Crypt Stem Cells Research Articles

A more physiological approach to lipid metabolism alterations in cancer: CRC-like organoids assessment.

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent “Warburg effect” blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.

Acetaminophen Intoxication Rapidly Induces Apoptosis of Intestinal Crypt Stem Cells and Enhances Intestinal Permeability.

Acetaminophen (APAP)‐induced liver injury is the most common cause of acute liver failure (ALF) in the Western world. APAP toxicity progresses to multiorgan dysfunction and thus has broader whole‐body implications. Importantly, greater 30‐day mortality has been observed in liver transplant recipients following ALF due to APAP‐related versus non‐APAP‐related causes. Reasons for this discrepancy have yet to be determined. Extrahepatic toxicities of APAP overdose may represent underappreciated and unaddressed comorbidities within this patient population. In the present study, rapid induction of apoptosis following APAP overdose was observed in the intestine, an organ that greatly influences the physiology of the liver. Strikingly, apoptotic cells appeared to be strictly restricted to the intestinal crypts. The use of leucine‐rich repeat‐containing G protein–coupled receptor 5 (LGR5) reporter mice confirmed that the LGR5‐positive (+) crypt base stem cells were disproportionately affected by APAP‐induced cell death. Although the apoptotic cells were cleared within 24 hours after APAP treatment, potentially long‐lived consequences on the intestine due to APAP exposure were indicated by prolonged deficits in gut barrier function. Moreover, small intestinal cell death was found to be independent of tumor necrosis factor receptor signaling and may represent a direct toxic insult to the intestine by exposure to high concentrations of APAP. Conclusion: APAP induces intestinal injury through a regulated process of apoptotic cell death that disproportionately affects LGR5+ stem cells. This work advances our understanding of the consequences of APAP toxicity in a novel organ that was not previously considered as a significant site of injury and thus presents potential new considerations for patient management.

Glycyrrhetinic Acid Maintains Intestinal Homeostasis via HuR.

Glycyrrhetinic acid (GA) is one of the main components of the traditional Chinese medicine of licorice, which can coordinate and promote the effects of other medicines in the traditional prescription. We found that GA could promote the proliferation, decrease the apoptotic rate, and attenuate DFMO-elicited growth arrest and delay in restitution after wounding in IEC-6 cells via HuR. GA failed to promote proliferation and to suppress apoptosis after silencing HuR by siRNA in IEC-6 cells. Furthermore, with the model of small intestinal organoids developed from intestinal crypt stem cells, we found that GA could increase HuR and its downstream ki67 levels to promote intestinal organoid development. In the in vivo assay, GA was shown to maintain the integrity of the intestinal epithelium under the circumstance of 48 h-fasting in rats via raising HuR and its downstream genes such as EGF, EGFR, and MEK. These results suggested that via HuR modulation, GA could promote intestinal epithelium homeostasis, and therefore contribute to the absorption of constituents from other medicines co-existing in the traditional prescription with licorice in the small intestine. Our results provide a new perspective for understanding the effect of licorice on enhancing the therapeutic effect of traditional prescriptions according to the traditional Chinese medicine theory.

Receptor subtype discrimination using extensive shape complementary designed interfaces.

Discriminating between closely related members of a protein family which differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe an approach for computationally designing binders targeting functional sites with large, shape complementary interfaces to ‘read out’ subtle sequence differences for sub-type specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.

776 – Deletion of Claudin-7 Leads to the Loss of Intestinal Crypt Stem Cells and Disruption of Epithelial Self-Renewal in Mouse Small Intestines

776 – Deletion of Claudin-7 Leads to the Loss of Intestinal Crypt Stem Cells and Disruption of Epithelial Self-Renewal in Mouse Small Intestines

Age-related dysfunction of the DNA damage response in intestinal stem cells

BackgroundSenescence increases the risks of inflammatory bowel diseases and colon cancer. Intestinal stem cells (ISCs) in crypts differentiate into epithelial cells and thereby maintain intestinal homeostasis. However, the influence of aging on the functions of ISCs is largely unknown. The mutation rate is highest in the small and large intestines. Numerous types of naturally occurring DNA damage are removed by the DNA damage response (DDR). This response induces DNA repair and apoptosis; therefore, its dysregulation leads to accumulation of damaged DNA and consequently cellular dysfunctions, including tumorigenesis. This study investigated whether aging affects the DDR in mouse ISCs.MethodsYoung (2–3-month-old) and old (> 19-month-old) Lgr5-EGFP-IRES-creERT2 mice were irradiated. The DDR in Lgr5-positive ISCs was compared between these mice by immunohistochemical analyses.ResultsInduction of DDR marker proteins (phosphorylated ATR and 53BP1), inflammatory factors (phosphorylated NF-κB and interleukin-6), and a mitochondrial biogenesis-associated gene (peroxisome proliferator-activated receptor-γ coactivator 1α) was lower in old ISCs than in young ISCs in vivo.ConclusionThe competence of the DDR in ISCs declines with age in vivo.

Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model.

Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children younger than 5 years. Our ability to combat bacterial pathogens has been hindered by antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri. An S. flexneri 2457T-specific bacteriophage was isolated and assessed through kill curve experiments and infection assays with colorectal adenocarcinoma HT-29 cells and a novel human intestinal organoid-derived epithelial monolayer model. In our treatment protocol, organoids were generated from intestinal crypt stem cells, expanded in culture, and seeded onto transwells to establish 2-dimensional monolayers that differentiate into intestinal cells. The isolated bacteriophage efficiently killed S. flexneri 2457T, other S. flexneri strains, and a strain of 2457T harboring an antibiotic resistance cassette. Analyses with laboratory and commensal Escherichia coli strains demonstrated that the bacteriophage was specific to S. flexneri, as observed under co-culture conditions. Importantly, the bacteriophage prevented both S. flexneri 2457T epithelial cell adherence and invasion in both infection models. Bacteriophages offer feasible alternatives to antibiotics for eliminating enteric pathogens, confirmed here by the bacteriophage-targeted killing of S. flexneri. Furthermore, application of the organoid model has provided important insight into Shigella pathogenesis and bacteriophage-dependent intervention strategies. The screening platform described herein provides proof-of-concept analysis for the development of novel bacteriophage therapies to target antibiotic-resistant pathogens.

Activation of G protein-coupled estrogen receptor protects intestine from ischemia/reperfusion injury in mice by protecting the crypt cell proliferation.

The intestinal ischemia/reperfusion (I/R) injury is a common clinical event related with high mortality in patients undergoing surgery or trauma. Estrogen exerts salutary effect on intestinal I/R injury, but the receptor type is not totally understood. We aimed to identify whether the G protein-coupled estrogen receptor (GPER) could protect the intestine against I/R injury and explored the mechanism. Adult male C57BL/6 mice were subjected to intestinal I/R injury by clamping (45 min) of the superior mesenteric artery followed by 4 h of intestinal reperfusion. Our results revealed that the selective GPER blocker abolished the protective effect of estrogen on intestinal I/R injury. Selective GPER agonist G-1 significantly alleviated I/R-induced intestinal mucosal damage, neutrophil infiltration, up-regulation of TNF-α and cyclooxygenase-2 (Cox-2) expression, and restored impaired intestinal barrier function. G-1 could ameliorate the impaired crypt cell proliferation ability induced by I/R and restore the decrease in villus height and crypt depth. The up-regulation of inducible nitric oxide synthase (iNOS) expression after I/R treatment was attenuated by G-1 administration. Moreover, selective iNOS inhibitor had a similar effect with G-1 on promoting the proliferation of crypt cells in the intestinal I/R model. Both GPER and iNOS were expressed in leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) positive stem cells in crypt. Together, these findings demonstrate that GPER activation can prompt epithelial cell repair following intestinal injury, which occurred at least in part by inhibiting the iNOS expression in intestinal stem cells (ISCs). GPER may be a novel therapeutic target for intestinal I/R injury.

Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling.

It has been well established that radiation-induced gastrointestinal injury is manifested through loss of intestinal crypt stem cells and disruption of the mucosal layers, resulting in diarrhoea, weight loss, electrolyte imbalance, infection and mortality. Podophyllotoxin and rutin in combination (G-003M) has been reported to regulate endogenous cellular antioxidant defense systems and inflammatory response. However, the mechanism by which G-003M ameliorates radiation-induced intestinal stem cell (ISC) injury remains unclear. Here, we hypothesize the radioprotective potential of G-003M would amplify the intestinal crypt stem cells through upregulation of Wnt/β-catenin signaling and accelerate the reconstitution of the irradiated intestine. Our results showed significant functional and structural intestine regeneration in irradiated animals following G-003M treatment which resulted in improved animal survival. Immunohistochemical examination revealed an enhancement in Lgr5+ ve crypt stem cells. Increased β-catenin nuclear translocation resulted in upregulation of β-catenin target genes that supported ISC renewal and expansion in G-003M-treated mice, as compared to IR-treated mice. However, G-003M could not rescue the Wnt knockdown cohorts (XAV939 treated) which exhibited greater incidence of intestinal apoptosis, DNA damage and crypt depopulation upon radiation exposure. These findings suggest the involvement of Wnt pathway during G-003M mediated amelioration of IR-induced ISC injury. G-003M also minimised acute inflammation by restricting the infiltration of immune cells into the intestinal venules. Furthermore, G-003M treated animals showed improved anti-tumor response compared to FDA approved Amifostine. Taken together, our findings suggest that G-003M may be used as a potential countermeasure for radiation injuries as well as an adjuvant during anti-cancer therapy.

Human Intestinal Enteroids With Inducible Neurogenin-3 Expression as a Novel Model of Gut Hormone Secretion.

Background & AimsEnteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but because of their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are biologically relevant in an in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, similar to the intestine, HIEs spontaneously produce few EECs, which limits their study.MethodsTo increase the number of EECs in HIEs, we used lentivirus transduction to stably engineer jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIEs). We examined the impact of NGN3 induction on EECs by quantifying the increase in the enterochromaffin cells and other EEC subtypes. We functionally assessed secretion of serotonin and EEC hormones in response to norepinephrine and rotavirus infection.ResultsTreating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, showing increased enterochromaffin cell differentiation. Despite increased ChgA-positive cells, other differentiated cell types of the epithelium remained largely unchanged by gene expression and immunostaining. RNA sequencing of doxycycline-induced tetNGN3-HIEs identified increased expression of key hormones and enzymes associated with several other EEC subtypes. Doxycycline-induced tetNGN3-HIEs secreted serotonin, monocyte chemoattractant protein-1, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin in response to norepinephrine and rotavirus infection, further supporting the presence of multiple EEC types.ConclusionsWe have combined HIEs and inducible-NGN3 expression to establish a flexible in vitro model system for functional studies of EECs in enteroids and advance the molecular and physiological investigation of EECs.

An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition.

C-terminal binding protein 2 (CtBP2) drives intestinal polyposis in the Apc min mouse model of human Familial Adenomatous Polyposis. As CtBP2 is targetable by an inhibitor of its dehydrogenase domain, understanding CtBP2’s role in adenoma formation is necessary to optimize CtBP-targeted therapies in Apc mutated human neoplasia. Tumor initiating cell (TIC) populations were substantially decreased in Apc min Ctbp2+/- intestinal epithelia. Moreover, normally nuclear Ctbp2 was mislocalized to the cytoplasm of intestinal crypt stem cells in Ctbp2+/- mice, both Apc min and wildtype, correlating with low/absent CD133 expression in those cells, and possibly explaining the lower burden of polyps in Apc min Ctbp2+/- mice. The CtBP inhibitor 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) also robustly downregulated TIC populations and significantly decreased intestinal polyposis in Apc min mice. We have therefore demonstrated a critical link between polyposis, intestinal TIC’s and Ctbp2 gene dosage or activity, supporting continued efforts targeting CtBP in the treatment or prevention of Apc mutated neoplasia.

Hyperbaric Oxygen Increases Stem Cell Proliferation, Angiogenesis and Wound-Healing Ability of WJ-MSCs in Diabetic Mice.

Hyperbaric oxygen therapy (HBOT) is effective for the medical treatment of diverse diseases, infections, and tissue injury. In fact, in recent years there is growing evidence on the beneficial effect of HBOT on non-healing ischemic wounds. However, there is still yet discussion on how this treatment could benefit from combination with regenerative medicine strategies. Here we analyzed the effects of HBOT on three specific aspects of tissue growth, maintenance, and regeneration: (i) modulation of adult rodent (Mus musculus) intestinal stem cell turnover rates; (ii) angiogenesis dynamics during the development of the chorio-allantoic membrane (CAM) in Gallus gallus embryos; (iii) and wound-healing in a spontaneous type II diabetic mouse model with a low capacity to regenerate skin. To analyze these aspects of tissue growth, maintenance, and regeneration, we used HBOT alone or in combination with cellular therapy. Specifically, Wharton Jelly Mesenchymal Stem cells (WJ-MSC) were embedded in a commercial collagen-scaffold. HBOT did not affect the metabolic rate of adult mice nor of chicken embryos. Notwithstanding, HBOT modified the proliferation rate of stem cells in the mice small intestinal crypts, increased angiogenesis in the CAM, and improved wound-healing and tissue repair in diabetic mice. Moreover, our study demonstrates that combining stem cell therapy and HBOT has a collaborative effect on wound-healing. In summary, our data underscore the importance of oxygen tension as a regulator of stem cell biology and support the potential use of oxygenation in clinical treatments.

Modeling Cell Dynamics in Colon and Intestinal Crypts: The Significance of Central Stem Cells in Tumorigenesis.

Colon and intestinal crypts have been widely chosen to study cell dynamics because of their fairly simple structures. In the colon and intestinal crypts, stem cells (SCs) are located at very bottom of the crypt, fully differentiated cells (FDs) are located in the top of the crypt, and transit-amplifying cells (TAs) are in the middle of the crypt between FDs and SCs. Recently, it has been discovered that there are two types of stem cells in the intestinal crypts: central stem cells (CeSCs) and border stem cells. To investigate dynamics of mutants in colon and intestinal crypts, we develop a four-compartmental stochastic model, which includes two SC compartments, and TAs and FDs compartments. We calculate the probability of the progeny of marked or mutant cells located at each of these compartments taking over the entire crypt or being washed out from the crypt. We found that the progeny of CeSCs will take over the entire crypt with a probability close to one. Interestingly, the progeny of advantageous mutant TAs and FDs will be washed out faster than disadvantageous mutants. Saliently, the model predicts that the time that the progeny of wild-type central stem cells will take over the mouse intestinal crypt is around 60 days, which is in perfect agreement with an experimental observation.

Marine microalgae bioengineered Schizochytrium sp. meal hydrolysates inhibits acute inflammation

Bioengineered marine microalgae Schizochytrium sp. is currently used to produce docosahexaenoic acid (DHA). However, following DHA extraction, the remaining protein-rich materials are not well utilized. In this study, we report that marine microalgae bioengineered Schizochytrium sp. hydrolysate (MESH), which exhibits a unique peptide profile as identified by Ultra Performance Liquid Chromatography coupled with Q-TOF mass spectrometry(UPLC/Q-TOF-MS), ameliorated bowel inflammation in mice. In a mouse model of experimentalcolitis induced by dextran sulfate sodium, compared with the control mice, the mice treated with MESH were highly resistant to colitis, as demonstrated by marked reductions in body weight loss, clinical colitis scores, colonic histological damage, and colonic inflammation. Mechanistically, MESH attenuated the induction of pro-inflammatory cytokines and increased the induction of anti-inflammatory cytokines. MESH also promoted the proliferation of colonic crypt stem cells and progenitor cells required for crypt repair. Collectively, these results reveal a previously unrecognized role of MESH as a potential anti-inflammatory treatment for colitis.

The Role of Nrf2 in the Response to Normal Tissue Radiation Injury

The transcription factor Nrf2 is an important modulator of antioxidant and drug metabolism, carbohydrate and lipid metabolism, as well as heme and iron metabolism. Regulation of Nrf2 expression occurs transcriptionally and post-transcriptionally. Post-transcriptional regulation entails ubiquitination followed by proteasome-dependent degradation. Additionally, Nrf2-mediated gene expression is subject to negative regulation by ATF3, Bach1 and cMyc. Nrf2-mediated gene expression is an important regulator of a cell's response to radiation. Although a majority of studies have shown that Nrf2 deficient cells are radiosensitized and Nrf2 over expression confers radioresistance, Nrf2's role in mediating the radiation response of crypt cells is controversial. The Nrf2 activator CDDO attenuates radiation-mediated crypt injury, whereas intestinal crypts in Nrf2 null mice are radiation resistant. Further investigation is needed in order to define the relationship between Nrf2 and radiation sensitivity in Lgr5+ and Bmi1+ cells that regulate regeneration of crypt stem cells. In hematopoietic compartments Nrf2 promotes the survival of irradiated osteoblasts that support long-term hematopoietic stem cell (LT-HSC) niches. Loss of Nrf2 in LT-HSCs increases stem cell intrinsic radiosensitivity, with the consequence of lowering the LD5030. An Nrf2 deficiency drives LT-HSCs from a quiescent to a proliferative state. This results in hematopoietic exhaustion and reduced engraftment after myoablative irradiation. The question of whether induction of Nrf2 in LT-HSC enhances hematopoietic reconstitution after bone marrow transplantation is not yet resolved. Irradiation of the lung induces pulmonary pneumonitis and fibrosis. Loss of Nrf2 promotes TGF-β/Smad signaling that induces ATF3 suppression of Nrf2-mediated target gene expression. This, in turn, results in elevated reactive oxygen species (ROS) and isolevuglandin adduction of protein that impairs collagen degradation, and may contribute to radiation-induced chronic cell injury. Loss of Nrf2 impairs ΔNp63 stem/progenitor cell mobilization after irradiation, while promoting alveolar type 2 cell epithelial-mesenchymal transitions into myofibroblasts. These studies identify Nrf2 as an important factor in the radiation response of normal tissue.

Predominant Asymmetrical Stem Cell Fate Outcome Limits the Rate of Niche Succession in Human Colonic Crypts

Stem cell (SC) dynamics within the human colorectal crypt SC niche remain poorly understood, with previous studies proposing divergent hypotheses on the predominant mode of SC self-renewal and the rate of SC replacement. Here we use age-related mitochondrial oxidative phosphorylation (OXPHOS) defects to trace clonal lineages within human colorectal crypts across the adult life-course. By resolving the frequency and size distribution of OXPHOS-deficient clones, quantitative analysis shows that, in common with mouse, long-term maintenance of the colonic epithelial crypt relies on stochastic SC loss and replacement mediated by competition for limited niche access. We find that the colonic crypt is maintained by ~5 effective SCs. However, with a SC loss/replacement rate estimated to be slower than once per year, our results indicate that the vast majority of individual SC divisions result in asymmetric fate outcome. These findings provide a quantitative platform to detect and study deviations from human colorectal crypt SC niche homeostasis during the process of colorectal carcinogenesis.

Epimorphin regulates the intestinal stem cell niche via effects on the stromal microenvironment.

Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin ( Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission, and small bowel length in vivo. Stem cell-derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim-/- and WT mice. Enteroids cocultured with ISEMFS had increased growth and crypt-like budding compared with enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared with cocultures with wild-type (WT) ISEMFs. In primary crypt cultures, Epim-/- enteroids had significantly increased surface area and budding compared with WTs. However, stem cell assays comparing the number of Epim-/- vs. WT colony-forming units after first passage showed no differences in the absence of ISEMF support. Epim-/- vs. WT ISEMFs had increased Wnt4 expression, and addition of Wnt4 to WT cocultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment. NEW & NOTEWORTHY The role of subepithelial intestinal myofibroblasts (ISEMFs) in the gut stem cell niche is controversial. We provide novel evidence supporting ISEMFs as important niche contributors. We show that the in vivo intestinal effects of deletion of myofibroblast Epim can be recapitulated in crypt stem cell cultures in vitro. ISEMFs support cocultured stem cell proliferation and enteroid growth, and these effects are augmented by deletion of Epim, a syntaxin that regulates myofibroblast cell secretion.

Sox9‐independent Control of Intestinal Paneth Cell Differentiation and Function

The genetic and signaling mechanisms controlling the generation and function of differentiated cell types are of continued interest. The intestinal Paneth cell (PC) is a post‐mitotic and dedicated secretory cell type with reported functions in supporting intestinal stem cell (ISC) maintenance in the crypt stem cell niche, and in secreting antimicrobial peptides to protect the crypt from potentially pathogenic luminal microbiota. Previous studies have reported a near‐complete loss of PCs (as defined by morphological and marker‐based criteria) in epithelium where the transcription factor Sox9 is genetically ablated, leading to the proposition that Sox9 is required for PC differentiation. Currently, however, the mechanisms through which Sox9 controls PC differentiation are not known. We have found evidence in Sox9 knockout (KO) small intestine (SI) that cells at the crypt base frequently express PC‐markers such as Lysozyme (Lyz) and Cryptdin, exhibit apical granules, and are post‐mitotic, all of which are features indicative of PC differentiation. The numbers of Lyz+ cells are highest in the distal Sox9 KO SI but are significantly reduced relative to control, and are essentially absent in the proximal SI, suggesting that regionally patterned signals may drive PC differentiation independent of Sox9. Apical granularity and expression levels of Lyz/Cryptdin are also altered, suggesting that while Sox9 KO ISCs can initiate PC differentiation, they are unable to reach full maturity. Antibody and qPCR based detection of PC gene products (including secreted antimicrobials, growth factors, and transcriptional regulators proposed to drive PC differentiation downstream of Sox9) are maintained in Sox9 KO cultured intestinal organoids and are elevated in organoids treated with pharmacological agents that drive the PC differentiation program. These results indicate that Sox9‐deficient ‘Paneth‐like’ cells are continually generated in culture and can be pushed toward PC differentiation with factors known to stimulate the PC differentiation program. We conclude that Sox9 is not required for the activation of at least sub‐sets of gene products associated with PC differentiation and function. Ongoing experiments are designed to investigate Sox9‐ dependent and independent pathways toward PC fate, and to characterize the functionality of Sox9−/− ‘Paneth‐like’ cells compared to controls.Support or Funding InformationRO1 DK091427‐05F32 DK115016‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of cells expressing OLFM4 and LGR5 mRNA by in situ hybridization in the yolk sac and small intestine of embryonic and early post-hatch chicks

The chicken yolk sac (YS) and small intestine are essential for nutrient absorption during the pre-hatch and post-hatch periods, respectively. Absorptive enterocytes and secretory cells line the intestinal villi and originate from stem cells located in the intestinal crypts. Similarly, in the YS, there are absorptive and secretory cells that presumably originate from a stem cell population. Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4) are 2 widely used markers for intestinal stem cells. The objective of this study was to map the distribution of putative stem cells expressing LGR5 and OLFM4 mRNA in the chicken small intestine from the late embryonic period to early post hatch and the YS during embryogenesis. At embryonic d 11, 13, 15, 17, and 19, the YS was collected (n = 3), and small intestine was collected at embryonic d 19, d of hatch (doh), and d 1, 4, and 7 post hatch (n = 3). Cells expressing OLFM4 and LGR5 mRNA were identified by in situ hybridization. In the YS, cells expressing only LGR5 and not OLFM4 mRNA were localized to the vascular endothelial cells lining the blood vessels. In the small intestine, cells in the intestinal crypt expressed both LGR5 and OLFM4 mRNA. Staining for OLFM4 mRNA was more intense than LGR5 mRNA, demonstrating that Olfm4 is a more robust marker for stem cells than Lgr5. At embryonic d 19 and doh, cells staining for OLFM4 mRNA were already present in the rudimentary crypts, with the greatest staining in the duodenal crypts. The intensity of OLFM4 mRNA staining increased from doh to d 7 post hatch. Dual label staining at doh for the peptide transporter PepT1 and Olfm4 revealed a population of cells above the crypts that did not express Olfm4 or PepT1 mRNA. These cells are likely progenitor transit amplifying cells. Thus, avians and mammals share similarity in the ontogeny of stem cells in the intestinal crypts.

Role of DDX3 in the pathogenesis of inflammatory bowel disease.

When crypt stem cells of the gastrointestinal tract become injured, the result is increased synthesis of pro-inflammatory cytokines and matrix metalloproteinases by their progeny – the colonic epithelium. Chronic inflammation of the gastrointestinal tract is a characteristic of inflammatory bowel disease, which includes Crohn’s Disease and Ulcerative Colitis. In our ongoing investigation to decipher the characteristic functions of a RNA helicase gene, DDX3, we identified high DDX3 expression by immunohistochemistry of colon biopsy samples, which included chronic/mild Morbus Crohn, active Morbus Crohn, Chronic/mild Colitis Ulcerosa and active Colitis Ulcerosa in epithelium and stromal compartments. We used a small molecule inhibitor of DDX3, RK-33, on two human colonic epithelial cell lines, HCEC1CT and HCEC2CT and found that RK-33 was able to decrease expression of MMP-1, MMP-2, MMP-3, and MMP-10. Moreover, forced differentiation of a human colonic cancer cell line, HT29, resulted in decreased DDX3 levels, indicating that DDX3 contributes to the modulation of colonic epithelium differentiation. In conclusion, our results revealed novel functions of DDX3 in inflammatory bowel disease and indicate a potential for using RK-33 as a systemic therapy to promote not only differentiation of transformed colonic epithelium but also to reduce MMP expression and thus elicit a decreased inflammatory response.