Akifumi Ootani

Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the γ-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein–coupled receptor-5–positive (Lgr5+) and B lymphoma moloney murine leukemia virus insertion region homolog-1–positive (Bmi1+) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5+ cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1+ ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1+ ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5+ ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (KrasG12D), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Clinical symptoms in endoscopic reflux esophagitis: evaluation in 8031 adult subjects.

This study aimed to evaluate the correlation between symptoms and endoscopic findings in reflux esophagitis. Subjects, 8031 persons without medication for gastrointestinal disease, were briefly asked about the presence of heartburn, dysphagia, odynophagia, and acid regurgitation by associated medical staff before endoscopy for assessment of esophagitis utilizing the Los Angeles Classification. Endoscopically, 1199 (14.9%) were classified as positive reflux esophagitis, and 2223 (27.7%) had heartburn, 1522 (19.0%) had dysphagia, 493 (6.1%) had odynophagia, and 1466 (18.3%) had acid regurgitation. Multivariate analysis indicated that the symptom most related to esophagitis was heartburn (odds ratio: 2.46), although ∼40% of subjects with grade C or D did not complain of heartburn. Regarding the other symptoms, less than 30% subjects with severe esophagitis complained of the symptoms and the odds ratio was ∼1. These results indicate that endoscopic esophagitis was not equivalent to any reflux symptoms from which subjects suffered in their daily lives.

Endoscopic closure of perforations caused by EMR in the stomach by application of metallic clips

BACKGROUND The number of complications associated with use of EMR for early-stage gastric cancer, including perforation, has increased with the increasing use of this procedure. Endoscopic clip application was performed in patients who sustained a perforation as a result of EMR for gastric neoplasm. PATIENTS AND METHODS Seven patients who underwent endoscopic application of metallic clips to close perforations were studied. The omental patch method was applied in one case with a large perforation. OBSERVATIONS In all patients, endoscopic clip application successfully closed the perforation of the stomach, which occurred after EMR. No patient required laparotomy. CONCLUSIONS The technique of endoscopic clip application might be useful for treatment of patients who sustain a perforation caused by EMR.

iNOS enhances rat intestinal apoptosis after ischemia-reperfusion.

The aim of this study was to demonstrate (i) the role of iNOS (inducible nitric oxide synthase) on apoptosis in the rat intestinal mucosa after ischemia-reperfusion, and (ii) the effect of iNOS on the release of cytochrome c from mitochondria. The superior mesenteric artery was occluded for 60 min and was followed by a 60 min reperfusion. Rats were pretreated with an intraperitoneal injection of the following iNOS inhibitors: N-nitro-L-arginine methyl ester, aminoguanidine, and (1S,5S,6R,7R)-7- chloro-3-imino-5-methyl-2-azabicyclo [4. 1. 0] heptane hydrochloride (ONO-1714). Apoptosis was evaluated and NO(X) in the portal vein was assayed. The amount of iNOS, caspase-3, and cytochrome c were determined by a Western blot analysis. Intestinal mucosal epithelial mitochondrial dehydrogenase activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoilium bromide. Ischemia-reperfusion increased intestinal mucosal apoptosis, NO(X) production in the portal vein, the amount of iNOS protein, and the release of cytochrome c, but not caspase-3. Inhibitors of iNOS significantly attenuated the induction of apoptosis, increased NO(X) production, and release of cytochrome c. Mitochondrial dysfunction was induced by ischemia-reperfusion, which was ameliorated by iNOS inhibitors. Our results indicate that iNOS is related to increased mucosal apoptosis in the rat small intestine after ischemia-reperfusion, which is partly explained by the release of cytochrome c from mitochondria to cytosols following mitochondrial dysfunction.

Non-equivalence of Wnt and R-spondin ligands during Lgr5 + intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium—an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1–RSPO4) engage distinct LGR4–LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

BackgroundGastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.ResultsBoth the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.ConclusionsWe document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

T cell deficiency leads to liver carcinogenesis in azoxymethane-treated rats

There is an increasing amount of evidence suggesting that T cell deficiency contributes to tumor development. However, it is unclear whether T cell deficiency leads to liver and colon carcinogenesis. The aim of this study was to investigate the role of T cells on liver and colon carcinogenesis. Athymic F344/N Jcl-rnu/- (nu/nu) rats and euthymic F344/N Jcl-rnu/+ (nu/+) rats were administered the carcinogen azoxymethane (AOM) at a dose of 15 mg/kg body wt once a week for 2 weeks. At 48 weeks after the second carcinogen treatment, the rats were sacrificed, and livers and colons were examined. Apoptosis and cell proliferation were evaluated by DNA fragmentation and proliferating cell nuclear antigen assays, respectively. Wild-type p53 and members of the Jun and Fos oncogene families were detected by Western blotting. AOM treatment induced 100% liver tumor and 63.6% colon tumor incidence in T cell–deficient nu/nu rats, compared with 0% and 38.5% incidence in nu/+ rats. T cell deficiency promoted the inhibitory action of AOM on apoptosis in both liver and colon at 48 weeks. In contrast, T cell deficiency increased cell proliferation after AOM treatment in both tissues. Wild-type p53 was reduced in both tissues of T cell–deficient rats. AOM treatment induced c-Jun and c-Fos expressions in the liver but increased only Fos B in the colon, whereas T cell deficiency enhanced c-Jun overexpression in the liver. These results suggest that T cell deficiency leads to liver carcinogenesis partly by a reduction in wild-type p53 and increasing c-Jun expression in AOM-treated rats.

Evaluation of endoscopic hemostasis with metallic hemoclips for bleeding gastric ulcer: comparison with endoscopic injection of absolute ethanol in a prospective, randomized study.

OBJECTIVE:Although metallic hemoclips have been used for hemostasis of bleeding ulcer, there have been few prospective trials to evaluate their efficacy. In this study, a prospective, randomized trial was performed to evaluate endoscopic hemoclipping for bleeding gastric ulcer in comparison with endoscopic injection of absolute ethanol.METHODS:During the period 1995–1998, 126 gastric ulcer patients with bleeding or nonbleeding visible vessel were considered for entry. They were randomly assigned to one of three groups: endoscopic hemostasis performed with injection of absolute ethanol (group I, n = 42), hemoclipping (group II, n = 42), and a combination of the two methods (group III, n = 42).RESULTS:The permanent hemostatic rate was 85.7% in group I, 90.5% in group II, and 92.9% in group III. The mean volume of blood transfusion was 313 ± 77 ml in group I, 274 ± 54 ml in group II, and 163 ± 42 ml in group III, which was significantly less than in groups I or II (p < 0.05). No patients required emergency surgery. Five patients died within a month after initial hemostasis as a result of unrelated conditions.CONCLUSIONS:Endoscopic hemostasis with hemoclips for bleeding gastric ulcer was as effective and safe as that with injection of absolute ethanol, and a combination of ethanol injection and hemoclips did not result in a great advantage over the two individual procedures.