD patients through their transplantation or transfusion. Recent advance in stem cell biology has added intestinal stem cells (ISCs) as a new player in this field. It has been shown that ISCs can be grown in vitro as organoids and that those ex-vivo cultured organoids can be employed as donor cells for transplantation studies. Further studies using mice colitis models have shown that ex-vivo cultured organoids can engraft onto the colitic ulcers and reconstruct the crypt-villus structures. Such transplantation of organoids may not only facilitate the regeneration of the refractory ulcers that may persist in IBD patients but may also reduce the risk of developing colitis-associated cancers. Endoscopy-assisted transplantation of organoids may, therefore, become one of the alternative therapies for refractory IBD patients. Keywords: Intestinal stem cells, Organoids, Ulcerative colitis, Crohn's disease, Organoid medicine

Background and aims The intestinal epithelium of inflammatory bowel disease [IBD] patients is exposed to various pro-inflammatory cytokines, most notably tumour necrosis factor alpha [TNF-α]. We have previously shown that the Notch signalling pathway is also upregulated in such an epithelium, contributing to intestinal epithelial cell [IEC] proliferation and regeneration. We aimed to reproduce such environment in vitro and explore the gene regulation involved. Methods Human IEC cell lines or patient-derived organoids were used to analyse Notch- and TNF-α-dependent gene expression. Immunohistochemistry was performed to analyse expression of ubiquitin D [UBD] in various patient-derived intestinal tissues. Results In human IEC cell lines, we found that Notch signalling and TNF-α-induced NFκB signalling are reciprocally regulated to promote expression of a specific gene subset. Global gene expression analysis identified UBD to be one of the most highly upregulated genes, due to synergy of Notch and TNF-α. The synergistic expression of UBD was regulated at the transcriptional level, whereas the UBD protein had an extremely short half-life due to post-translational, proteasomal degradation. In uninflamed intestinal tissues from IBD patients, UBD expression was limited to IECs residing at the crypt bottom. In contrast, UBD-expressing IECs were seen throughout the crypt in inflamed tissues, indicating substantial induction by the local inflammatory environment. Analysis using patient-derived organoids consistently confirmed conserved Notch- and TNF-α-dependent expression of UBD. Notably, post-infliximab [IFX] downregulation of UBD reflected favourable outcome in IBD patients. Conclusion We propose that UBD is a novel inflammatory-phase protein expressed in IECs, with a highly rapid responsiveness to anti-TNF-α treatment.

Notch signaling is activated in the intestinal epithelial cells (IECs) of patients with inflammatory bowel disease (IBD), and contributes to mucosal regeneration. Our previous study indicated that TNF-α and Notch signaling may synergistically promote the expression of the intestinal stem cell (ISC) marker OLFM4 in human IECs. In the present study, we investigated the gene regulation and function of OLFM4 in human IEC lines. We confirmed that TNF-α and Notch synergistically upregulate the mRNA expression of OLFM4. Luciferase reporter assay showed that OLFM4 transcription is regulated by the synergy of TNF-α and Notch. At the protein level, synergy between TNF-α and Notch promoted cytoplasmic accumulation of OLFM4, which has potential anti-apoptotic properties in human IECs. Analysis of patient-derived tissues and organoids consistently showed cytoplasmic accumulation of OLFM4 in response to NF-κB and Notch activation. Cytoplasmic accumulation of OLFM4 in human IECs is tightly regulated by Notch and TNF-α in synergy. Such cytoplasmic accumulation of OLFM4 may have a cell-protective role in the inflamed mucosa of patients with IBD., Highlights • Notch and TNF-α signaling is important in IECs of patients with IBD. • Notch and TNF-α signaling promotes the cytoplasmic accumulation of OLFM4. • OLFM4 accumulation may have anti-apoptotic properties. • OLFM4 could protect against mucosal inflammation in IBD.

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.