Whole genome characterization of in vitro generated mouse colonic monolayers
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE116541 | GEO | 2019/01/23
REPOSITORIES: GEO
Similar Datasets
Project description:Whole genome characterization of in vitro generated mouse colonic monolayers
| PRJNA478953 | ENA
Project description:Transcriptional profiling of microscopically laser dissected murine colonic tissue from 3 separate normal crypts, wound associated epithelium (WAE), normal epithelium, and regenerating crypts (day 6 only) at days 2,4,and 6 after colonic mucosal injury. Injury model performed as described in: H. Seno et al., Efficient colonic mucosal wound repair requires Trem2 signaling. Proc. Natl. Acad. Sci. USA 106, 256-261 (2009)
2012-06-18 | E-MTAB-1175 | biostudies-arrayexpress
Project description:There is a gradient of M-NM-2-catenin expression along the colonic crypt axis with the highest levels at the crypt bottom. However, it remains unclear whether different levels of canonical Wnt signaling exert distinct roles in the colonic epithelium. In the present study, we first showed that the canonical Wnt signaling is active in the proliferative compartment of normal colonic crypts by separating actively proliferating progenitor cells from non-proliferating cells in the colon using transgenic mice expressing a histone H2B-green fluorescent protein (GFP) fusion protein under the control of a tetracycline responsive regulatory element. Subsequently, we investigated the dose-dependent effect of canonical Wnt activation on colonic epithelial differentiation by controlling the expression levels of stabilized M-NM-2-catenin using a doxycycline-inducible transgenic system in mice. We show that elevated levels of Wnt signaling induce the amplification of Lgr5+ cells, which is accompanied by crypt fission and a reduction in cell proliferation among progenitor cells. In contrast, lower levels of M-NM-2-catenin induction enhanced cell proliferation rates of epithelial progenitors without affecting crypt fission rates. Notably, slow-cycling cells produced by M-NM-2-catenin activation exhibit activation of Notch signaling and the treatment of M-NM-2-catenin expressing mice with a Notch inhibitor turned such slow-cycling cells into actively proliferating cells. Our results indicate that the activation of the canonical Wnt signaling pathway is sufficient for de novo crypt formation, and suggest that different levels of canonical Wnt activations, in cooperation with Notch signaling, establish a hierarchy of slower-cycling stem cells and faster-cycling progenitor cells characteristic for the colonic epithelium. To separate actively proliferating cells from non-proliferating cells in normal colon, we used transgenic mice expressing a histone H2B-GFP fusion protein under the control of a tetracycline responsive regulatory element (H2B-GFP mice). In H2B-GFP mice, all colonic crypt cells exhibited strong nuclear GFP signal 7 days after doxycycline administration. When doxycycline was withdrawn for 2 days after the labeling period, nuclear GFP signal was diluted in proliferating cells whereas non-proliferating cells retained GFP. GFPhigh non-proliferating and GFPlow proliferating epithelial cells were sorted from the isolated crypts by FACS for microarray analysis. In addition, to investigate the effects of acute Wnt activation in colon, we generated doxycycline-inducible M-NM-2-catenin mice. Colonic crypts were isolated from untreated mice and mice fed doxycycline in the drinking water (2.0 mg/ml) for 5 days and subjected to microarray analysis.
2013-01-22 | E-GEOD-41688 | biostudies-arrayexpress
Project description:Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis, increasing morbidity and mortality. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxin application to apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human gut epithelial lineages. Transcriptomics informed timing of stem cell differentiation to achieve in vitro colonocyte maturation like that observed in vivo. Transepithelial electrical resistance (TEER) and fluorescent dextran permeability assays measured cytotoxicity as barrier loss post-toxin exposure. Leaky epithelial barriers were induced with diclofenac. scRNAseq demonstrated broad and variable toxin receptor expression across human gut lineages. Absorptive colonocytes displayed generally enhanced toxin receptor, Rho GTPase, and cell junction expression. 22-day differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes. hCE monolayers exhibited high barrier function after 1-day differentiation. Basal TcdA/B application to monolayers caused greater toxicity and apoptosis. Diclofenac induced leaky hCE monolayers and enhanced toxicity of apical TcdB exposure. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude of basal toxicity. Leaky paracellular junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive culture system to evaluate the impact of microbial toxins on gut epithelium.
2023-02-13 | GSE223605 | GEO
Project description:Human mucosa was collected from two different individuals undergoing colectomy. After treatment with EDTA, colonic crypts were isolated and further dis-aggregated using Dispase. Next, cells were stained using antibodies directed against the extracellular domain of EpCAM, PTK7, CD11, CD31, and CD45. Cells were analyzed and sorted via flow cytometry (BD Aria). After excluding non-epithelial cells (Epcam. CD11, CD31, and CD45 negative), the EpCAM positive fraction was divided into fractions expressing either high, medium, low, or negative levels of PTK7. Sorted cells were transferred to Trizol for RNA extraction and RNA was purified using the Qiagen RNA Mini Kit. Colonic crypts were isolated from normal human mucosa derived from individuals undergoing colectomy. Single cell fractions from these crypts were sorted and isolated RNA processed and hybridized to Affymetrix PrimeView Arrays
2016-01-12 | E-GEOD-68340 | biostudies-arrayexpress
Project description:Escherichia coli of the B2 phylotype reside in human and animal intestines. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the localization of phosphatidylinositol-4,5-bisphosphate (PIP2) abundance in the plasma membrane. In Caco-2 monolayers, transepithelial electrical resistance was measured to determine barrier function. Cell morphologyproliferation and dissemination was assessed microscopically. Cell signaling and polarity regulation was analyzed by RNA-Seq.
2021-09-01 | GSE169213 | GEO
Project description:Transcriptome comparison of small and large intestine-derived monolayers and murine intestinal crypts
| PRJNA789342 | ENA
Project description:There is a gradient of β-catenin expression along the colonic crypt axis with the highest levels at the crypt bottom. However, it remains unclear whether different levels of canonical Wnt signaling exert distinct roles in the colonic epithelium. In the present study, we first showed that the canonical Wnt signaling is active in the proliferative compartment of normal colonic crypts by separating actively proliferating progenitor cells from non-proliferating cells in the colon using transgenic mice expressing a histone H2B-green fluorescent protein (GFP) fusion protein under the control of a tetracycline responsive regulatory element. Subsequently, we investigated the dose-dependent effect of canonical Wnt activation on colonic epithelial differentiation by controlling the expression levels of stabilized β-catenin using a doxycycline-inducible transgenic system in mice. We show that elevated levels of Wnt signaling induce the amplification of Lgr5+ cells, which is accompanied by crypt fission and a reduction in cell proliferation among progenitor cells. In contrast, lower levels of β-catenin induction enhanced cell proliferation rates of epithelial progenitors without affecting crypt fission rates. Notably, slow-cycling cells produced by β-catenin activation exhibit activation of Notch signaling and the treatment of β-catenin expressing mice with a Notch inhibitor turned such slow-cycling cells into actively proliferating cells. Our results indicate that the activation of the canonical Wnt signaling pathway is sufficient for de novo crypt formation, and suggest that different levels of canonical Wnt activations, in cooperation with Notch signaling, establish a hierarchy of slower-cycling stem cells and faster-cycling progenitor cells characteristic for the colonic epithelium.
2013-01-22 | GSE41688 | GEO
Project description:The study describes miRNA expression in colonic epithelium of chronic SIV-infected rhesus macaques. We profiled and characterized miRNA/mRNA expression exclusively in colonic epithelium (CE) of 12 chronically SIV-infected and 8 control rhesus macaques (RMs). About 46 miRNAs were differentially expressed (DE) (20-up and 26-down) in CE during chronic SIV infection. Using TargetScan, we bioinformatically crossed the predicted targets of DE miRNAs to genome-wide transcriptomic data and identified several critical miRNA-mRNA pairings that suggested miRNA-mediated regulation of aberrant epithelial gene expression in CE. Immunofluorescence, luciferase reporter and miRNA overexpression studies confirmed the ability of miR-130a and miR-212 to bind the 3’ UTR and downregulate protein expression of occludin (OCLN) and peroxisome proliferator activator receptor gamma (PPAR), respectively, two proteins with pivotal roles in epithelial barrier function. MiR-130a and miR-212 overexpression in Caco-2 cells significantly reduced transepithelial electrical resistance (TEER). Interestingly, delta-9-tetrahydrocannabinol (9-THC) treatment restored TEER to levels observed with control miRNA mimic. Finally, ex-vivo 9-THC treatment of colon tissue from chronically SIV-infected RMs significantly increased PPAR gene expression. Our findings suggest that dysregulated miR-130a and miR-212 expression in CE during chronic HIV/SIV infection can facilitate epithelial barrier disruption by downregulating OCLN and PPAR protein expression. Most importantly, our results highlight the beneficial effects of cannabinoids on epithelial barrier function in not just HIV/SIV but potentially other chronic intestinal inflammatory diseases.
2018-02-01 | GSE104767 | GEO
Project description:The goal of this study was to evaluate the transcriptional response of 4 human duodenal enteroid lines on monolayers to norovirus infections (GII.4). Enteroids were plated as monolayers in Intesticult (Stem Cell Technologies) proliferation medium. After 1 day of cell growth as a monolayer, the proliferation medium was changed with differentiation medium for 5 days. Five-day-differentiated monolayers were washed and were either mock-infected or inoculated with human norovirus supplemented with 500 μM glycochenodeoxycholic acid (GCDCA), for 1 to 2 h at 37°C. Total RNA was extracted using the Qiagen RNeasy kit and paired-end Illumina sequencing was performed.
2021-10-01 | GSE183223 | GEO