Crypt Base Research Articles

Arachidonic Acid Promotes Intestinal Regeneration by Activating WNT Signaling.

SummaryIntestinal regeneration is crucial for functional restoration after injury, and nutritional molecules can play an important role in this process. Here, we found that arachidonic acid (AA) serves as a direct proliferation promoter of intestinal epithelial cells that facilitates small intestinal regeneration in both three-dimensional cultured organoids and mouse models. As shown in the study, during post-irradiation regeneration, AA positively regulates intestinal epithelial cell proliferation by upregulating the expression of Ascl2 and activating WNT signaling, but negatively regulates intestinal epithelial cell differentiation. AA acts as a delicate regulator that efficiently facilitates epithelial tissue repair by activating radiation-resistant Msi1+ cells rather than Lgr5+ cells, which are extensively considered WNT-activated crypt base stem cells. Additionally, short-term AA treatment maintains optimal intestinal epithelial homeostasis under physiological conditions. As a result, AA treatment can be considered a potential therapy for irradiation injury repair and tissue regeneration.

The M1 muscarinic acetylcholine receptor in the crypt stem cell compartment mediates intestinal mucosal growth.

Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

Cycling Stem Cells Are Radioresistant and Regenerate the Intestine

SUMMARYA certain number of epithelial cells in intestinal crypts are DNA damage resistant and contribute to regeneration. However, the cellular mechanism underlying intestinal regeneration remains unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are right above Lgr5high cells in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radiosensitive crypt base columnar stem cells (CBCs). This enables an efficient repopulation of the intestinal epithelium at early stage when Lgr5high cells are not emerging. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are cycling ISCs that maintain and regenerate the intestinal epithelium.

Visualizing Human Colorectal Cancer Intratumor Heterogeneity with Phylogeography.

Phylogeography combines ancestry with location and can be translated to intratumor heterogeneity (ITH) to visualize how tumors spread. ITH is common in human tumors, with many genetic and phenotypic differences between regions. The roles of ITH in progression are uncertain because many subclones lack discernable driver mutations. ITH can be visualized by mapping mutations onto microscopic sections, where subclones are directly associated with phenotypes, especially the deeper areas with the more invasive cells that confer worst clinical outcomes. Instead of a stepwise hierarchy where subclones segregate by phenotype with later branching subclones in more invasive areas, multiple subclones share superficial and invasive phenotype and are jigsaw arrayed in vertical columns. Phylogeography shows that both early and late subclones extend from the surface to the invasive front, suggesting that founder cells start with phenotypic plasticity and essentially all the drivers necessary to rapidly grow into large invasive tumors.

Intestinal stem cells promote crypt fission during postnatal growth of the small intestine

ObjectiveWnt-β-catenin signalling is essential for intestinal stem cells. Our aim was to investigate the relationship between intestinal stem cells and crypt fission which peaks during infancy.DesignDuodenal biopsies were obtained during...

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5+ Intestinal Stem Cells.

Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.

Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling.

BackgroundDachshund homologue 1 (DACH1) is highly expressed in LGR5+ intestinal stem cells and colorectal tumours. However, the roles of DACH1 in intestinal cell stemness and colorectal tumorigenesis remain largely undefined.MethodsWe used immunohistochemistry, western blotting and quantitative real-time PCR to analyse DACH1 expression in colorectal cancer (CRC) samples. CRISPR/Cas9 gene editing and lentiviral vector-mediated overexpression and shRNA-mediated knockdown of DACH1 were utilized to modulate DACH1 expression in cell lines and organoids. An intestinal organoid-based functional model was analysed, and cancer cell colony formation, sphere formation assays and murine xenotransplants were performed to reveal the role of DACH1 in CRC cell proliferation, stemness and tumorigenesis. Immunofluorescence, co-immunoprecipitation, RNA interference and microarray data analyses were conducted to demonstrate the association between DACH1 and the bone morphogenetic protein (BMP) signalling pathway.FindingsDACH1 is specifically expressed in discrete crypt base cells, and increased DACH1 expression was found in all stages of CRC. Moreover, the high expression of DACH1 independently predicted poor prognosis. In colon cancer cells, shRNA-mediated suppression of DACH1 inhibited cell growth in vitro and in vivo. By studying the intestinal organoid-based functional model, we found that depletion of DACH1 reduced the organoid formation efficiency and tumour organoid size. DACH1 overexpression stimulated both colonsphere formation and tumour organoid formation in the context of dysregulated BMP signalling. Mechanistic characterizations indicated that overexpression of DACH1 affects a subset of stem cell signature genes implicated in stem cell proliferation and maintenance through the suppression of BMP signalling via SMAD4.InterpretationTogether, our study highlights DACH1 as an integral regulator of BMP signalling during intestinal tumorigenesis, and DACH1 could be a potential prognostic marker and therapeutic target for colorectal cancer patients.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER‐Grem1 flx/flx) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7–13 days). Post‐mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Dissecting the cellular responses of immune cells and Lgr5+ stem cells in the inflamed mouse colon with the RNAscope in situ hybridization technology

Abstract Due to its exposure to a harsh luminal environment, the intestinal epithelium has a remarkably fast turnover rate that is facilitated by a resident intestinal stem cell (ISC) population present at the base of the intestinal crypt. These ISCs, marked by the GPCR Lgr5, allow the intestinal epithelium to adapt to different types of damage, such as inflammation. Chronic intestinal inflammation is a hallmark of the inflammatory bowel diseases (IBD) yet the interplay between inflammatory immune cells and ISCs remains to be elucidated. Therefore, we utilized the single-molecule RNA in situ hybridization (ISH) technology RNAscope to visualize the expression of multiple immune and ISC markers within the inflamed intestinal tissue environment. To interrogate the expression pattern of inflammatory immune cell and ISC markers within the intestinal crypt, we performed the assay on colons from either control or TNBS-treated mice. We visualized the location of each intestinal cell population, including the resident Lgr5+ ISC population, within the crypt. The impact of inflammation on the Lgr5+ ISC population, as well as the Wnt/β-catenin pathway, was also examined. Using a multiplex assay, we assessed infiltration of regulatory T cells and Th17 cells in the inflamed region. Lastly, we examined the expression of several receptor-ligand pairs for cytokines and ISC markers. Taken together, these results demonstrate the ability of the RNAscope assay to visualize the ISCs within the morphological context of the intestinal crypt and in relationship to inflammatory immune cells. This work may help understand mechanisms behind the pathogenesis of IBD and other inflammatory diseases and also aid in the development of potential therapies.

Expression Profile and Prognostic Significance of EPHB3 in Colorectal Cancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) is expressed in cells at the base of intestinal crypts, acting as a cellular guide in the maintenance of intestinal crypt architecture. We aimed to investigate the expression profile of EPHB3 in colorectal precancerous lesions and colorectal cancers (CRCs), and assess its prognostic value. EPHB3 expression was higher in CRCs than in normal mucosa and was associated with the intestinal stem cell markers EPHB2, OLFM4, LRIG1, and a proposed cancer stem cell marker, CD44. Enhanced EPHB3 expression significantly declined during the transformation from adenoma to carcinoma and as the tumor invaded into deeper tissue layers. Namely, a substantial reduction of EPHB3 expression was observed in the budding cancer cells at the invasive tumor fronts, which was more extensive than E-cadherin downregulation. In an azoxymethane/dextran sulfate sodium-induced, colitis-associated, CRC model, EPHB3 expression increased along with tumor development. In a large cohort of CRC patients, EPHB3 positivity was observed in 24% of 610 CRCs and was negatively correlated with tumor differentiation, lympho-vascular invasion, and tumor, node, and metastasis stages. EPHB3 was positively associated with microsatellite instability but was associated with neither CpG island methylation, nor with KRAS and BRAF mutations. Notably, EPHB3 positivity was associated with better clinical outcomes, although it was not an independent prognostic marker. Overexpression of EPHB3 in the colon cancer cell line, DLD1, led to decreased cell growth and migration and reduced mitogen-activated protein kinase signaling. Taken together, our data demonstrate the suppressive role of EPHB3 in CRC progression.

Enteric Glial Networks Visualized using SOX10 Fluorescent Reporter in Optically‐Cleared Full Thickness Intestinal Tissues

Enteric glia are important components of the enteric nervous system (ENS) that control critical functions of the alimentary canal including motility, fluid and ion transport, and immune barrier. Anatomical assessments of enteric glia to date have utilized microdissected whole‐mount tissue or thin sections to visualize cells in the optically dense matrix of the bowel. The aim of this study was to assess the morphological features of enteric glia throughout the small and large intestines of the mouse in intact full‐thickness preparations. B6.Cg‐Polr2atm1(CAG‐GCaMP5g,‐tdTomato)Tvrd/J mice were bred to homozygosity with B6.Cg‐Tg(SOX10‐cre/ERT2)Vpa mice which were maintained as hemizygous. Mice received a gavage of tamoxifen (5mg in 0.2ml corn oil) for five days at seven to eight weeks of age to induce tdTomato (tdT) expression in glia. Mice were euthanized by CO2 inhalation at 10–20 weeks of age. Jejunum and mid colon segments were opened on the mesenteric border, fixed in 4% paraformaldehyde, immunostained for anti‐S100β, and cleared with Ce3D solution. Tissues were imaged using confocal microscopy and 3D reconstructions were made using Analyze. SOX10‐tdT cells were found in the bowel throughout all layers. In the muscularis externa of both the jejunum and colon, there were extensive networks within myenteric ganglia, intraganglionic fiber tracts, within the circular muscle layer, along fiber tracts ascending through the circular muscle, and rare cells were observed in the longitudinal muscle layer. In the submucosal layer of both the jejunum and colon, SOX‐tdT cells were in submucosal ganglia, along intraganglionic fibers and formed a regular network of processes that extended within the lamina propria surrounding the base of intestinal crypts. In the mucosa, SOX10‐tdT distribution differed between the jejunum and colon. In the colonic mucosa, there were two distinct bands of SOX10‐tdT networks oriented perpendicular to and surrounding all colonic glands which were approximately 120μm in height. These bands were located approximately 20μm and 60μm from the base of the glands. SOX10‐tdT cells were very rarely observed near the luminal surface of the colonic glands. In the jejunal mucosa, there were extensive SOX10‐tdT networks throughout the lamina propria surrounding crypts and extending to the tips of villi. Overlap of SOX10‐tdT and S100β immunoreactivity was nearly 100% throughout all glial populations with two exceptions, only 10–20% of SOX10‐tdT cells had S100β immunoreactivity in the jejunum mucosa and there were submucosal neurons that expressed S100β that did not contain SOX10‐tdT. Collectively, these data demonstrate significant complexity of the glial networks of the intestines with distinct morphological differences between mucosal glia of the colon and jejunum that may implicate different functions of enteric glia in the small and large intestines.Support or Funding InformationSupported by NIH grants R01DK106011, P30DK084567 and DP2AT010875 and Department of Defense grant W81XWH‐18‐1‐0218.Representative SOX10‐tdT fluorescence in projections of 409 or 139 1.22μm‐thick confocal images through the full thickness of either jejunum (left) or colon (right), respectively.Figure 1

A29 HOPX LABELS A COLONIC STEM CELL THAT CONTRIBUTES TO COLONIC REGENERATION BUT NOT COLONIC TUMORS

Abstract Background Colorectal cancer is the 2nd leading cause of cancer death in Canada. In rapidly dividing tissues such as the intestine or colon, only long-lived, multipotent, self-renewing tissue stem cells have longevity to accumulate mutations and serve as the cellular origin of cancer. In the small intestine, genetic fate mapping studies have demonstrated that there are at least two principal stem cell pools: actively cycling, crypt base cells expressing Lgr5, and quiescent cells situated above the crypt base. Clevers and colleagues have previously shown that Lgr5-expressing cells can give rise to cancer upon mutation. Interestingly, when Lgr5+ stem cells are selectively “killed”, intestinal integrity remains intact and other stem cells restore homeostasis. To determine whether another stem cell population can give rise to cancer in the colon, we examined whether the atypical homeobox protein Hopx, marks stem cells in the colon and whether these cells can give rise to colon cancer. Aims In the present study, we aim to determine whether Hopx-expressing cells are colonic stem cells that contribute to gut healing and can give rise to colonic tumours following the loss of APC. Methods To determine whether Hopx expressing cells show stemness, we crossed Hopx-CreERT mice to R26-TdTomato reporter mice. We then conducted genetic lineage tracing studies in the colon during homeostasis and following dextran sodium sulphate (DSS)-induced colitis. To test the function of Hopx expressing cells, Hopx-CreERT mice were also crossed to R26DTR mice and treated with diphtheria toxin (DT) following tamoxifen. These mice were then exposed to either normal drinking water or DSS to determine the role of Hopx+ cells in colonic regeneration. To test whether Hopx expressing cells can serve as a cellular origin for colon cancer, Hopx-CreERT mice were crossed to Apcf/f (floxed) mice. Results Consistent with the labeling of a stem cell, following tamoxifen, Hopx+ cells expressing tdTomato expanded to lineage trace full colonic crypts within 7 days, and labelling was persistent for greater than 6 months. Interestingly, ablation of Hopx+ cells with DT did not alter weight, histological damage or survival during normal homeostasis, however, Hopx+ cell ablation in mice treated with DSS resulted in increased histological damage. Surprisingly, loss of APC in Hopx-expressing cells did not induce colonic adenomas even after 8 months following tamoxifen administration. Conclusions These findings prove that Hopx expressing cells identify a novel colonic stem cell pool that is redundant in homeostasis, but in the context of injury, are essential for epithelial regeneration. Interestingly, Hopx+ cells do not have the capacity to give rise to colorectal adenomas upon loss of the APC gene. Funding Agencies CIHR

A83 INTESTINAL EPITHELIAL CELL STEMNESS AND DIFFERENTIATION ARE REGULATED BY THE HIPPO PATHWAY EFFECTOR YAP1

Abstract Background The high rate of cell turnover in the intestinal epithelium is supported by the LGR5+ crypt base columnar (CBC) stem cells, which are located at the lower part of the gland. Among of the various factors and signals like Wnt and Notch, YAP1 (yes associated protein) also plays an important role in stemness of CBC stem cells. YAP1 is the effector of the Hippo pathway. Hippo Pathway restricts the cells proliferation, tissues overgrowth and cancer formation through the phosphorylation and inactivation of the YAP1 protein. When active, YAP1 transfers into nucleus, forms the complex with TEADs transcription factors and promotes the transcription of genes involved in cell growth and proliferation. Aims In the present study, we investigated the role of the YAP1 in the colorectal cancer multipotent HT29 cell line, which contain cancer stem cells (CSC). Methods For approaching to this goal, YAP1 expression was knocked down using shRNAs in HT29 cells. Then stem cells and intestinal cell lineages (secretory goblet, Paneth and enteroendocrine and absorptive) markers expression was analyzed using qPCR and Western blot. Results The results showed the reduction of the expression of stem cells markers including LGR5 in YAP1 knockdown HT29 cells compare with control. Expression of the goblet cells markers (MUC2 and trefoil factor 3) and absorptive cells markers (sucrase-isomaltase and dipeptidylpeptidase IV) were significantly increased in YAP1 knockdown cells but Paneth (DEFA5 and lysozyme) and enteroendocrine (CHGA) were not detected. Finally, examination of the main transcription factors for intestinal differentiation revealed an increase in CDX2 expression. Conclusions These results suggest that YAP1 is involved in the maintenance of colorectal cancer stem cells while preventing intestinal differentiation in both secretory and absorptive lineages through the repression of CDX2. Funding Agencies CIHR

ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids

ObjectiveEnteroendocrine cells (EECs) survey the gut luminal environment and coordinate hormonal, immune and neuronal responses to it. They exhibit well-characterised physiological roles ranging from the control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in the human gut. The small molecule isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation. MethodsWe investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real-time quantitative polymerase chain reaction (RT-qPCR), fluorescent-activated cell sorting, immunostaining and single-cell RNA sequencing. ResultsISX-9 increased the number of neurogenin3-RFP (Ngn3)-positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors that play roles in mouse EEC specification. Single-cell analysis showed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased toward serotonin-producing enterochromaffin (EC) cells. Further, we observed enrichment of organoids with functional EC cells that was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types. ConclusionOur data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to the mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling

BackgroundGegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium. PurposeThis study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models. MethodsAcute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4−/−) acute UC mice were also used in this study. ResultsGQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4−/− UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells. ConclusionsGQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.

Dietary restriction increases protective gut bacteria to rescue lethal methotrexate-induced intestinal toxicity

ABSTRACT Methotrexate (MTX) is a typical chemotherapeutic drug that is widely used in the treatment of various malignant diseases as well as autoimmune diseases, with gastrointestinal toxicity being its most prominent complication which could have a significant effect on the prognosis of patients. Yet effective ways to alleviate such complications remains to be explored. Here we show that 30% dietary restriction (DR) for 2 weeks dramatically increased the survival rate of 2-month-old female mice after lethal-dose MTX exposure. DR significantly reduced intestinal inflammation, preserved the number of basal crypt PCNA-positive cells, and protected the function of intestinal stem cells (ISCs) after MTX treatment. Furthermore, ablating intestinal microbiota by broad-spectrum antibiotics completely eliminated the protective effect achieved by DR. 16S rRNA gene deep-sequencing analysis revealed that short-term DR significantly increased the Lactobacillus genus, with Lactobacillus rhamnosus GG gavage partially mimicking the rescue effect of DR on the intestines of ad libitum fed mice exposed to lethal-dose MTX. Together, the current study reveals that DR could be a highly effective way to alleviate the lethal injury in the intestine after high-dose MTX treatment, which is functionally mediated by increasing the protective intestinal microbiota taxa in mice. Keywords: Dietary restriction, Methotrexate, Gut microbiota, Intestinal stem cells, intestinal toxicity

Effects of Receptor for Advanced Glycation End-Products (RAGE) Signaling on Intestinal Ischemic Damage in Mice

Objective: Superior mesenteric artery ischemia and nonocclusive mesenteric ischemia are representative diseases of the vascular emergency known as irreversible transmural intestinal necrosis (ITIN). The receptor for advanced glycation end-products (RAGE) belongs to the immunoglobulin superfamily of extracellular ligands, which also includes high-mobility group box 1 (HMGB-1) and proteins of the S100 family. The HMGB-1 ligands have been implicated in the pathogenesis of various inflammatory disorders. This study was designed to investigate the relation between RAGE and ITIN in a murine acute intestinal ischemic model. Materials and Methods: ITIN was induced by clipping the cranial mesenteric artery and the peripheral blood vessels. Mucosal and blood samples were collected and analyzed by reverse-transcription PCR and immunohistochemistry for mucosal inflammation and levels of RAGE-related proteins. The influence of RAGE signaling on intestinal cell reproduction was investigated using the cell scratch test, an in vitro wound-healing assay. Finally, RAGE-related proteins and their respective inhibitors were administered intraperitoneally to ITIN model mice to determine their effects. Results: RAGE-expressing cells were located at the base of the intestinal crypts at day 0. As ITIN progressed, most of the damaged intestinal cells expressed RAGE, and ligands of RAGE such as HMGB-1, S100 A8/A9, and S100β were present in the crypt cells from the bottom to the top. The quantities of S100 A8/A9 and S100β were particularly high, above the levels found in other diseases. When S100 A8/A9 and S100β were applied to small intestinal epithelial cells in vitro, regeneration was significantly impeded. Inflammatory Gr1+ neutrophils and F4/80+ macrophages are involved in tissue ischemia. S100 A8/A9 enhances inflammatory myeloid cell influx. Conclusions: RAGE-related proteins are elevated in ITIN model mice and impede intestinal regeneration in vitro. RAGE-related proteins may be a new therapeutic target or a new marker for ITIN.

NCoR1 Protects Mice From Dextran Sodium Sulfate-Induced Colitis by Guarding Colonic Crypt Cells From Luminal Insult.

Background & AimsColonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy.MethodsWe performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1F/F]) and intestinal epithelial cell–specific NCoR1-deficient mice (NCoR1ΔIEC). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor α treatments.ResultsNCoR1ΔIEC mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1ΔIEC mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor α co-treatment, which was accompanied by increased histone acetylation.ConclusionsNCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153.

Cross-talk between Hippo and Wnt signalling pathways in intestinal crypts: Insights from an agent-based model

Intestinal crypts are responsible for the total cell renewal of the lining of the intestines; this turnover is governed by the interplay between signalling pathways and the cell cycle. The role of Wnt signalling in cell proliferation and differentiation in the intestinal crypt has been extensively studied, with increased signalling found towards the lower regions of the crypt. Recent studies have shown that the Wnt signalling gradient found within the crypt may arise as a result of division-based spreading from a Wnt ‘reservoir’ at the crypt base. The discovery of the Hippo pathway’s involvement in maintaining crypt homeostasis is more recent; a mechanistic understanding of Hippo pathway dynamics, and its possible cross-talk with the Wnt pathway, remains lacking. To explore how the interplay between these pathways may control crypt homeostasis, we extended an ordinary differential equation model of the Wnt signalling pathway to include a phenomenological description of Hippo signalling in single cells, and then coupled it to a cell-based description of cell movement, proliferation and contact inhibition in agent-based simulations. Furthermore, we compared an imposed Wnt gradient with a division-based Wnt gradient model. Our results suggest that Hippo signalling affects the Wnt pathway by reducing the presence of free cytoplasmic β-catenin, causing cell cycle arrest. We also show that a division-based spreading of Wnt can form a Wnt gradient, resulting in proliferative dynamics comparable to imposed-gradient models. Finally, a simulated APC double mutant, with misregulated Wnt and Hippo signalling activity, is predicted to cause monoclonal conversion of the crypt.

Hemagglutinating Virus of Japan Envelope (HVJ-E)-Guided Gene Transfer to the Intestinal Epithelium.

The rapidly self-renewing epithelium of the small intestine represents an exquisite model for the stem cell-driven tissue renewal and tumorigenesis. Intestinal stem cells (ISCs) are located in the crypt base, where they produce rapidly dividing progenitors that undergo cell-cycle arrest and terminal differentiation upon several rounds of cell division. So far, genetic studies in mice have played a central role in analyzing function of genes during the stem cell-driven renewal of the intestinal epithelium. However, generation and maintenance of genetically engineered mice are a time-consuming endeavor, which limits the progress in intestinal biology. Recently, we have established a novel method that serves as an alternative to mouse genetics in intestinal biology. The method, termed intestine-specific gene transfer (iGT), enables rapid and efficient delivery of small molecules, such as siRNAs and plasmids, into the intestinal epithelium of living mice by utilizing the hemagglutinating virus of Japan envelope (HVJ-E). Here, we describe a detailed protocol for iGT and discuss how this method can accelerate progress in intestinal biology and elucidate the mechanisms of intestinal epithelium self-renewal.