DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact ...[more]
Project description:The purpose of this study was to characterise iPSC-derived human intestinal epithelial organoids (iPSCo) by comparing these cultures with primary purified intestinal epithelial cells (IEC). Intestinal epithelial organoid (IEO) cultures were derived from at least three different lines of iPSCs, RNA was extracted and gene expression was profiled using RNA-sequencing. We compared these profiles with datasets we have previously derived from purified IEC from mature terminal ileum (TI) and sigmoid colon (SC) as well as human fetal proximal gut (FPG) and fetal distal gut (FDG).
Project description:The purpose of this study was to characterise iPSC-derived human intestinal epithelial organoids (iPSCo) by comparing these cultures with primary purified intestinal epithelial cells (IEC). Intestinal epithelial organoid (IEO) cultures were derived from at least three different lines of iPSCs, DNA was extracted and gene expression was profiled using Illumina Infinium HumanMethylation450Beadarray. We compared these profiles with datasets we have previously derived from purified IEC from mature terminal ileum (TI) and sigmoid colon (SC) as well as human fetal proximal gut (FPG) and fetal distal gut (FDG).
Project description:This study investigated alterations in the transcriptomic profiles of intestinal epithelial cells in Inflammatory Bowel Diseases (IBD), i.e. Crohn's Disease and Ulcerative Colitis. Biopsies were taken from treatment-naive paediatric patients at diagnostic endoscopy from terminal ileum (TI), ascending colon (AC) and sigmoid colon (SC). Intestinal epithelial cells were purified using enzyme digestion and magnetic bead separation. RNA extraction was performed on EpCAM-positive cells, using AllPrep DNA/RNA mini kit. An Agilent Bioanalyzer was used to check RNA integrity following the manufacturer’s guidelines. mRNA was sequenced at the University of Kiel, Germany.
Project description:Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome wide association studies and experimental animal models point at a central role of the IL-10 axis in Inflammatory Bowel Diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1hi-expressingmacrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages, but resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning factor in the colon and define intestinal CX3CR1hi macrophages as a decisive factor that determines gut health or inflammation. Microarray of resident macrophages sorted from colons of Interleukin-10 deficeint mice and macrophage-restricted interleukin-10 receptor deficient mice versus colonic resident macrophages of wild type mice
Project description:We analyzed the transcriptional profile of colon and small-intestinal (SI) tissues in response to ex-vivo colonization with members of the gut microbiota. Tissues were dissected from SPF or GF mice, and connected to the ex-vivo gut organ culture system. Then, microbial cultures or fecal samples were infused into the lumen, and tissues were processed in different time points, as indicated below. Overall design: A small 3-5 mm segment of the proximal colon or SI terminal ileum of mice was placed directly into TRIzol.
Project description:Gut dysbiosis is closely involved in the pathogenesis of inflammatory bowel disease (IBD). However, it remains unclear whether IBD-associated gut dysbiosis plays a primary role in disease manifestation or is merely secondary to intestinal inflammation. Here, we established a humanized gnotobiotic (hGB) mouse system to assess the functional role of gut dysbiosis associated with two types of IBD - Crohn's disease (CD) and ulcerative colitis (UC). In order to explore the functional impact of dysbiotic microbiota in IBD patients on host immune responses, we analyzed gene expression profiles in colonic mucosa of hGB mice colonized with healty (HC), CD, and UC microbiota. Overall design: WT-GF and GB mice (HC, CD, UC) were sacrificed after 2 weeks from human microbiota reconstitution and collected colon tissue for RNA extraction and hybridization on Affymetrix microarrays. WT-GF: n=2, HC-Mb GB mice: n=4, CD-Mb GB mice: n=5, UC-Mb GB mice: n=5.