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Gene expression by RNA-seq in purified paneth cells in antibiotics microbiome depleded mice

ABSTRACT: The study of how gene expression in paneth cells changes after TNF admistration in mice were the microbiome has been depleted with broad spectrum antibiotics. The depletion of the microbiome excludes the occurence of indirect effects mediated through it by TNF on PC gene expression, only direct TNF effecs on crypt cells are measured

ORGANISM(S): Mus musculus

PROVIDER: GSE237588 | GEO | 2023/12/22

REPOSITORIES: GEO

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Single cell RNA sequencing analysis of germ free (GF) mouse Paneth cells and microbiome-transferred exGF mouse Paneth cells.

Project description:The single cell level Paneth cell transcriptome under gnotobiotic condition has not been resolved. How gut microbiome modulates Paneth cell transcriptome at single cell level in germ-free mice was not known. We used a flow cytometry method to isolate highly pure ileal Paneth cells from germ free (GF) Paneth cell reporter mice (Lyz1-3'UTR-IRES-CreER) and from exGF Paneth cell reporter mice that were transplanted with wild type C57B/l6 mouse gut microbiota (exGF+B6M). These isolated Paneth cells were subjected to single cell RNA sequencing by 10xGenomics.

2023-09-01 | GSE237328 | GEO

Proteomic profiling of mouse Paneth cells microdissected from small intestinal tissue

Project description:Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their co-existence with stem cells making it difficult to culture Panth cells alone in vitro. Using a simplied toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region and surfactant assisted one pot protein digestion, we identified more than 1,300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues, a comparable proteomic coverage can be achieved with 3,600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplied workflow enables profiling of Paneth cell associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.

2022-10-12 | PXD034143 | Pride

transcriptome of purified PC and non-PC fractions: GR-WT & GR-KO

Project description:Paneth Cells (PCs) are secretory cells located in the crypts of Lieberkühn of the small intestine. They produce antimicrobial peptides (AMPs) and are thought to keep the microbiome under control. The cytokine TNF, which functions mainly via its TNF receptor 1 (TNFR1 or P55), is a driving force in many inflammatory bowel disease (IBD) patients, but its impact on PCs is poorly known. We have generated intestinal glucocorticoid receptor knockout mice (GR villin KO) and investigated the diffrences between WT and KO paneth and non-paneth cell fractionf from the cell sorting.

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Gene expression by RNA-seq in purified paneth cells in antibiotics microbiome depleded mice

Project description:Gene expression by RNA-seq in purified paneth cells in antibiotics microbiome depleded mice

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Gene expression by RNA-seq in purified paneth cells of WT and panetch cell specific P55 KO mice 3 hours after TNF

Project description:The study of how gene expression in paneth cells changes 3 hours after TNF admistration in mice were the TNFR1 (P55) genes was deleted in paneth cells using Defa6-cre.

2023-12-15 | GSE237759 | GEO

RNA-seq of crypt and villus cells from small intestinal epithelium from mice treated with antibiotics with an intestinal-epithelial specific deletion of LSD1 compared to wild type

Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts and villus from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (cKO) (Villin-Cre+; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. RNA was directly isolated from the crypt and villus, and this was used for RNAseq. Gene expression analysis of cKO derived crypt and villus provides a spatially restricted outlook on the maturation status of the intestinal epithelium in the villi and the absence of Paneth cells in the crypt. Additionally, these mice were treated with antibiotics to study epithelium intrinsic changes related to LSD1 deletion but independent of the bacterial microbiome.

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Shotgun proteomics on murine paneth cells after TNF-alpha treatment

Project description:Mouse were injected with TNF-alpha prior isolation of paneth cells with flow cytometry.

2024-07-18 | PXD053044 | Pride

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

2010-11-29 | GSE25109 | GEO

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Project description:Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGF?, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. We used intestinal cell fractions from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations: stem cells were sorted based on high level of GFP expression (GFPhi) and Paneth cells were sorted based on high level of CD24 expression (CD24hi) and high side-scatter (SSChi). Differentially labelled cRNA from GFPhi and CD24hi/SSChi cells from two different sorts (each combining ten individual mice) were hybridized on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.

2010-11-29 | E-GEOD-25109 | biostudies-arrayexpress

Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

Project description:We performed 7 single-cell RNAseq experiments to identify progenitor cell populations in the small intestine. To also capture rare intestinal cell populations such as stem cells and secretory cells we not only analyzed crypt cells from wildtype mice but also used two reporter mouse lines: i) the FltpZV/+ mouse line of which we mixed live crypt cells with Fltp Venus reporter (FVR) positive cells at different ratios and ii) Foxa2-Venus fusion (FVF) reporter mice. The FVF-enriched samples are part of another manuscript (link to GEO# will be provided). Using this FACS-based enrichment strategy, we could identify a Paneth cell-primed ISC population and potential progenitor populations for all intestinal lineages. To assess the role of Wnt/PCP signaling for enteroendocrine and Paneth cell differentiation, we performed 4 single-cell RNAseq experiments from crypt cells of Celsr1crsh/+; FltpZV/ZV compound mutant mice. When comparing control and mutant cells we found specific transcriptional alterations in the Paneth cell lineage.

2020-09-11 | GSE152325 | GEO

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