Background Gastrointestinal diseases often involve cellular damage, degeneration or dysfunction in the tract, frequently requiring surgical interventions risking complications and lowered quality of life. Regenerative medicine holds great promise in improving patient care and providing novel treatment options for previously irreparable and untreatable tissues. Despite the clinical potential of intestinal organoids as a resource for regenerative cell therapy and bioengineering, the lack of clinical-grade cultures has hampered further development. Moreover, strategies to efficiently and reliably expand clinical-grade cultures at the scale required for application is limited. Methods A GMP-compliant protocol was developed to generate patient-derived colonic organoids from endoscopic biopsies. Clinical-grade colonic organoids cultured and expanded in Type-I collagen were compared to conventional Matrigel cultured organoids. To improve the culture-, cost-, and time-efficiency of culture expansion, several strategies were developed including organoid area-based passaging, one well plate culture, and the incorporation of Wnt activating peptide, PG-008. Conventional recombinant WNT3A culture was compared to the peptide PG-008 culture using single cell RNA sequencing. Results Clinical-grade collagen cultured organoids exhibited similar culture efficiency to Matrigel. Organoid establishment rate from 60 patients using the GMP-compliant protocol was 82%. The incorporation of PG-008 significantly enhanced organoid growth and stabilized patient-patient variability through intestinal stem cell (ISC) enrichment. Single cell RNA sequencing revealed that PG-008 resulted in remarkably pure culture consisting of ISCs and transit amplifying cells, suitable for rapid and consistent expansion. Intriguingly, our GMP-grade colonic organoids contained LGR5(+) ISCs, and injury-induced LGR5(-) regenerative ISCs, both enriched in peptide culture. Conclusions Our study establishes clinical-grade colonic organoids for further application, including autologous transplantations and bioengineering. Further, collagen cultured organoids can be a valuable model facilitating in vitro investigations into regenerative stem cell induction and intestinal regeneration.

The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of VPS35L, VPS26C and VPS29, together with the CCC complex comprising CCDC22, CCDC93, and COMMD proteins, plays a crucial role in this process. The precise mechanisms underlying Retriever assembly and its interaction with CCC have remained elusive. Here, we present the first high-resolution structure of Retriever determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog, Retromer. By combining AlphaFold predictions and biochemical, cellular, and proteomic analyses, we further elucidate the structural organization of the entire Retriever-CCC complex and uncover how cancer-associated mutations disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with Retriever-CCC-mediated endosomal recycling.

Background A combination of endoscopic and histological evaluation is important in the management of patients with ulcerative colitis. We aimed to adapt our previous deep neural network system (deep neural ulcerative colitis [DNUC]) to full video colonoscopy and evaluate its validity in the real-time detection of histological mucosal inflammation. Methods In this multicentre, cross-sectional study, we prospectively enrolled consecutive patients (>= 15 years) with ulcerative colitis who had an indication for colonoscopy at five hospitals in Japan. Patients in clinical remission were randomly assigned (1:2) to study 1 and study 2. Those with clinically active disease were assigned to study 2 only. Study 1 assessed the validity of real-time histological assessment using DNUC and study 2 validated the consistency of endoscopic scoring between DNUC and experts. The primary endpoint for study 1 was comparison of the results judged by DNUC (healing or active) with biopsy specimens evaluated by pathologists. In study 2, the primary endpoint was the ability of DNUC to determine the Ulcerative Colitis Endoscopic Index of Severity score compared with centrally evaluated scoring by inflammatory bowel disease endoscopy experts. Findings From April 1, 2020, to March 31, 2021, 770 patients (180 in study 1 and 590 in study 2) were enrolled. Using real-time histological evaluation, DNUC was able to evaluate the presence or absence of histological inflammation in 729 (81%) of 900 biopsy specimens. For predicting histological remission, the DNUC had a sensitivity of 97.9% (95% CI 97.0-98.5) and a specificity of 94.6% (91.1-96.9). Moreover, its positive predictive value was 98.6% (97.7-99.2) and negative predictive value was 92.1% (88.7-94.3). The intradass correlation coefficient between DNUC and experts for endoscopic scoring was 0.927 (95% CI 0.915-0.938). Interpretation DNUC provided consistently accurate endoscopic scoring and showed potential for reducing the number of biopsies required. This system is an objective and consistent application for video colonoscopy that has potential for use in various medical situations. Copyright (C) 2021 Elsevier Ltd. All rights reserved.

Several studies have reported a positive correlation between serum drug concentrations and endoscopic remission in patients with Crohn's disease. To examine the association between the concentrations of cytokine blockers (infliximab, adalimumab and ustekinumab) and endoscopic remission of small bowel lesions. This was a cross-sectional study conducted at a single tertiary referral centre. Patients with Crohn's disease who received maintenance cytokine blocker therapy were recruited from April 2018 to May 2020. We performed balloon-assisted enteroscopy and collected serum samples to measure drug concentrations. The primary endpoint was the relationship between the concentrations of cytokine blockers and endoscopic remission in the small bowel. We enrolled 143 patients, 66, 44 and 33 of whom were receiving infliximab, adalimumab and ustekinumab, respectively. Enteroscopic findings showed that the rate of endoscopic remission of small bowel lesions was significantly lower than that of colonic lesions ( P < 0.01). For each agent, the mean drug concentration in patients exhibiting endoscopic remission in the small bowel was higher than that observed in patients with endoscopic remission in the colon (but not in the small bowel) or with any active disease (either in the small bowel, colon or both). Patients with infliximab, adalimumab and ustekinumab concentrations >5, 14 and 4 µg/mL were nearly 5.3-, 9.4- and 14.7-times more likely to exhibit endoscopic remission of the small bowel, respectively. Cytokine blocker treatment was less efficacious for small bowel inflammation than colonic inflammation. Higher serum concentrations were needed to achieve endoscopic remission of small bowel lesions.

Copper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under lowor high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.

SummaryBackgroundMucosal healing is an important treatment target in patients with ulcerative colitis.AimsTo explore the optimal colonoscopic strategy to determine the risk for clinical relapse in patients with ulcerative colitis.MethodsWe enrolled 325 consecutive patients with ulcerative colitis in clinical and biochemical remission from April 2018 to March 2019. Five colonic segments were endoscopically and histologically assessed systematically. For endoscopic evaluation, we used three different modes of the Ulcerative Colitis Endoscopic Index of Severity (UCEIS): “original,” “worst affected,” and “pancolonic.” The Geboes score was used for histological evaluation. We prospectively followed up the patients and defined clinical relapse as the primary endpoint.ResultsWithin 1 year after colonoscopy, 18.2% of patients experienced a clinical relapse. Receiver operating characteristic curve analysis showed areas under the curve of 0.755, 0.817, and 0.852 for the “original,” “worst affected,” and “pancolonic” groups, respectively; hence, pancolonic UCEIS obtained the highest predictive value. Using the pancolonic UCEIS cutoff value of 3, Kaplan–Meier curve analysis showed that patients with endoscopic activity had a significantly lower relapse‐free rate than those with endoscopic remission (P < 0.01). Multivariate analysis demonstrated endoscopic (pancolonic UCEIS >3) and histological (Geboes >3.0) activities as independent risks for relapse (HR: 3.96 and 3.48, respectively). Combining pancolonic UCEIS ≤3 and Geboes score ≤3.0 to provide 1‐year relapse avoidance was 92.0% sensitive and 97.0% specific.ConclusionEvaluating disease remission by complete colonoscopy is relevant, and the combination of pancolonic endoscopic and histological evaluations may appropriately evaluate mucosal healing.

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.

Many diseases that affect modern humans fall in the category of complex diseases, thus called because they result from a combination of multiple aetiological and pathogenic factors. Regardless of the organ or system affected, complex diseases present major challenges in diagnosis, classification, and management. Current forms of therapy are usually applied in an indiscriminate fashion based on clinical information, but even the most advanced drugs only benefit a limited number of patients and to a variable and unpredictable degree. This 'one measure does not fit all' situation has spurred the notion that therapy for complex disease should be tailored to individual patients or groups of patients, giving rise to the notion of 'precision medicine' [PM]. Inflammatory bowel disease [IBD] is a prototypical complex disease where the need for PM has become increasingly clear. This prompted the European Crohn's and Colitis Organisation to focus the Seventh Scientific Workshop on this emerging theme. The articles in this special issue of the Journal address the various complementary aspects of PM in IBD, including what PM is; why it is needed and how it can be used; how PM can contribute to prediction and prevention of IBD; how IBD PM can aid in prognosis and improve response to therapy; and the challenges and future directions of PM in IBD. This first article of this series is structured on three simple concepts [what, why, and how] and addresses the definition of PM, discusses the rationale for the need of PM in IBD, and outlines the methodology required to implement PM in IBD in a correct and clinically meaningful way.

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.

Paneth cells (PCs) are small intestinal epithelial cells that secrete antimicrobial peptides and growth factors, such as Wnt ligands. Intriguingly, the context in which PC-derived Wnt secretion is relevant in vivo remains unknown as intestinal epithelial ablation of Wnt does not affect homeostatic proliferation or restitution after irradiation injury. Considering the importance of growth factors in tumor development, we explored here the role of PCs in intestinal carcinogenesis using a genetic model of PC depletion through conditional expression of diphtheria toxin-α subunit. PC depletion in Apc Min mice impaired adenoma development in the small intestine and led to decreased Wnt3 expression in small bowel adenomas. To determine if PC-derived Wnt3 was required for adenoma development, we examined tumor formation after PC-specific ablation of Wnt3 We found that this was sufficient to decrease small intestinal adenoma formation; moreover, organoids derived from these tumors displayed slower growth capacity. Overall, we report that PC-derived Wnt3 is required to sustain early tumorigenesis in the small bowel and identify a clear role for PC-derived Wnt production in intestinal pathology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 115 UC who had started TOF treatments between May and November 2018. The observation periods lasted up to 52 weeks after TOF administration. Patients were refractory to multiple treatments, such as corticosteroids, biologic agents, and a calcineurin inhibitor. TOF was initiated at 20 mg a day and decreasing to 10 mg a day after 8 weeks or more. All immunesuppressive agents, such as corticosteroids, immunomodulators, biologic agents, and calcineurin inhibitors, were withdrawn before starting TOF treatment. Disease activity was evaluated using the partial Mayo score (pMS), and laboratory data were collected at 4, 8, 12, 24, and 52 weeks after TOF administration. Our primary outcome was the clinical remission rate at 52 weeks; we also analyzed treatment efficacy and adverse events. We defined remission as pMS ≤ 2 without individual subscores > 1, response as a decrease of pMS by at least 2, and meeting one of the following conditions: (1) a decrease of the bleeding subscore by at least 1; (2) bleeding subscore < 1; or (3) above-defined remission induced. Failure was defined as TOF discontinuation due to symptom recurrence in patients in remission, nonresponse to TOF treatment, and serious adverse events. Differences in medians between the groups were compared using a non-parametric (Mann-Whitney), and comparisons between categorical variables were performed using chi-square test. TOF discontinuation-free survival was calculated. Kaplan-Meier curves were drawn for each subgroup and compared with log-rank (Mantel-Cox) test. SPSS version 26 (IBM Corp., Armonk, NY, USA) was used for statistical analysis, and the significant threshold was 0.05. pISSN 1598-9100 • eISSN 2288-1956 https://doi.org/10.5217/ir.2020.00030 Intest Res 2021;19(1):115-118

In this protocol, we describe our methods to isolate crypts from patients' biopsy samples and to culture human intestinal stem cells as it’s called “organoid.” Beyond that, we describe how to dissociate organoids cells into single cells for single-cell analysis as a further application. This protocol should provide investigators sufficient tools to generate human organoids from biopsy samples and to accomplish a stable in-vitro assay system.

BACKGROUND & AIMS: There are intra- and interobserver variations in endoscopic assessment of ulcerative colitis (UC) and biopsies are often collected for histologic evaluation. We sought to develop a deep neural network system for consistent, objective, and real-time analysis of endoscopic images from patients with UC. METHODS: We constructed the deep neural network for evaluation of UC (DNUC) algorithm using 40,758 images of colonoscopies and 6885 biopsy results from 2012 patients with UC who underwent colonoscopy from January 2014 through March 2018 at a single center in Japan (the training set). We validated the accuracy of the DNUC algorithm in a prospective study of 875 patients with UC who underwent colonoscopy from April 2018 through April 2019, with 4187 endoscopic images and 4104 biopsy specimens. Endoscopic remission was defined as a UC endoscopic index of severity score of 0; histologic remission was defined as a Geboes score of 3 points or less. RESULTS: In the prospective study, the DNUC identified patients with endoscopic remission with 90.1% accuracy (95% confidence interval [CI] 89.2%-90.9%) and a kappa coefficient of 0.798 (95% CI 0.780-0.814), using findings reported by endoscopists as the reference standard. The intraclass correlation coefficient between the DNUC and the endoscopists for UC endoscopic index of severity scoring was 0.917 (95% CI 0.911-0.921). The DNUC identified patients in histologic remission with 92.9% accuracy (95% CI 92.1%93.7%); the kappa coefficient between the DNUC and the biopsy result was 0.859 (95% CI 0.841-0.875). CONCLUSIONS: We developed a deep neural network for evaluation of endoscopic images from patients with UC that identified those in endoscopic remission with 90.1% accuracy and histologic remission with 92.9% accuracy. The DNUC can therefore identify patients in remission without the need for mucosal biopsy collection and analysis.

Chronic diarrhea is one of the major symptoms in gastroenterology. However, this may be caused by pathologic conditions for which the diagnosis is critical. Villous atrophy, as an endoscopic lesion, accompanied by chronic diarrhea can occasionally be observed in the patients with inflammatory diseases of the gastrointestinal (GI) tract. Herein, we present a case with persistent diarrhea accompanied by intestinal wall thickening without any other significant endoscopic features other than villous atrophy in the jejunum and the ileum, where we diagnosed as an indolent T cell lymphoproliferative disorder (T-LPD) of the GI tract, defined in the 2016–2017 revised World Health Organization classification, via single-balloon enteroscopy (SBE). Interestingly, we found the same lymphocyte infiltration from the distal third portion of the duodenum, where gastroscopy could not reach, via SBE, even though no endoscopic findings were observed such as villous atrophy. Since infiltrating cells in the intestinal tissues were CCR4 + , mogamulizumab was administered with resulting durable symptomatic remission for more than 2 years. Patients with persistent diarrhea may have serious small intestinal disorder including not only chronic inflammatory diseases but also lymphoid neoplasmic conditions including T-LPD of GI tract.

Inflammatory bowel disease (IBD) is an idiopathic, multi-etiological disease characterized by inflammation and mucosal destruction of the gastrointestinal tract. Despite the remarkable advance in immunomodulating therapies, there still remains a certain population of patients who are refractory to conventional as well as biologic therapies and fail to achieve mucosal healing. To improve the prognosis of those patients, at least 2 types of stem cells have been tested for their potential therapeutic use. Transplantation of hematopoietic stem cells or mesenchymal stem cells have been tested in several clinical studies, but their beneficial effect still remains controversial. In this review, we would like to overview the recent clinical challenges of stem cell-based therapies in IBD and also introduce our new therapeutic plan of intestinal stem cell transplantation for IBD, based on our ex vivo intestinal organoid culture technique.

Background and Aim A missense variant of the nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene (R139C) predisposes Asian patients with inflammatory bowel disease (IBD) to thiopurine-induced leukopenia. This study evaluates the long-term effect of NUDT15 R139C heterozygosity on hematological parameters during thiopurine administration. Methods We enrolled 83 Japanese IBD patients who were on anti-tumor necrosis factor-alpha agents and had used thiopurine. NUDT15 R139C was genotyped by polymerase chain reaction. We retrospectively reviewed patient clinical charts to collect data on white blood cell (WBC) count, mean corpuscular volume (MCV), hemoglobin, and platelet count during the 24 months following thiopurine initiation. Results The included patients had either Crohn's disease (54; 65.1%) or ulcerative colitis (29; 34.9%). Genotyping of NUDT15 R139C identified 62 patients (74.7%) of genotype C/C and 21 (25.3%) of genotype C/T. The median dose of thiopurine was lower in the C/T group than in the C/C group after starting thiopurine. At 6 months, the mean WBC count of the C/T group became significantly lower than that of the C/C group (P = 0.008) and remained lower through the 24 months. The C/T group developed grade 2-4 leukopenia by 6 months, which persisted through 12-24 months. The mean MCV in the C/T group became higher than that of the C/C group after 3 months. Conclusions NUDT15 R139C heterozygosity affected the WBC count and MCV for 24 months after thiopurine administration. Our results indicate that careful monitoring of leukopenia and dose adjustment are necessary throughout treatment in IBD patients heterozygous for the NUDT15 R139C.

BACKGROUND & AIMS: It is important to objectively assess Crohn's disease (CD) activity in patients treated with antibodies against tumor necrosis factor (anti-TNF). Detection of healing by endoscopy (endoscopic healing) associates with patient outcome, based on evidence from studies of ileocolonoscopy. We assessed endoscopic healing after treatment, based on findings from balloon-assisted enteroscopy (BAE), in patients with CD. METHODS: We performed a post-hoc analysis of data from a clinical trial from 116 patients with CD (46 with ileal and 70 with ileocolonic type) who received induction and then maintenance therapy with anti-TNF agents from January 2013 through March 2018 at a single center in Japan. We compared findings from BAE before induction therapy and then again during maintenance therapy (median 13 months later). Endoscopic healing was defined as the modified simple endoscopic score for CD below 5. We also collected data on previous treatments, makers of inflammation, and disease type. RESULTS: Before treatment, small bowel ulcerations were present in 114 patients (98%); 42 patients (60%) with ileocolonic disease had colon ulcerations. During maintenance therapy, 41/114 patients (36%) had small bowel endoscopic healing; all the patients with small bowel endoscopic healing also had colonic endoscopic healing. Colonic endoscopic healing was observed in 33/42 patients (79%). The proportion of patients with small bowel endoscopic healing was significantly lower than that of colonic endoscopic healing (P <.001). Among all patients, failure to achieve small bowel endoscopic healing was significantly associated with structuring or penetrating disease (P =.014), lack of concomitant treatment with immunomodulators (P =.015), and having received previous treatment with an anti-TNF agents (P =.018). CONCLUSIONS: In a post-hoc analysis of patients with CD treated with anti-TNF agents, we found small bowel ulcerations, detected by BAE, to be more difficult to heal than colon ulcerations.