Intestinal Epithelial Stem Cells Research Articles

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency.

Primary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined. In this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH -/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy). Results from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively. Multivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

Non-professional efferocytosis of Salmonella-infected intestinal epithelial cells in the neonatal host.

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.

THE DEVELOPMENT OF HUMAN PRIMARY CULTURE MODELS TO MODEL INFLAMMATORY SIGNALING IN CROHN’S DISEASE AND ULCERATIVE COLITIS

Abstract Intestinal inflammation is a major pathological feature associated with gastrointestinal disorders such as Crohn’s disease and ulcerative colitis (UC). To unravel the complex mechanisms underlying these conditions and to develop effective therapeutic strategies, it is crucial to recapitulate intestinal inflammatory signaling using accurate and biologically relevant experimental systems. To establish primary cell-based models that reprise the inflammatory signaling in Crohn’s and UC, we developed the RepliGut platform. Briefly, we established a biobank of primary intestinal stem cells, derived from healthy full-length intestines from a range of donors of different sexes, ages, and ethnicities. From each donor, intestinal epithelial stem cells were isolated from each region of the small intestine and colon (duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon). The intestinal stem cells were passed to generate large and quality-controlled cell lots of “ready-to-use” vials which are cryopreserved and can be thawed directly onto 12- or 96-well Transwell inserts, generating the RepliGut platform. The RepliGut culture conditions allow for investigation into the physiology of intestinal epithelial cells as either proliferative stem or differentiated cell types and enable studies on focused regional differences in the intestinal tract, while the Transwell platform allows apical and basal media access that facilitates barrier function measures and convenient bi-lateral media sampling. Using this system, Crohn’s and UC-like inflammation can be produced via treatment with the most clinically and therapeutically validated cytokines associated with disease pathophysiology, TNF-α and IFN-γ. Circulating concentrations of both TNF-α and IFN-γ are observed to be elevated in patients with inflammatory bowel diseases and therapeutic antagonists including adalimumab (TNF-α) and JAK inhibitors such as tofacitinib (IFN-γ) represent the current standard of care. On the RepliGut platform, both TNF-α and IFN-γ produced a dose-dependent loss in epithelial barrier function, increased cytokine secretion, and caused cellular death, all of which were mitigated by adalimumab and tofacitinib, respectively. Moreover, at clinically relevant concentrations, combinations of TNF-α and IFN-γ produced synergistic inflammatory response on the intestinal epithelial cultures. In summary, the RepliGut platform provides a convenient and high-throughput platform that reproduces cytokine-induced intestinal inflammation for mechanistic studies and drug screening.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells

BackgroundIntestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture.MethodsWe established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.ResultsThe proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.ConclusionsThe high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

PSV-8 Early Life Adversity Impacts Intestinal Epithelial Development and Stem Cells Activities in Pigs

Abstract Early life adversity significantly impacts organ development and predisposes animals towards greater risk of diseases later in life. In the previous research in pigs, long lasting intestinal barrier dysfunctions have been found to be associated with early weaning stress. However, the underlying mechanism is not fully elucidated. Intestinal epithelial functions are associated with the number and maturation of multicellular components, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (EEC). Intestinal epithelial stem cells (IESC) give rise to the whole epithelium within 5 to 7 days. Such rapid turnover rate allows intestinal adaptation to stress conditions. We hypothesized that the early life stress impacted stem cells activities which would contribute to the long-lasting post-stress functional changes in intestinal epithelium. The objective of the present study is to evaluate the epithelium component cells and IESC proliferation and differentiation activities in an early weaning stress model in pigs. Piglets (n = 18) were obtained from commercial litters and were pair-weaned at the age of d 15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were co-housed and raised under the same conditions. At the age of d 35, ileal tissues were isolated and fixed for morphological measurements. Intestinal crypts were isolated and cultured in the Matrigel matrix to form enteroids. The proliferation activities of IESCs were measured by enteroids morphology; differentiation capacity was measured by quantification of epithelial cell markers with and without chemicals-induced enteroids differentiation. Gene expressions and (or) staining of specific markers for IESCs (Olfm4, BMI1), enterocytes (ALP1), goblet cells (Muc2), Paneth cells (LYZ), and EECs (ChgA) in ileal tissue and ileal enteroids were also quantified. Data were analyzed by the student’s t-test. Our results showed that EW significantly (P < 0.05) reduced ileal villus width and crypt fission rate, while no changes were found on villus height and crypt depth compared with LW control. The alterations of ileal epithelium components in EW pigs were indicated by significantly fewer number of EECs, greater number of the goblet cells, and reduced ALP1 marker expression. The enteroids formation and budding rates were both significantly suppressed in EW pigs, which indicated a reduced IESC proliferating activity. In the fully developed enteroids, gene expressions of LYZ, ALP1, BMI1, Olfm4, and Hes1 were least in EW, but Muc2 expression was greater compared with control. In the differentiation culture media, the enteroids from EW pigs exhibited significantly enhanced potential for goblet cell differentiation but reduced capacity for enterocyte differentiation compared with LW pigs. Overall, the results indicate that early weaning stress affects the programming and activity of IESCs, which leads to an altered epithelium cell population and intestinal functions. Further understanding of mechanisms regulating IESCs activity will shed light on novel strategies mitigating stress-associated intestinal dysfunctions.

Glutamine Regulates Gene Expression Profiles to Increase the Proliferation of Porcine Intestinal Epithelial Cells and the Expansion of Intestinal Stem Cells.

The intestinal epithelium is known for its rapid self-renewal, and glutamine is crucial in providing carbon and nitrogen for biosynthesis. However, understanding how glutamine affects gene expression in the intestinal epithelium is limited, and identifying the essential genes and signals involved in regulating intestinal epithelial cell growth is particularly challenging. In this study, glutamine supplementation exhibited a robust acceleration of intestinal epithelial cell proliferation and stem cell expansion. RNA sequencing indicated diverse transcriptome changes between the control and glutamine supplementation groups, identifying 925 up-regulated and 1152 down-regulated genes. The up-regulated DEGs were enriched in the KEGG pathway of cell cycle and GO terms of DNA replication initiation, regulation of phosphatidylinositol 3-kinase activity, DNA replication, minichromosome maintenance protein (MCM) complex, and ATP binding, whereas the down-regulated DEGs were enriched in the KEGG pathway of p53 signaling pathway, TNF signaling pathway, and JAK-STAT signaling pathway and GO terms of inflammatory response and intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress. Furthermore, GSEA analysis revealed a significant up-regulation of the cell cycle, DNA replication initiation, ATP-dependent RNA helicase activity, and down-regulation of the TNF signaling pathway. The protein-protein association network of the intersecting genes highlighted the significance of DNA replication licensing factors (MCM3, MCM6, and MCM10) in promoting intestinal epithelial growth in response to glutamine. Based on these findings, we propose that glutamine may upregulate DNA replication licensing factors, leading to increased PI3K/Akt signaling and the suppression of TNF, JAK-STAT, and p53 pathways. Consequently, this mechanism results in the proliferation of porcine intestinal epithelial cells and the expansion of intestinal stem cells.

Mettl14-mediated m6 A modification regulates the abnormal differentiation of small intestinal epithelial stem cells in diabetic state.

Diabetes mellitus (DM) and its related complications are a global epidemic characterized by high morbidity and mortality. However, little is known about diabetic enteropathy (DE) and its the potential underlying mechanism. Intestinal epithelial stem cells (IESCs) were harvested from experimental mice, and the levels of dominant N6-methyladenosine (m6 A)-related enzyme were detected by RT-PCR, Western blotting, immunohistochemistry. The role of Mettl14 in the abnormal differentiation of intestinal epithelial cells (IECs) during DM was confirmed by knockdown experiments. RT-PCR, MeRIP, and bioinformatics analysis were carried out to confirm the downstream target of Mettl14. Through bioinformatics analysis, RT-PCR, and Western blotting, we further analyzed the differentiation-related gene in the IECs from mice with DM. In this study, the levels of Mettl14 and m6 A were higher in db/db mice than that in control mice. And abnormal differentiation of IECs in DM was associated with Mettl14 overexpression. Additionally, Mettl14 is a major determinant of IESCs identity and organoid-forming upon DM state. Mechanistically, we revealed that the candidate binding target of Mettl14 was Fzd2 mRNA and affected Fzd2 stability. Moreover, Mettl14 downregulation was observed to attenuate the abnormal differentiation of IECs through modulating Fzd2 m6A modification in DM state. Together, our results provide definitive evidence for the essential role of Mettl14 in differentiation of IESCs in DM state.

Mesenchymal Stem Cell-Derived Exosomes are Effective for Radiation Enteritis and Essential for the Proliferation and Differentiation of Lgr5+ Intestinal Epithelial Stem Cells by Regulating Mir-195/Akt/β-Catenin Pathway.

Radiation enteritis (RE) is a common complication of abdominal or pelvic radiotherapy, which when severe, could be life-threatening. Currently, there are no effective treatments. Studies have shown that mesenchymal stem cells (MSCs)-derived exosomes (MSC-exos) exhibit promising therapeutic effects in inflammatory diseases. However, the specific role of MSC-exos in RE and the regulatory mechanisms remain elusive. In vivo assay was carried out by injecting MSC-exos into the total abdominal irradiation (TAI)-induced RE mouse model. For in vitro assay, Lgr5-positive intestinal epithelial stem cells (Lgr5+ IESC) were extracted from mice, followed by irradiation along with MSC-exos treatment. HE staining was performed to measure histopathological changes. mRNA expression of inflammatory factors TNF-α and IL-6 and stem cell markers LGR5, and OCT4 were quantified by RT-qPCR. EdU and TUNEL staining was performed to estimate cell proliferation and apoptosis. MiR-195 expression in TAI mice and radiation-induced Lgr5+ IESC was tested. We found that the injection of MSC-exos inhibited inflammatory reaction, increased stem cell marker expression, and maintained intestinal epithelial integrity in TAI mice. Furthermore, MSC-exos treatment increased the proliferation and simultaneously suppressed apoptosis in radiation-stimulated Lgr5+ IESC. MiR-195 expression increased by radiation exposure was decreased by MSC-exos therapy. MiR-195 overexpression facilitated the progress of RE by counteracting the effect of MSC-exos. Mechanistically, the Akt and Wnt/β-catenin pathways inhibited by MSC-exos were activated by miR-195 upregulation. MSC-Exos are effective in treating RE and are essential for the proliferation and differentiation of Lgr5+ IESCs. Moreover, MSC-exos mediates its function by regulating miR-195 Akt β-catenin pathways.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Heavy metal-laden particulate dust disrupts intestinal epithelial stem cells and lineage differentiation

Chronic heavy metals exposure has been associated with intestinal inflammation and higher incidences of colorectal cancer, however there is no molecular evidence that metal laden-particulates can cause injury to the gut epithelia. Communities that live adjacent to abandoned uranium mines have higher incidence of metabolic and digestive diseases; however, the cause is unknown. The goal of this study is to characterize particulate dust containing non-fissile uranium as an environmental toxicant that damages intestinal epithelia and characterize the subsequent transcriptomic changes. We hypothesized that non-fissile uranium will alter the differentiation pattern of progenitor cells in the crypt biasing them towards the secretory lineage. Human colonoids derived from healthy adult colonic biopsies (UNM IRB approved study 18-626 and 18-171) were established and shown to physiologically model the intestinal epithelia. Colonoids were acutely exposed (24h) to non-fissile uranium dust obtained near the Jackpile uranium mine in New Mexico, one of the largest open pit uranium mines in the US, located on the Laguna Pueblo. Control and dust-exposed colonoids (n=3 unique donors) were dissociated into single cells and processed for droplet-based single cell sequencing (scRNAseq). scRNAseq identified significant changes in the secretory lineage in uranium dust-exposed colonoids. Specifically, there was a four-fold expansion of enteroendocrine cells (EEC) with increases in EEC hormones serotonin and PYY in all colonoid lines. Proliferative pathways, such as Wnt signaling and negative regulation of apoptosis were upregulated in these EECs. These transcriptomic changes in EECs and proliferative cells and increased basolateral secretion of secretory granules and serotonin was validated in colonoids (˃5 biologically unique human donors) via RNA FISH, immunofluorescence and ELISAs. Our results demonstrate that acute uranium dust exposure directly induces transcriptomic and cellular changes in intestinal epithelia independently of microbiota, stroma, and immune cells. These results indicate that adult human colonoids are a relevant model for investigating environmental toxicants on intestinal epithelia and provide an improved understanding of uranium dust as a model of intestinal injury. This study was funded by NIDDK (K01DK106323), NIEHS (R56ES034400, P42ES025589), and NIGMS (P20GM121176, P20GM130422). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ecological niches for colorectal cancer stem cell survival and thrival.

To date, colorectal cancer is still ranking top three cancer types severely threatening lives. According to cancer stem cell hypothesis, malignant colorectal lumps are cultivated by a set of abnormal epithelial cells with stem cell-like characteristics. These vicious stem cells are derived from intestinal epithelial stem cells or transformed by terminally differentiated epithelial cells when they accumulate an array of transforming genomic alterations. Colorectal cancer stem cells, whatever cell-of-origin, give rise to all morphologically and functionally heterogenous tumor daughter cells, conferring them with overwhelming resilience to intrinsic and extrinsic stresses. On the other hand, colorectal cancer stem cells and their daughter cells continuously participate in constructing ecological niches for their survival and thrival by communicating with adjacent stromal cells and circulating immune guardians. In this review, we first provide an overview of the normal cell-of-origin populations contributing to colorectal cancer stem cell reservoirs and the niche architecture which cancer stem cells depend on at early stage. Then we survey recent advances on how these aberrant niches are fostered by cancer stem cells and their neighbors. We also discuss recent research on how niche microenvironment affects colorectal cancer stem cell behaviors such as plasticity, metabolism, escape of immune surveillance as well as resistance to clinical therapies, therefore endowing them with competitive advantages compared to their normal partners. In the end, we explore therapeutic strategies available to target malignant stem cells.

Changes in equine intestinal stem/progenitor cell number at resection margins in cases of small intestinal strangulation.

Intestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression. Intestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post-operative complications, or case outcome. Retrospective cohort study. Intestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post-operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one-way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post-operative complications negatively related to outcome were determined using linear regression modelling. Thirty-six cases of SIS were evaluated. Ki67+ cell counts were decreased in the proximal (mean=15.45 cells; 95% CI= 10.27-20.63; SD=4.17; p= 0.02) and distal resection margins (mean=15.05; 95% CI=8.46-21.64; SD=4.141; p= 0.03) in horses euthanised postoperatively compared to control (mean=23.62 cells; 95% CI=19.42-27.83; SD=5.883). In the distal resection margin, an increase in SOX9+ Ki67+ cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236-1.123; p= 0.008, SE=0.1393). Small population size. Proliferating cell and ISC numbers may be associated with case outcome.

Monogenic deficiency in murine intestinal Cdc42 leads to mucosal inflammation that induces crypt dysplasia

CDC42 controls intestinal epithelial (IEC) stem cell (IESC) division. How aberrant CDC42 initiates intestinal inflammation or neoplasia is unclear. We utilized models of inflammatory bowel diseases (IBD), colorectal cancer, aging, and IESC injury to determine the loss of intestinal Cdc42 upon inflammation and neoplasia. Intestinal specimens were collected to determine the levels of CDC42 in IBD or colorectal cancer. Cdc42 floxed mice were crossed with Villin-Cre, Villin-CreERT2 and/or Lgr5-eGFP-IRES-CreERT2, or Bmi1-CreERT2 mice to generate Cdc42 deficient mice. Irradiation, colitis, aging, and intestinal organoid were used to evaluate CDC42 upon mucosal inflammation, IESC/progenitor regenerative capacity, and IEC repair. Our studies revealed that increased CDC42 in colorectal cancer correlated with lower survival; in contrast, lower levels of CDC42 were found in the inflamed IBD colon. Colonic Cdc42 depletion significantly reduced Lgr5+ IESCs, increased progenitors' hyperplasia, and induced mucosal inflammation, which led to crypt dysplasia. Colonic Cdc42 depletion markedly enhanced irradiation- or chemical-induced colitis. Depletion or inhibition of Cdc42 reduced colonic Lgr5+ IESC regeneration. In conclusion, depletion of Cdc42 reduces the IESC regeneration and IEC repair, leading to prolonged mucosal inflammation. Constitutive monogenic loss of Cdc42 induces mucosal inflammation, which could result in intestinal neoplasia in the context of aging.

The lncRNA Tincr Regulates the Abnormal Differentiation of Intestinal Epithelial Stem Cells in the Diabetic State Via the miR-668-3p/Klf3 Axis.

Diabetes mellitus (DM) is among the most common chronic diseases, and diabetic enteropathy (DE), which is a complication caused by DM, is a serious health condition. Long noncoding RNAs (lncRNAs) are regulators of DE progression. However, the mechanisms of action of multiple lncRNAs involved in DE remain poorly understood. Reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization were used to analyze terminal differentiation-induced lncRNA (Tincr) expression in intestinal epithelial cells (IECs) in the DM state. Microarray analysis, bioinformatics analysis, and luciferase reporter assays were used to identify the genes targeted by Tincr. The role of miR-668-3p was then explored by up- and down-regulating its expression in vitro and in vivo. In this study, we observed that the level of lncRNA Tincr was increased in IECs in the DM state. More importantly, Tincr was associated with abnormal intestinal epithelial stem cell (IESC) differentiation in DM. Our mechanistic study demonstrated that Tincr is a major marker of Lgr5+ stem cells in DM. In addition, we investigated whether Tincr directly targets miR-668-3p and whether miR-668-3p targets Klf3. Our findings showed that Tincr sponged miR-668-3p, which attenuated abnormal IESC differentiation in DM by regulating Klf3 expression. This study presents evidence of an essential role for Tincr in IESC differentiation in DM.

Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications

BackgroundEquine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids (“mini-guts”) derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques.ResultsIntestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75–14.25)) than control (median 25, 15–28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2–109) compared to crypts (median 21.2%, 20–21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids.ConclusionsEquine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.

Intestinal epithelial stem cell transplants as a novel therapy for cerebrovascular stroke

Almost 2/3rds of stroke survivors exhibit vascular cognitive impairment and a third of stroke patients will develop dementia 1–3 years after stroke. These dire consequences underscore the need for effective stroke therapies. In addition to its damaging effects on the brain, stroke rapidly dysregulates the intestinal epithelium, resulting in elevated blood levels of inflammatory cytokines and toxic gut metabolites due to a ‘leaky’ gut. We tested whether repairing the gut via intestinal epithelial stem cell (IESC) transplants would also improve stroke recovery. Organoids containing IESCs derived from young rats transplanted into older rats after stroke were incorporated into the gut, restored stroke-induced gut dysmorphology and decreased gut permeability, and reduced circulating levels of endotoxin LPS and the inflammatory cytokine IL-17A. Remarkably, IESC transplants also improved stroke-induced acute (4d) sensory-motor disability and chronic (30d) cognitive-affective function. Moreover, IESCs from older animals displayed senescent features and were not therapeutic for stroke. These data underscore the gut as a critical therapeutic target for stroke and demonstrate the effectiveness of gut stem cell therapy.

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 &lt;em&gt;Ex Vivo&lt;/em&gt;

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.

Butyrate induces development-dependent necrotizing enterocolitis-like intestinal epithelial injury via necroptosis.

The accumulation of short-chain fatty acids (SCFAs) from bacterial fermentation may adversely affect the under-developed gut as observed in premature newborns at risk for necrotizing enterocolitis (NEC). This study explores the mechanism by which specific SCFA fermentation products may injure the premature newborn intestine mucosa leading to NEC-like intestinal cell injury. Intraluminal injections of sodium butyrate were administered to 14- and 28-day-old mice, whose small intestine and stool were harvested for analysis. Human intestinal epithelial stem cells (hIESCs) and differentiated enterocytes from preterm and term infants were treated with sodium butyrate at varying concentrations. Necrosulfonamide (NSA) and necrostatin-1 (Nec-1) were used to determine the protective effects of necroptosis inhibitors on butyrate-induced cell injury. The more severe intestinal epithelial injury was observed in younger mice upon exposure to butyrate (p = 0.02). Enterocytes from preterm newborns demonstrated a significant increase in sensitivity to butyrate-induced cell injury compared to term newborn enterocytes (p = 0.068, hIESCs; p = 0.038, differentiated cells). NSA and Nec-1 significantly inhibited the cell death induced by butyrate. Butyrate induces developmental stage-dependent intestinal injury that resembles NEC. A primary mechanism of cell injury in NEC is necroptosis. Necroptosis inhibition may represent a potential preventive or therapeutic strategy for NEC. Butyrate induces developmental stage-dependent intestinal injury that resembles NEC. A primary mechanism of cell injury caused by butyrate in NEC is necroptosis. Necroptosis inhibitors proved effective at significantly ameliorating the enteral toxicity of butyrate and thereby suggest a novel mechanism and approach to the prevention and treatment of NEC in premature newborns.