Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA134501 | ENA |
REPOSITORIES: ENA
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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
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, TGFα, 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.
2010-11-29 | GSE25109 | GEO
Project description:Lgr5+ stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF and Notch signals to neighboring Lgr5+ stem cells. While the colon lacks Paneth cells, Deep Crypt Secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report Reg4 as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon mini-gut formation. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. Stem cells are forced to generate DCS cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche.
2016-10-05 | GSE63256 | GEO
Project description:Paneth cells (PCs) are long-lived secretory cells that reside at the bottoms of small intestinal crypts. Besides serving as niche cells for the neighboring Lgr5-positive stem cells, PCs secrete granules containing a broad spectrum of antimicrobial proteins, including lysozymes and defensins1. Here, we have used single-cell RNA sequencing to explore PC differentiation. We found a maturation gradient from early secretory progenitors to mature PCs, capturing the full maturation path of PCs. Moreover, differential expression of a subset of defensin genes in lysozyme-high PCs, e.g. Defa20, reveals at least two distinct stages of maturation. We traced Lgr5+ stem cells from Lgr5-CreERT2 C57Bl6/J mice bred to a Rosa26LSL-YFP reporter mice and sorted YFP+ cells 5 days, 3 weeks and 8 weeks after tamoxifen injection.
2016-06-23 | E-GEOD-76408 | biostudies-arrayexpress
Project description:Gene inactivation of the orphan G protein-coupled receptor Lgr4, a paralog of the epithelial stem cell marker Lgr5, results in 50% decrease of epithelial cell proliferation and 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, Lgr4-deficient crypts or progenitors, but not Lgr5-deficient progenitors, die rapidly with dramatic downregulation of stem cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by LiCl addition to the culture medium, but not by Wnt agonists. Our results identify Lgr4 as a permissive factor of the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy. Microarray hybridization was performed on LGR4 KO intestinal crypts at day 0, day 0.5 and day 1 versus wild-type crypts. The effects of LiCl treatment on LGR4 KO crypts at day1 versus control cells were investigated. After amplification and labelling, sample pairs were hybridized onto Mouse Exonic Evidence Based Oligonucleotide (MEEBO) arrays containing on average 38784 mouse 70mer oligonucleotide probes (Stanford University, US). Hybridizations were replicated with dye swap.
2012-03-10 | E-GEOD-27904 | biostudies-arrayexpress
Project description:At the base of the intestinal crypt, long-lived Lgr5+ stem cells are intercalated by Paneth cells that provide essential niche signals for stem-cell maintenance. This unique epithelial anatomy makes the intestinal crypt one of the most accessible models for the study of adult stem cell biology. The glycosylation patterns of this compartment are poorly characterized and the impact of glycans on stem cell differentiation remains largely unexplored. We found that Paneth cells, but not Lgr5+ stem cells, express abundant terminal N-acetyllactosamine (LacNAc). Employing an enzymatic method to edit glycans in cultured crypt organoids, we assessed the functional role of LacNAc in the intestinal crypt. We show that blocking access to LacNAc on Paneth cells leads to hyperproliferation of the neighbouring Lgr5+ stem cells, which is accompanied by the down-regulation of genes that are known as negative regulators of proliferation
2017-12-27 | GSE108512 | GEO
Project description:Gene inactivation of the orphan G protein-coupled receptor Lgr4, a paralog of the epithelial stem cell marker Lgr5, results in 50% decrease of epithelial cell proliferation and 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, Lgr4-deficient crypts or progenitors, but not Lgr5-deficient progenitors, die rapidly with dramatic downregulation of stem cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by LiCl addition to the culture medium, but not by Wnt agonists. Our results identify Lgr4 as a permissive factor of the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.
2012-03-10 | GSE27904 | GEO
Project description:The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of approximately six cycling Lgr5+ stem cells at the bottoms of small intestinal crypts1. We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. Keywords: expression profiling Freshly isolated small intestinal crypts from two mice were divided into two parts. RNA was directly isolated from one part (RNeasy Mini Kit, Qiagen), the other part was cultured for one week according to the conditions described in the associated paper, followed by RNA isolation. We prepared labeled cRNA following the manufacturer’s instruction (Agilent Technologies). Differentially labelled cRNA from small intestinal crypts and organoids were hybridised separately for the two mice on a 4X44k Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.
2009-04-03 | E-GEOD-14594 | biostudies-arrayexpress
Project description:Paneth cells (PCs) are long-lived secretory cells that reside at the bottoms of small intestinal crypts. Besides serving as niche cells for the neighboring Lgr5-positive stem cells, PCs secrete granules containing a broad spectrum of antimicrobial proteins, including lysozymes and defensins1. Here, we have used single-cell RNA sequencing to explore PC differentiation. We found a maturation gradient from early secretory progenitors to mature PCs, capturing the full maturation path of PCs. Moreover, differential expression of a subset of defensin genes in lysozyme-high PCs, e.g. Defa20, reveals at least two distinct stages of maturation.
2016-06-23 | GSE76408 | GEO
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