Intestinal Crypt Research Articles

Transient HR enhancement by RAD51-stimulatory compound confers protection on intestinal rather than hematopoietic tissue against irradiation in mice

DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS1 pretreatment could increase the survival and regeneration of irradiated intestinal organoid in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.

Glycyrrhizic acid and patchouli alcohol in Huoxiang Zhengqi attenuate intestinal inflammation and barrier injury via regulating endogenous corticosterone metabolism mediated by 11β-HSD1

Ethnopharmacological relevanceUlcerative colitis (UC), a chronic inflammatory bowel disease, has become a significant public health challenge due to the limited effectiveness of available therapies. Huoxiang Zhengqi (HXZQ), a well-established traditional Chinese formula, shows potential in managing UC, as suggested by clinical and pharmacological studies. However, the active components and mechanisms responsible for its effects remain unclear. Aim of studyThis study aimed to identify the bioactive components of HXZQ responsible for its therapeutic effects on UC and to elucidate their underlying mechanisms. Materials and methodsThe effect of HXZQ against dextran sodium sulfate (DSS) -induced colitis was investigated. Ingredients in HXZQ were characterized and analyzed in colitic mice using liquid chromatography–mass spectrometry (LC-MS) and gas chromatography–mass spectrometry (GC-MS). In vitro, biological activity of compounds was assessed using lipopolysaccharide (LPS)-induced Ana-1 cells and bone marrow-derived macrophages (BMDMs), tumor necrosis factor-alpha-induced Caco-2 cells, and isolated intestinal crypts from colitic mice. These results were confirmed in vivo. The targets of the components were identified through bioinformatics analysis and validated via molecular docking, enzyme inhibition assays, and in vivo experiments. Hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative real-time polymerase chain reaction (qPCR) were employed to confirm the pharmaceutical effects. ResultsA clinical equivalent dose of HXZQ (2.5 mL/kg) effectively treated DSS-induced colitis. 113 compounds were identified in HXZQ, with 35 compounds detected in colitic mice. Glycyrrhizic acid (GA) and patchouli alcohol (PA) emerged as key contributors to the anti-colitic effects of HXZQ. Further investigation revealed that HXZQ and its active components decreased the levels of pro-inflammatory cytokines TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6) in colon, likely by inhibiting nuclear factor kappa-B (NF-κB) signaling pathway. This inhibition indirectly activated the intestinal farnesoid X receptor (FXR) signaling pathway, correcting bile acid imbalances caused by colitis. Additionally, these components significantly enhanced the expression of tight junction proteins ZO-1 and Occludin, as well as the adhesion protein E-cadherin, and reduced goblet cell loss, thereby repairing intestinal barrier injury. Mechanistically, GA and PA were found to inhibit 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activity, leading to increased local active corticosterone levels in the intestine to exert anti-inflammatory effects. Notably, the inhibition of 11β-HSD1 with the selective inhibitor BVT ameliorated colitis in mice. ConclusionsHXZQ exhibits therapeutic effects on UC, primarily through GA and PA inhibiting 11β-HSD1. This suggests new natural therapy approaches for UC and positions 11β-HSD1 as a potential target for colitis treatment.

Features of the growth and histostructure of the duodenum of japanese quails (Coturnix japonica) of the meat and universal direction

The article presents data on the relationship between the live weight, weight of the gastrointestinal tract and the productivity type of Japanese quail (Coturnix japonica domestica). It is shown that during the first week of the experiment, quails of the universal (dual-purpose) productivity type developed faster than the meat quails in terms of live weight, however, by the age of 28 days, this indicator becomes higher in meat quails. This pattern persists until the end of rearing. The relative weight of the gastrointestinal tract, as well as the duodenum, was higher in meat quails in the first week, and later it became less than in a universal type quail.The histological structure of the duodenum was also studied. On histological slides, the preservation of all layers of the duodenum is observed. The lamina propria of the mucosa is rich in lymphoid tissue. The submucosa is poorly distinguishable. The thickness of the mucosa of the duodenum prevailed throughout the experiment in quails of the universal type. In the first 7 days, the length of the duodenum villi was significantly higher than that of meat quails. The ratio of the length of the villi to the crypt increases with age in all groups of quails. In the first 7 days, this indicator was higher in quails of the egg-meat type, but by the 56th day it gradually leveled off. The depth of crypts (common intestinal glands) in the first week of growth prevailed in the intestines of meat quails (p ≤ 0.05).

BMP suppresses Wnt signaling via the Bcl11b-regulated NuRD complex to maintain intestinal stem cells.

Lgr5+ intestinal stem cells (ISCs) are crucial for the intestinal epithelium renewal and regeneration after injury. However, the mechanism underlying the interplay between Wnt and BMP signaling in this process is not fully understood. Here we report that Bcl11b, which is downregulated by BMP signaling, enhances Wnt signaling to maintain Lgr5+ ISCs and thus promotes the regeneration of the intestinal epithelium upon injury. Loss of Bcl11b function leads to a significant decrease of Lgr5+ ISCs in both intestinal crypts and cultured organoids. Mechanistically, BMP suppresses the expression of Bcl11b, which can positively regulate Wnt target genes by inhibiting the function of the Nucleosome Remodeling and Deacetylase (NuRD) complex and facilitating the β-catenin-TCF4 interaction. Bcl11b can also promote intestinal epithelium repair after injuries elicited by both irradiation and DSS-induced inflammation. Furthermore, Bcl11b deletion prevents proliferation and tumorigenesis of colorectal cancer cells. Together, our findings suggest that BMP suppresses Wnt signaling via Bcl11b regulation, thus balancing homeostasis and regeneration in the intestinal epithelium.

The GTPase RAB6 is required for stem cell maintenance and cell migration in the gut epithelium.

Intestinal epithelial cells, which are instrumental in nutrient absorption, fluid regulation, and pathogen defense, undergo continuous proliferation and differentiation within the intestinal crypts, migrating towards the luminal surface where they are eventually shed. RAB GTPases are key regulators of intracellular vesicular trafficking and are involved in various cellular processes, including cell migration and polarity. Here, we investigated the role of RAB6 in the development and maintenance of the gut epithelium. We generated conditional knockout mice with RAB6 specifically deleted in the gut epithelium. We found that deletion of the Rab6a gene resulted in embryonic lethality. In adult mice, RAB6 depletion led to altered villus architecture and impaired junction integrity without affecting the segregation of apical and basolateral membrane domains. Further, RAB6 depletion slowed down cell migration and adversely affected both cell proliferation and stem cell maintenance. Notably, the absence of RAB6 resulted in a diminished number of functional stem cells, as evidenced by the rapid death of isolated crypts from Rab6a KO mice when cultured as 3D organoids. Together, these results underscore the essential role of RAB6 in maintaining gut epithelial homeostasis.

Intestinal Tissue, Digestive Enzyme, and Antioxidant Enzyme Activities in the Early Development Stage of Endangered Brachymystax tsinlingensis.

This work explores the digestive system characteristics of Brachymystax tsinlingensis during early developmental stages and aims to solve the problem of high lethality of fry during the transgression period, which is crucial for the artificial propagation and population conservation of endangered fishes. This study was carried out on intestinal tissue, digestive enzymes, and antioxidant enzyme activities in the early development stage of Brachymystax tsinlingensis. Ten random samples during endogenous nutrition (7, 10, and 11 days after hatching), mixed nutrition (13 and 19 DAH), and exogenous nutrition (31, 33, 39, 45, and 73 DAH) were collected by histological and biochemical analysis methods. The results showed that the intestine of Brachymystax tsinlingensis already has four layers initially at 7 DAH, and the intestinal gland tissue is evident at 73 DAH. The contents of total protein (TP) and the activities of lipase (LPS) and trypsin (TPS) were maximal at 39 DAH, and the activities were 3.20 ± 0.26 mg/mL, 2.52 ± 0.69 U/g, and 2717.45 ± 295.26 U/mg, respectively. Catalase (CAT) and glutathione peroxidase (GSH-PX) activities both showed the lowest values at 39 DAH, which were 0.57 ± 0.11 U/mg and 3.35 ± 0.94 U/mg, respectively. The activity of amylase (AMS) and the content of malonaldehyde (MDA) increased, and the highest values were reached at 45 DAH (1.32 ± 0.41 U/mg) and 73 DAH (1.29 ± 0.43 nmoL/mg), respectively. Superoxide dismutase (SOD) and GSH-PX activities both showed a peak value at 7 DAH (126.58 ± 20.13 U/mg and 6.47 ± 1.86 U/mg). Overall, the changes in intestinal tissue, digestive enzymes, and antioxidant enzyme activities at 39 DAH of Brachymystax tsinlingensis are inseparable from different vegetative stages during the developmental period, and these results can provide a reference for the proliferation and cultivation of Brachymystax tsinlingensis resources.

Thai Fermented Soybean (Thua-Nao) Prevents Early Stages of Colorectal Carcinogenesis Induced by Diethylnitrosamine and 1,2-Dimethylhydrazine Through Modulations of Cell Proliferation and Gut Microbiota in Rats.

Thua-nao is a traditional fermented soybean product widely consumed in the northern areas of Thailand. There has been little research on the biological activity of Thua-nao, particularly its anticancer properties. The objective of this study was to examine the cancer chemopreventive effects of dried Thua-nao on liver and colorectal carcinogenesis induced by carcinogens in rats. Rats were injected with diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH) to induce preneoplastic lesions. Rats orally received dried Thua-nao for 13 weeks. The preneoplastic lesions, including glutathione S-transferase placental form (GST-P)-positive foci and aberrant crypt foci (ACF), were evaluated in the liver and colon, respectively. The cancer chemopreventive mechanisms of dried Thua-nao on liver and colorectal carcinogenesis were examined. Dried Thua-nao administration suppressed colorectal aberrant crypt foci. Moreover, dried Thua-nao reduced proliferation cell nuclear antigen (PCNA)-positive cells in the colon. Interestingly, dried Thua-nao modulated the gut microbiota in DEN- and DMH-induced rats. Isoflavones, including genistein and daidzein, represent promising chemopreventive agents in dried Thua-nao. In conclusion, these results highlight the cancer chemopreventive effect of dried Thua-nao in DEN and DMH-induced colorectal carcinogenesis through cell proliferation reduction and gut microbiota modulation.

Large Animal Models Enhance the Study of Crypt-Mediated Epithelial Recovery from Prolonged Intestinal Ischemia Reperfusion Injury.

Intestinal ischemia and reperfusion injury (IRI) is a deadly and common condition. Death is associated with sepsis due to insufficient epithelial repair, requiring stem cell-driven regeneration, typically beginning 48 hours after injury. Animal models are critical to advancing this field. To effectively study epithelial healing, models must survive clinically relevant intestinal ischemic injury extending to the crypt. Though mouse models are indispensable to intestinal research, their application for studying epithelial repair following severe IRI may be limited. Ischemic injury was induced in mouse and porcine jejunum for up to 3 hours, with up to 72 hours of reperfusion. Histologic damage was scored by Chiu-Park grade and animal survival was assessed. Findings were compared between species. A mouse IRI literature review was performed to evaluate the purported degree of injury, duration of recovery, and reported survival rates. In mice and pigs, 3 hours of ischemia induced severe, reliable injury extending into the crypt. However, at 48 hours, mouse survival was only 23.5% compared to 100% survival in pigs. In literature, ischemia was induced for >1 hour in only 4 of 102 mouse studies and none to 3 hours. Recovery was attempted for 48 hours in only 6 reports. 47 studies reported intestinal crypt injury. Of those that featured histologic intestinal crypt damage, survival rates at 48 hours ranged from 10-50% (median 30%). Mouse models are not ideal to study intestinal stem cell mediated recovery from severe IRI. Alternative large animal models, like pigs, are recommended.

ChiA: a major player in the virulence of Crohn’s disease-associated adherent and invasive Escherichia coli (AIEC)

ABSTRACT We investigated the role of ChiA and its associated polymorphisms in the interaction between Crohn’s disease (CD)-associated adherent-invasive Escherichia coli (AIEC) and intestinal mucosa. We observed a higher abundance of chiA among the metagenome of CD patients compared to healthy subjects. In dextran sulfate sodium-induced colitis mice model, AIEC-LF82∆chiA colonization was reduced in ileal, colonic and fecal samples compared to wild-type LF82. The binding of ChiA to recombinant human CHI3L1 or mucus was higher with the pathogenic polymorphism. The strength of ChiA-mucin interaction was 300-fold stronger than ChiA-rhCHI3L1. ChiA was able to degrade mucin to promote its growth and enabled LF82 to get closer to epithelial cells. The pathogenic polymorphism of ChiA had a stronger impact on mucus degradation than on the binding capability of AIEC to adhere to the intestinal epithelium. We observed that ChiA could favor an efficient bacterial invasion of intestinal crypts, and that ChiA, especially its pathogenic polymorphism, gives LF82 an advantage to uptake within Peyer’s patches, macrophages and mesenteric lymph nodes. All together, these data support the role of ChiA in the virulence of AIEC and show that it could be a promising target to reduce AIEC colonization in patients with CD.

PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family.

MAGIC matrices: freeform bioprinting materials to support complex and reproducible organoid morphogenesis.

Organoids are powerful models of tissue physiology, yet their applications remain limited due to their relatively simple morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports optimal organoid morphogenesis following freeform 3D bioprinting of cell slurries at tissue-like densities. The material is designed with two temperature regimes: at 4 °C it exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We first characterize the rheological properties of MAGIC matrices that optimize organoid morphogenesis, including low stiffness and high stress relaxation. Next, we combine this material with a custom piezoelectric printhead that allows more reproducible and robust self-organization from uniform and spatially organized tissue "seeds." We apply MAGIC matrix bioprinting for high-throughput generation of intestinal, mammary, vascular, salivary gland, and brain organoid arrays that are structurally similar to those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and efficiency of morphogenesis. The flexibility of this method and material enabled fabrication of fully 3D microphysiological systems, including perfusable organoid tubes that experience cyclic 3D strain in response to pressurization. Furthermore, the reproducibility of organoid structure increased the statistical power of a drug response assay by up to 8 orders-of-magnitude for a given number of comparisons. Combined, these advances lay the foundation for the efficient fabrication of complex tissue morphologies by canalizing their self-organization in both space and time.

Xenotransplanted human organoids identify transepithelial zinc transport as a key mediator of intestinal adaptation

Short bowel syndrome (SBS) leads to severe morbidity and mortality. Intestinal adaptation is crucial in improving outcomes. To understand the human gene pathways associated with adaptation, we perform single-cell transcriptomic analysis of human small intestinal organoids explanted from mice with experimental SBS. We show that transmembrane ion pathways, specifically the transepithelial zinc transport pathway genes SLC39A4 and SLC39A5, are upregulated in SBS. This discovery is corroborated by an external dataset, bulk RT-qPCR, and Western blots. Oral zinc supplementation is shown to improve survival and weight gain of SBS mice and increase the proliferation of intestinal crypt cells in vitro. Finally, we identify the upregulation of SLC39A5 and associated transcription factor KLF5 in biopsied intestinal tissue specimens from patients with SBS. Thus, we identify zinc supplementation as a potential therapy for SBS and describe a xenotransplantation model that provides a platform for discovery in other intestinal diseases.

Dietary and metabolic effects on intestinal stem cells in health and disease.

Diet and nutritional metabolites exhibit wide-ranging effects on health and disease partly by altering tissue composition and function. With rapidly rising rates of obesity, there is particular interest in how obesogenic diets influence tissue homeostasis and risk of tumorigenesis; epidemiologically, these diets have a positive correlation with various cancers, including colorectal cancer. The gastrointestinal tract is a highly specialized, continuously renewing tissue with a fundamental role in nutrient uptake and is, in turn, influenced by diet composition and host metabolic state. Intestinal stem cells are found at the base of the intestinal crypt and can generate all mature lineages that comprise the intestinal epithelium and are uniquely influenced by host diet, metabolic by-products and energy dynamics. Similarly, tumour growth and metabolism can also be shaped by nutrient availability and host diet. In this Review, we discuss how different diets and metabolic changes influence intestinal stem cells in homeostatic and pathological conditions, as well as tumorigenesis. We also discuss how dietary changes and composition affect the intestinal epithelium and its surrounding microenvironment.

Regulatory effects of the p38 mitogen-activated protein kinase-myosin light chain kinase pathway on the intestinal epithelial mechanical barrier and the mechanism of modified Pulsatilla decoction in the treatment of ulcerative colitis.

To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo. We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules. In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue. The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

To sense or not to sense, Paneth cell regulation of mucosal immunity

Paneth cells located within intestinal crypts support epithelial stem cells and immunity through growth factors and antimicrobial peptides. In this issue of Cell Host & Microbe, Wallaeys etal. report that TNF sensing by Paneth cells disrupts the unfolded protein response and decreases antimicrobial peptides, causing bacterial translocation and sepsis.

CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis

BackgroundColony stimulating factor 1 (CSF1) is generally expressed by immune cells in response to pro-inflammatory stimuli. The CSF1 receptor (CSFR) is activated by CSF1, and plays a key role in macrophage homeostasis. Furthermore, the CSF1R+ macrophages maintain homeostasis in the intestinal epithelium. The aim of this study was to explore the functions of CSF1-expressing and CSF1R+ macrophages in necrotizing enterocolitis (NEC), which commonly affects the ileum of neonates.MethodsIn-situ CSF1 expression in the intestines of neonates with NEC or intestinal atresia (n = 4 each) was detected by immunofluorescence staining. The CSF1 levels in the intestinal crypt-derived organoid cultures were measured by ELISA. Peripheral blood monocyte-derived Mφ macrophages were co-cultured with the organoids and stimulated with lipopolysaccharide (LPS) to mimic the inflamed state of the ileum in NEC patients.ResultsCSF1 was expressed in the intestinal epithelial cells of the fetal and neonatal samples, but suppressed in the NEC samples. Furthermore, CSF1 expression was downregulated in the intestinal crypt-derived organoids by LPS. CSF1R+ macrophages were detected near the intestinal crypts in the non-inflamed intestines but were absent in tissues obtained from pediatric NEC patients. Peripheral blood monocyte-derived macrophages promoted intestinal organoid proliferation in vitro following CSF1 stimulation. Finally, low concentrations of LPS slightly enhanced the proliferation of organoids co-cultured with the macrophages, whereas higher doses had a significant inhibitory effect.ConclusionsIntestinal epithelial cells express CSF1 to regulate the resident macrophages, maintain epithelial homeostasis, and resist infection. The abundant CSF1R+ macrophages in the fetal intestine may overexpress TNF-α upon activation of the TLR4/NF-κB pathway, resulting in epithelial damage and NEC induction.

A Ctnnb1 enhancer transcriptionally regulates Wnt signaling dosage to balance homeostasis and tumorigenesis of intestinal epithelia.

The β-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here, we identified an upstream enhancer of Ctnnb1 - the coding gene for β-catenin, named ieCtnnb1 (intestinal enhancer of Ctnnb1), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/β-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4α and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia.

A Ctnnb1 enhancer transcriptionally regulates Wnt signaling dosage to balance homeostasis and tumorigenesis of intestinal epithelia

The β-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here, we identified an upstream enhancer of Ctnnb1 – the coding gene for β-catenin, named ieCtnnb1 (intestinal enhancer of Ctnnb1), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/β-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4α and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia.

Age-Dependent Differences in Radiation-Induced DNA Damage Responses in Intestinal Stem Cells.

Age at exposure is a critical modifier of the risk of radiation-induced cancer. However, the effects of age on radiation-induced carcinogenesis remain poorly understood. In this study, we focused on tissue stem cells using Lgr5-eGFP-ires-CreERT2 mice to compare radiation-induced DNA damage responses between Lgr5+ and Lgr5- intestinal stem cells. Three-dimensional immunostaining analyses demonstrated that radiation induced apoptosis and the mitotic index more efficiently in adult Lgr5- stem cells than in adult Lgr5+ stem cells but not in infants, regardless of Lgr5 expression. Supporting this evidence, rapid and transient p53 activation occurred after irradiation in adult intestinal crypts but not in infants. RNA sequencing revealed greater variability in gene expression in adult Lgr5+ stem cells than in infant Lgr5+ stem cells after irradiation. Notably, the cell cycle and DNA repair pathways were more enriched in adult stem cells than in infant stem cells after irradiation. Our findings suggest that radiation-induced DNA damage responses in mouse intestinal crypts differ between infants and adults, potentially contributing to the age-dependent susceptibility to radiation carcinogenesis.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.