Project description:Continuous regeneration of digestive enzyme (zymogen) secreting chief cells is a normal aspect of stomach function that is disrupted in pre-cancerous lesions. Regulation of zymogenic cell (ZC) differentiation is poorly understood. Here we profile Parietal, Pit, and Zymogenic cells for comparison and study. Experiment Overall Design: Samples were obtained through Laser-capture microdisection of gastric epithelium. The three samples come from enrichments for Zymogenic Cells (ZC), Parietal Cells (PC), and Mucus Pit Cells (Pit). Experiment Overall Design: Individual parietal cells (visualized by DBA-positivity and autofluorescence) from well-oriented gastric units were dissected (PixCell II LCM apparatus, 7.5-µm spot diameter; CapSure HS LCM caps, Arcturus, Mountain View, CA) one-at-a-time from the pit zone and collected for GeneChips. Pit cells (E-cadherin-positive, DBA-negative) were then collected from the same gastric units. Only the pit cells in a 2-3-cell-thick region at the apex of the gastric unit â?? but not yet upon the gastric surface â?? were taken. ZCs (E-cadherin-positive, GSII/DBA-negative cells in the base zones) were collected from different slides in corpus gastric units after potentially contaminating basal parietal cells had first been dissected and discarded. 3000-5000 individual cells from each cell lineage were isolated from 4-5 individual mice, and RNA was purified by PicoPure kit (Arcturus). RNA integrity was verified, and RNAs for each lineage were pooled from multiple mice and multiple dissections to make 20 ng total RNA, which was then amplified, labeled, and fragmented (by the Arcturus RiboAmp HS kit followed by the RNA Amplification and Labeling Kit from Enzo Life Sciences). The resulting biotinylated cRNA probes were hybridized to Affymetrix (Santa Clara, CA) GeneChip® Mouse Genome 430 2.0 microarrays.
Project description:Continuous regeneration of digestive enzyme (zymogen) secreting chief cells is a normal aspect of stomach function that is disrupted in pre-cancerous lesions. Regulation of zymogenic cell (ZC) differentiation is poorly understood. Here we profile Parietal, Pit, and Zymogenic cells for comparison and study. Keywords: Bhlhb8, Mist1, mucous neck cell, laser-capture microdissection, zymogenic, gastric cell differentiation
Project description:In this experiment, mucous neck cells from the gastric epithelium of normal, adult C57/B6 mice were laser-capture microdissected to determine gene expression in neck cells relative to pit cells, parietal cells, and zymogenic cells, whose expression profiles were previously deposited in GEO.
Project description:A dozen of cells expressing various marker genes was defined as adult gastric epithelial stem cells. Do these cells represent different cell types or they are just the same cell with cycling expression of different marker genes? The origin of the adult stem cells could answer this question. Yet, the identity of the embryonic gastric progenitors was not known. We performed single cell RNA-sequencing to characterize cellular composition of the embryonic stomach at the time of initiation of cytodifferentiation. Our analysis of the scRNA-seq data led to identification of two different embryonic gastric epithelial progenitors. Further, we have defined signals that control their maintenance and differentiation along four main lineages, such as mucous pit, zymogenic, parietal and endocrine cells.
Project description:Mice were infected with Helicobacter pylori strain SS1 for 2, 7, 14 or 28 days or left uninfected. They were sacrificed at the appropriate times, the stomachs were removed and one half was plated for colony counts. The other half was embedded in OCT and cryosections were produced for 6-7 animals per timepoint. The sections were stained using the Histogene dye and roughly 500-1000 cells per gastric epithelial cell type were harvested by laser microdissection. RNA was prepared from these samples, subjected to two rounds of amplification, labelled and hybridized to 40.000 element murine cDNA arrays. The array names are composed as follows: "LCM" stands for laser capture microdissection; "mock" or "SS" stands for mock-infected or SS1-infected, respectively; "2,7,14 and 28" refers to the length of infection; "1-4" corresponds to the numbers given to every animal.The cellular origin of the RNA is represented by "mucus producing" or pit cell, "parietal" or acid-producing cell and "chief" or zymogenic cell. Between 5 and 7 samples were harvested per timepoint and cell type. Where the first labelling reaction and hybridization did not produce sufficiently good data (less than half of all spots made a regression correlation cutoff of >0.6), both were repeated (indicated by the addition of "relabeled" to the array name). The differential regulation of genes can be analyzed in both a time and cell type specific manner using publicly available software. Cell Type: "mucus producing" or pit cell (pit), acid-producing cell (parietal) and zymogenic cell (chief) time_series_design
Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:In this experiment, mucous neck cells from the gastric epithelium of normal, adult C57/B6 mice were laser-capture microdissected to determine gene expression in neck cells relative to pit cells, parietal cells, and zymogenic cells, whose expression profiles were previously deposited in GEO. In this screening experiment, only a single replicate Mouse Genome 430 2.0 GeneChip was generated from laser-capture microdissected cells in the neck region of the gastric unit (identified by positive staining with AlexaFluor-488-GS-II lectin positivity and/or position in the middle of the gastric unit with low labeling with E-cadherin and seconndary Alexafluor-488). RNA was purified by PicoPure kit, RNA integrity verified on Agilent Bioanalyzer, and total RNA pooled from multiple dissections and multiple mice to make ~100 ng RNA, which was then amplirifed, labeled, and fragmented (by Arctureus RiboAmp HS kit followed by the RNA Amplification and Labeling Kit from Enzo Life Sciences). Biotinylated cRNA probes were hybridized to the GeneChips. Given the inevitable contamination of the RNA with RNA from parietal cells, this GeneChip is used only in comparisons with laser-captured parietal cells described in GSE5018 as a baseline reference.
Project description:To describe the protein profile in hippocampus, colon and ileum tissue’ changing after the old faeces transplants, we adopted a quantitative label free proteomics approach.
Project description:Mice were infected with Helicobacter pylori strain SS1 for 2, 7, 14 or 28 days or left uninfected. They were sacrificed at the appropriate times, the stomachs were removed and one half was plated for colony counts. The other half was embedded in OCT and cryosections were produced for 6-7 animals per timepoint. The sections were stained using the Histogene dye and roughly 500-1000 cells per gastric epithelial cell type were harvested by laser microdissection. RNA was prepared from these samples, subjected to two rounds of amplification, labelled and hybridized to 40.000 element murine cDNA arrays. The array names are composed as follows: "LCM" stands for laser capture microdissection; "mock" or "SS" stands for mock-infected or SS1-infected, respectively; "2,7,14 and 28" refers to the length of infection; "1-4" corresponds to the numbers given to every animal.The cellular origin of the RNA is represented by "mucus producing" or pit cell, "parietal" or acid-producing cell and "chief" or zymogenic cell. Between 5 and 7 samples were harvested per timepoint and cell type. Where the first labelling reaction and hybridization did not produce sufficiently good data (less than half of all spots made a regression correlation cutoff of >0.6), both were repeated (indicated by the addition of "relabeled" to the array name). The differential regulation of genes can be analyzed in both a time and cell type specific manner using publicly available software. Cell Type: "mucus producing" or pit cell (pit), acid-producing cell (parietal) and zymogenic cell (chief)