Intestinal Stem Cell Populations Research Articles

Impaired intestinal FXR signaling is involved in aberrant stem cell function leading to intestinal failure-associated liver disease in pediatric patients with short bowel syndrome.

Intestinal failure-associated liver disease (IFALD) is a serious complication of long-term parenteral nutrition in patients with short bowel syndrome (SBS), and is the main cause of death in SBS patients. Prevention of IFALD is one of the major challenges in the treatment of SBS. Impairment of intestinal barrier function is a key factor in triggering IFALD, therefore promoting intestinal repair is particularly important. Intestinal repair mainly relies on the function of intestinal stem cells (ISC), which require robust mitochondrial fatty acid oxidation (FAO) for self-renewal. Herein, we report that aberrant LGR5+ ISC function in IFALD may be attributed to impaired farnesoid X receptor (FXR) signaling, a transcriptional factor activated by steroids and bile acids. In both surgical biopsies and patient-derived organoids (PDOs), SBS patients with IFALD represented lower population of LGR5+ cells and decreased FXR expression. Moreover, treatment with T-βMCA in PDOs (an antagonist for FXR) dose-dependently reduced the population of LGR5+ cells and the proliferation rate of enterocytes, concomitant with decreased key genes involved in FAO including CPT1a. Interestingly, however, treatment with Tropifexor in PDOs (an agonist for FXR) only enhanced FAO capacity, without improvement in ISC function and enterocyte proliferation. In conclusion, these findings suggested that impaired FXR may accelerate the depletion of LGR5 + ISC population through disrupted FAO processes, which may serve as a new potential target of preventive interventions against IFALD for SBS patients.

MTGR1 is required to maintain small intestinal stem cell populations

Undifferentiated intestinal stem cells (ISCs) are crucial for maintaining homeostasis and resolving injury. Lgr5+ cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis where they differentiate into specialized cell types. Coordinated execution of complex transcriptional programs is necessary to allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1−/− mice, we have assessed the effects of MTGR1 loss specifically in ISC biology. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5, the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic and functional analyses revealed deficiencies in Mtgr1-null ISCs, including deregulated ISC-associated transcriptional programs. Ex vivo, intestinal organoids established from Mtgr1-null mice were unable to survive and expand due to aberrant differentiation and loss of stem and proliferative cells. Together, these results indicate that the role of MTGR1 in intestinal differentiation is likely stem cell intrinsic and identify a novel role for MTGR1 in maintaining ISC function.

A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection.

The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and associated virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprised of 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells and cells enriched for mitochondrial genes, suggesting enhanced replication in these populations. In contrast, proliferating intestinal stem cell (ISC) populations had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in viral control. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.

Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.

Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.

Abstract 6016: A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells

Abstract Background: Gut microbial colonization, represents a critical time period for establishing the overall health of an individual. The intestine is a highly dynamic tissue with high turnover of intestinal epithelial cells throughout the life span of an individual making intestinal stem cells (ISCs) critical for normal gut homeostasis. Currently, there exists a lack of knowledge in how the gut microbiota regulates DNA methylation to influence the behavior of these ISCs. Aberrant DNA methylation is now documented to be critical in the disease pathogenesis of intestinal disorders such as colorectal cancer, thus understanding how the gut microbiome influences DNA methylation and ISCs is fundamental for improving human health. Methods: We utilized the Lgr5-GFP mouse model in which endogenous GFP expression is driven by the bonafide intestinal stem cell marker Lgr5. These mice were maintained under two different housing conditions, conventional and germ free. We collected mice under the two conditions and performed dissociation of the colon and small intestine epithelial cells using EDTA dissociation. We then analyzed cell populations using flow cytometry, and collected two cell populations: the differentiated cell population (EpiCam +, GFP -) and intestinal stem cell population (EpiCam +, GFP +). To perform a genome wide comprehensive DNA methylation analysis we performed Whole Genome Bisulfite Sequencing (WGBS) which included both stem cell and differentiated cell populations of females and males at the two age groups points (postnatal day 21 (p21) and p100). Results: There was no significant difference in CpG methylation at the global level between the two housing conditions. Next, we analyzed age matched samples from germ free and conventional mice to determine if there are differentially methylated regions (DMRs) associated with the presence of intestinal microbiota. We have identified DMRs that are maintained in germ free stem cells from p21 to p100 compared to conventional mice, as well as DMRs in the differentiated cell population at both ages. Interestingly in our analysis several genes displayed robust hypomethylation of control mice versus germ free mice at the p100 time point in both the stem cell and differentiated cell populations. One such gene, Ifitm3, has been heavily associated with chronic inflammation in the colonic epithelium and a poor risk factor for colonic cancer (Li D et. Al, Clin Cancer Res. 2011). We validated Ifitm3 methylation and expression changes between cohorts using pyrosequencing and RT-PCR respectively. Conclusions: This comprehensive analysis has yielded top candidate genes for studying functional consequences of DNA methylation changes that have been previously linked to severe patient diseases including cancer. Citation Format: Robert Craig Peery, Jiejun Shi, Leah Farmer, Li Yang, Emily Lawrence, Yumei Li, Wei Li, Lanlan Shen. A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6016.

Abstract WMP119: Novel Therapeutic Potential Of Intestinal Stem Cell Transplants For Ameliorating The Long Term Effects Of Cerebrovascular Stroke

Background: Stroke is a leading cause of disability and dementia with limited treatment options. Evidence suggests that stroke rapidly dysregulates the intestinal epithelium, causing a ‘leaky gut’ and elevated blood levels of inflammatory cytokines. Hypothesis: In view of the emerging evidence of the gut-brain connection, we tested whether early repair of the gut after ischemic stroke would prevent stroke-induced cognitive impairment. Methods: Adult and middle-aged Sprague-Dawley female and male rats were used for this study and assigned to the following groups: Control (sham), stroke with sham transplant (vehicle), or stroke with IESC transplantation. Rats were subjected to stereotaxic middle cerebral artery occlusion (MCAo) using Endothelin-1 (vasoconstrictor). Primary IESCs were isolated from young female and male rats to prepare organoid cultures. Dispersed organoid cells were labeled with PKH67 and injected iv 4h/24h/48h to middle aged hosts after stroke. Behavioral assessments were performed before stroke and 4 weeks after stroke. This included tests for depressive-like behaviors(Burrowing test and the Social Interaction test). Cognitive performance (Novel object Recognition test and Barnes maze test), which assess declarative and visuospatial memory respectively. Results: Vehicle-treated MCAo animals displayed depressive-like behaviors compared to their pre-stroke performance. In contrast, stroke animals that received IESC transplants showed were no different from their pre-stroke performance and no different from sham (no-stroke animals). A similar pattern was seen in tests of cognition. MCAo+vehicle animals showed poor ability to discriminate the novel object and displayed impaired learning and recall on the Barnes maze. In both tests, MCAo animals + IESC transplants animals were not impaired and had similar performance to the Sham (no stroke) group. MCAo caused a persistent elevation of the gut metabolite LPS and the inflammatory cytokine IL-17A, which was abated by IESC treatment. Conclusion: Overall, these data show that an intestinal stem cell population derived from a healthy young animal transferred to an older host after stroke can significantly improve long term behavior stroke outcomes.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function <em>Ex Vivo</em>

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Age disparities in intestinal stem cell quantities: a possible explanation for preterm infant susceptibility to necrotizing enterocolitis.

Preterm infants are more susceptible to necrotizing enterocolitis (NEC) than term Queryinfants. This may be due to a relative paucity of Lgr5+ or Bmi1+-expressing intestinal stem cells (ISCs) which are responsible for promoting intestinal recovery after injury. We hypothesized that the cellular markers of Lgr5+ and Bmi1+, which represent the two distinct ISC populations, would be lower in younger mice compared to older mice. In addition, we hypothesized that experimental NEC would result in a greater loss of Lgr5+ expression compared to Bmi1+ expression. Transgenic mice with EGFP-labeled Lgr5 underwent euthanasia at 10 different time points from E15 to P56 (n = 8-11/group). Lgr5+-expressing ISCs were quantified by GFP ELISA and Bmi1+ was assessed by qPCR. In addition, Lgr5EGFP mice underwent experimental NEC via formula feeding and hypoxic and hypothermic stress. Additional portions of the intestine underwent immunostaining with anti-GFP or anti-Bmi1+ antibodies to confirm ELISA and PCR results. For statistical analysis, p < 0.05 was significant. Lgr5+ and Bmi1+expression was lowest in embryonal and early postnatal mice and increased with age in all segments of the intestine. Experimental NEC was associated with loss of Lgr5+-expressing ISCs but no significant change in Bmi1+ expression. Lgr5+ and Bmi1+ expression increase with age. Lgr5+-expressing ISCs are lower following experimental necrotizing enterocolitis while Bmi1+ expression remains relatively unchanged. Developing a targeted medical therapy to protect the low population of ISCs in preterm infants may promote tissue recovery and regeneration after injury from NEC.

Ribosome impairment regulates intestinal stem cell identity via ZAKɑ activation

The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5-negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity.

Autophagic State Confers Facultative Stem Cell Capacity in The Intestinal Epithelium

BackgroundThe intestinal epithelium is a highly proliferative tissue that undergoes complete turnover in 3 to 5 days. The presence of intestinal stem cells expressing the canonical Wnt target gene Lgr5, known as active intestinal stem cells (a‐ISCs), are responsible for generating daughter cells that differentiate into specialized epithelial cells to carryout various functions in the intestine. The proliferative nature of the intestinal epithelium renders it susceptible to DNA damage‐inducing injury, such as high‐dose irradiation and chemotherapy. Although a‐ISCs and early daughter cells are killed by these insults, a radio‐resistant population of facultative intestinal stem cells (f‐ISCs) can regenerate lost a‐ISCs to restore homeostasis following injury. The functional characteristics of f‐ISCs are becoming increasingly understood, however, whether any epithelial cell can act as an f‐ISC is less clear. One reason for this ambiguity is a heavy reliance on Cre‐driven reporter mouse models with low recombination efficiency. Our objective was to find a functional marker to identify f‐ISCs based on cellular state rather than gene expression. The autophagy pathway has been shown to protect against DNA damage and irradiation‐induced apoptosis in the intestinal epithelium. Furthermore, recent studies demonstrate a requirement for the autophagy pathway during cellular de‐differentiation in both gastric chief and pancreatic acinar cells following metaplasia‐inducing injury. Given that autophagy plays roles in both radio‐resistance and cellular plasticity, two integral features of f‐ISCs, we hypothesized that autophagic activity could serve to identify cells with f‐ISC capacity.Methods and ResultsUsing the autophagic vesicle tracer dye CytoID combined with fluorescence‐activated cell sorting, we demonstrate that intestinal epithelial cells with high levels of CytoID exhibit increased organoid‐formation efficiency (OFE) compared to CytoID ‘low’ cells. Single cell sequencing reveals that the CytoID high population is largely composed of secretory cells including Paneth, Goblet, Tuft, and Enteroendocrine cells, whereas the CytoID low population consisted mainly of a‐ISCs and absorptive Enterocytes. Using reporter mice and antibodies against cell surface receptors, we observe that CytoID can identify cells with high OFE within Enteroendocrine, Paneth, and Goblet cell lineages. Finally, we demonstrate that autophagy is required for the enhanced organoid‐formation observed in CytoID high cells by plating these cells in the presence of the lysosomal inhibitor Bafilomycin A1.ConclusionsOur new data support the notion that epithelial cells in a high autophagic state are biased to secretory lineages and that autophagy status functions as a lineage agnostic marker of f‐ISCs. Furthermore, our data suggests that autophagy is required for organoid formation in these lineages.

The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells

ErbB3 is a RTK family member known to regulate proliferation and growth in the intestinal epithelium. Previous work by our lab determined that targeted Erbb3 deletion from the intestinal epithelium resulted in precocious Paneth cell development and an expanded population in mice. This was associated with increased expression of intestinal stem cell (ISC) markers. Conversely, ileal enteroids treated with the ErbB3 ligand neuregulin (NRG)‐1β had significantly reduced expression of ISC markers, including Lgr5 and Bmi1. These data suggest that ErbB3 restricts secretory cell differentiation and stemness in the intestinal crypt. However, the underlying mechanisms are largely unknown. BMI1 is a proto‐oncogene and member of the PRC complex. Bmi1+ intestinal epithelial cells represent a slow‐cycling ISC population that can regenerate the epithelium following injury and loss of Lgr5+ ISCs. Recent evidence determined that Bmi1+ ISCs are enriched in enteroendocrine markers and can give rise to goblet cells, suggesting BMI1 may be critical for differentiation of secretory lineages. We hypothesized that ErbB3 restricts secretory cell differentiation through regulation of BMI1.AimsTo define ErbB3‐dependent regulation of Bmi1 and determine if ErbB3 restriction of Paneth cell development and differentiation is through suppression of Bmi1.MethodsMucosal scrapings were collected from Vil‐Cre;Erbb3flox/flox mice and Erbb3flox/flox littermate controls for protein and gene expression analysis. Ileal enteroids and HT‐29 colorectal adenocarcinoma cells were treated with vehicle DMSO, the PI3K inhibitor LY294002 (10 μM), the MEK inhibitor U0126 (5 μM), the BMI1 inhibitor PTC‐209 (1 μM), and/or recombinant NRG‐1β (10 ng/mL). Enteroids and cell monolayers were harvested and prepared for gene expression analysis. RT‐qPCR for Bmi1 and Lyz1 was performed relative to Hprt as a loading control. Statistical analysis by one‐way ANOVA was conducted using Prism 9.ResultsConsistent with previous data, BMI1 protein and gene expression were significantly increased in small and large intestinal mucosal scrapings from Vil‐Cre;Erbb3flox/flox mice versus littermate controls. To determine if Bmi1 expression is sensitive to PI3K/Akt or MAPK signaling downstream of ErbB3, HT‐29 cells were treated with inhibitors followed by NRG‐1β after 1 hour, then harvested 24 hours later. PI3K and MAPK inhibition significantly increased Bmi1 expression. NRG‐1β treatment was able to overcome MAPK inhibition to reduce BMI1 expression. Similarly, in ileal enteroids, PI3K and MAPK blockade increased Bmi1 expression and NRG‐1β treatment reversed this effect. To determine whether BMI1 regulates expression of the Paneth cell marker LYZ1, HT‐29 cells were treated with the BMI1 inhibitor PTC‐209. BMI1 inhibition decreased LYZ1 expression.ConclusionsOur data demonstrate that ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling in both human and mouse intestinal cells. Furthermore, BMI1 activity promotes LYZ1 expression. Together, these results support our hypothesis that ErbB3 regulates secretory cell differentiation through BMI1 in the intestinal epithelium.

CDC42 controlled apical-basal polarity regulates intestinal stem cell to transit amplifying cell fate transition via YAP-EGF-mTOR signaling.

SUMMARYEpithelial polarity is controlled by a polarity machinery that includes Rho GTPase CDC42 and Scribble/PAR. By using intestinal stem cell (ISC)-specific deletion of CDC42 in olfactomedin-4 (Olfm4)-internal ribosome entry site (IRES)-EGFP/CreERT2;CDC42flox/flox mice, we find that CDC42 loss initiated in the ISCs causes a drastic hyperproliferation of transit amplifying (TA) cells and disrupts epithelial polarity. CDC42-null crypts display expanded TA cell and diminished ISC populations, accompanied by elevated Hippo signaling via YAP/TAZ-Ereg (yes-associated protein/WW domain-containing transcription regulator protein 1-epiregulin) and mechanistic target of rapamycin (mTOR) activation, independent from canonical Wnt signaling. YAP/TAZ conditional knockout (KO) restores the balance of ISC/TA cell populations and crypt proliferation but does not rescue the polarity in CDC42-null small intestine. mTOR or epidermal growth factor receptor (EGFR) inhibitor treatment of CDC42 KO mice exhibits similar rescuing effects without affecting YAP/TAZ signaling. Inducible ablation of Scribble in intestinal epithelial cells mimics that of CDC42 KO defects, including crypt hyperplasia and Hippo signaling activation. Mammalian epithelial polarity regulates ISC/TA cell fate and proliferation via a Hippo-Ereg-mTOR cascade.

Immune-Mediated Reprogramming of Intestinal Stem Cells Drives STAT1-Dependent Myc Expression and Epithelial Regeneration in GI-Gvhd

Crypt apoptosis and regeneration are characteristic findings in GI-GVHD. Intestinal stem cells (ISCs) are critical for maintaining the intestinal epithelium, but their frequencies are reduced in experimental GVHD, and the mechanisms driving crypt regeneration in this context are poorly understood.To better understand the impact of GVHD on individual epithelial components, we performed single cell RNA sequencing (scRNAseq) of purified small intestine crypts from B6 mice during homeostasis and five days after B6 into B6 syngeneic (syn) or B10.Br into B6 allogeneic (allo) BMT. Sequenced cells were first partitioned into distinct clusters using PhenoGraph, and the cluster identities were annotated based on their gene expression profiles. Unsupervised clustering indicated multiple subpopulations within the various recognized crypt components (Fig 1A). While secretory lineages clustered similarly across experimental conditions, there was highly distinct clustering among ISC populations and striking dissimilarity between allo and syn ISCs. Quantification by computing the subpopulation's phenotypic distance, a composite score integrating both the number and amplitude of differentially expressed genes, indicated that ISCs demonstrated the greatest transcriptional change in response to GVHD among all crypt lineages (Fig 1B). Gene Set Enrichment Analysis (GSEA) highlighted activation of the interferon-γ (IFNγ) pathway in allo ISCs, and differential gene expression indicated that STAT1 was their most highly upregulated transcription factor. We then performed MHC-mismatched allo-BMT into STAT1-floxed x villin-Cre recipients. Consistent with a role in IFNγ-related toxicity, STAT1-deficient recipients initially demonstrated reduced GVHD pathology, as well as a reduction in proliferating Ki67 + cells (Fig 2A). However, the pathology reduction in STAT1-deficient recipients was transient, while reduction in crypt proliferation persisted. STAT1-deficient recipients ultimately demonstrated increased mortality after allo-BMT, indicating a complex response to epithelial STAT1 signaling in GVHD.While IFNγ can induce epithelial apoptosis and kill organoid cultures, organoid exposure to IFNγ augmented size even at IFNγ concentrations that did not impair viability in a STAT1-dependent manner, and co-culture with allo T cells augmented organoid size (Fig 2B, C). Moreover, cell cycle analysis showed augmentation of cell cycle in ISCs after IFNγ treatment in association with upregulation of cyclin D1 (Fig 2D), and human organoids also showed increased size in response to IFNγ treatment, further suggesting that this growth promotion was not simply a secondary response to tissue injury.In addition to the IFNγ signaling, GSEA of allo ISCs indicated activation of the Myc pathway. scRNAseq data showed specific upregulation of Myc in allo ISCs (Fig 3A). Myc expression within individual ISCs indicated that greater STAT1 expression and IFNγ signaling directly correlated with Myc expression in the same ISCs, providing a potential direct link between T-cell-derived IFNγ and ISC-dependent regeneration (Fig 3B). Additionally, intestinal organoid qPCR showed that Myc expression was upregulated after IFNγ treatment, and scRNAseq of IFNγ-treated organoids indicated this Myc upregulation was restricted to the ISC/progenitor compartment. Although Myc is downstream of Wnt signaling, which is critical for ISC maintenance, expression of the representative Wnt target gene Axin2 was downregulated after IFNγ treatment, and Irf1, a representative IFNγ/STAT1 target gene, was upregulated, suggesting that IFNγ/STAT1 signaling could replace Wnt/β-catenin as a driver of ISC Myc expression. We next examined Myc function and found that treatment with the Myc inhibitor 10058-F4 suppressed IFNγ-dependent organoid growth (Fig 3C). Finally, immunofluorescent staining showed Myc protein expression in intestinal crypts after allo-BMT in a STAT1-dependent manner (Fig 3D), and Myc inhibitor treatment in vivo suppressed crypt epithelial proliferation in mice with GVHD.In summary, we found that epithelial STAT1 contributes to crypt regeneration in GVHD by transmitting T-cell-derived JAK/STAT cytokine signaling to activate Myc expression in ISCs. Clinical use of JAK inhibitors in GVHD may inhibit this regenerative response, necessitating concurrent interventions aimed at restoring it. [Display omitted] DisclosuresHanash: Evive Biotech: Ended employment in the past 24 months.

TNFα Induces LGR5+ Stem Cell Dysfunction In Patients With Crohn's Disease.

Background & AimsTumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn’s disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids.MethodsHuman enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated.ResultsTNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing.ConclusionsTNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD.

How to Obtain a Mega-Intestine with Normal Morphology: In Silico Modelling of Postnatal Intestinal Growth in a Cd97-Transgenic Mouse

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Taking a Step Back: Insights into the Mechanisms Regulating Gut Epithelial Dedifferentiation.

Despite the environmental constraints imposed upon the intestinal epithelium, this tissue must perform essential functions such as nutrient absorption and hormonal regulation, while also acting as a critical barrier to the outside world. These functions depend on a variety of specialized cell types that are constantly renewed by a rapidly proliferating population of intestinal stem cells (ISCs) residing at the base of the crypts of Lieberkühn. The niche components and signals regulating crypt morphogenesis and maintenance of homeostatic ISCs have been intensely studied over the last decades. Increasingly, however, researchers are turning their attention to unraveling the mechanisms driving gut epithelial regeneration due to physical damage or infection. It is now well established that injury to the gut barrier triggers major cell fate changes, demonstrating the highly plastic nature of the gut epithelium. In particular, lineage tracing and transcriptional profiling experiments have uncovered several injury-induced stem-cell populations and molecular markers of the regenerative state. Despite the progress achieved in recent years, several questions remain unresolved, particularly regarding the mechanisms driving dedifferentiation of the gut epithelium. In this review, we summarize the latest studies, primarily from murine models, that define the regenerative processes governing the gut epithelium and discuss areas that will require more in-depth investigation.

A149 TRP53 LOSS IN KRT15+ INTESTINAL STEM CELLS SEVERELY AFFECTS SECRETORY CELL FATE AND SELF-RENEWAL

Abstract Background Intestinal epithelium homeostasis is maintained by two main populations of stem cells: Lgr5+ and reserve stem cells. Under injury, cell plasticity has been observed in progenitor and differentiated cells. We recently reported that Krt15+ cells are present in small intestinal and colon epithelia, and harbor self-renewal, multipotent and regenerative capacities. As in Lgr5+and reserve stem cells, hyperactivation of Wnt/b-catenin pathway in Krt15+ stem cells lead to tumor formation in the intestinal epithelium. While these intestinal stem cell populations can act as tumor-initiating cells in sporadic colon cancer, little is known about the cell-of-origin of colitis-associated colon cancer (CAC). TP53 alteration is reported as an early event in CAC. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ stem cells will perturb the epithelial homeostasis and lead to tumor formation. Aims Identify if Krt15+ cells may act as the cell-of-origin in colitis-associated colorectal cancer Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15-CrePR1;Trp53fl/fl (Krt15△Trp53) mice, induced Cre recombination by injecting RU486 (PR agonist) and euthanized the mice at different time points following recombination. Results Results Twelve-month following Cre recombination, adenoma formation was observed in a small proportion of Krt15△Trp53 mice. Though, Trp53 loss in Krt15+ cells severely perturbed the small intestinal morphology in every mouse studied. Increased crypt length and villi width was observed in Krt15△Trp53 vs control mice without any changes in cell proliferation. We also observed an increased number of Tuft cells and goblet cells in the villi of experimental mice. In the crypt, higher number of Paneth cells and aberrant presence of goblet cells were noted in Krt15△Trp53mice. Interestingly, we also observed crypt cells expressing goblet and Paneth cell markers and decreased Notch pathway activation suggesting dysregulation of secretory cell fate. Krt15△Trp53 mice display higher number of fibroblasts in the villi and the submucosa, as well as thickening of the muscularis layer. Interestingly, similar observations (accumulation of secretory cells and fibrosis) have been reported in IBD patients, supporting the possible role of Krt15+ cells in CAC. Furthermore, crypts isolated from Krt15△Trp53 mice rapidly die when seeded as organoids vs crypts from control mice, suggesting that the alterations observed in vivo in Paneth cells might interfere with the stem cell niche and therefore reduce self-renewal of Krt15+ cells. Conclusions Trp53 loss specifically in Krt15+ cells impaired cell fate decision, induced secretory cell hyperplasia, affected self-renewal ability, and initiated adenoma formation supporting the possible role of Krt15+ cells in gut inflammation and cancer. Funding Agencies Canada Research Chair, Cancer Research Society, CFI