Engineered glycocalix regulates stem cell proliferation in murine crypt organoids
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE108512 | GEO | 2017/12/27
SECONDARY ACCESSION(S): PRJNA427529
REPOSITORIES: GEO
Similar Datasets
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: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: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:The Lgr5+ intestinal stem cell, Paneth and transit-amplifying cell compartment constitute the intestinal crypt which is the constant source of differentiated epithelial cells that replenish the intestinal villi ensuring organ maintenance and regeneration. The Lgr5+ crypt-based columnar (CBC) cells have been identified as the intestinal stem cells (ISCs) and, importantly, as cells-of-origin of intestinal cancer. We used microarrays to detail the global program of gene expression in normal ISCs describing a mild downregulation of the sumoylation (post-translational modification (PTM) of proteins by SUMO) imposed by the loss of one allele of the unique E2-conjugating enzyme (Ubc9) of the pathway.
2020-09-14 | GSE146106 | 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
Project description:The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these so called Crypt Base Columnar (CBC) cells. One of the genes within this stem cell signature is the Wnt target Ascl2. Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and de novo crypt formation on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the CBC stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate. Experiment Overall Design: For the stem cell signature we used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations, one expressing GFP at high levels (GFPhi) and the other expressing GFP at low levels (GFPlo). For the analysis of Ascl2 target genes RNA was isolated from intestinal epithelial cells of Ah-Cre/Ascl2floxed/floxed animals and Ah-Cre/Ascl2floxed/wt control animals 3 and 5 days post induction. Differentially labelled cRNA from GFPhi and GFPlo cells from two different sorts (each combining three different mice) were hybridised on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays. For the Ascl2 target gene analysis we analyzed the 3 and 5 days PI experiments in two dye swap experiments, resulting in four individual arrays.
2009-03-25 | E-GEOD-14201 | biostudies-arrayexpress
Project description:The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these so called Crypt Base Columnar (CBC) cells. One of the genes within this stem cell signature is the Wnt target Ascl2. Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and de novo crypt formation on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the CBC stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate. Keywords: expression profiling
2009-03-13 | GSE14201 | GEO
Project description:Wnt/b-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (PorcnDel/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of PorcnDel organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable, and that stromal production of Wnts can fully support normal murine intestinal homeostasis. Microarray was performed on samples enriched for stromal or epithelial cells from small intestine from Porcn(Del)/Villin-Cre and Porcn(WT)/Villin-Cre male C57Bl/6 mice.
2014-04-19 | E-GEOD-56911 | biostudies-arrayexpress
Project description:Paneth cells are a highly specialized population of intestinal epithelial cells located in the crypt adjacent to Lgr5+ stem cells, from which they differentiate through a process that requires downregulation of the Notch pathway. Their ability to store and release antimicrobial peptides protects the host from intestinal pathogens and controls intestinal inflammation. Here we show that PKCλ/ι is required for Paneth cell differentiation at the level of Atoh1 and Gfi1, through the control of EZH2 stability by direct phosphorylation. The selective inactivation of PKCλ/ι in epithelial cells results in the loss of mature Paneth cells, increased apoptosis and inflammation, and enhanced tumorigenesis. Importantly, PKCλ/ι expression in human Paneth cells decreases with progression of Crohn's disease. Kaplan-Meier survival analysis of CRC patients revealed that low PRKCI levels correlated with significantly worse patient survival. Therefore, PKCλ/ι is a negative regulator of intestinal inflammation and cancer through its role in Paneth cell homeostasis.
2017-08-23 | GSE84118 | GEO
Project description:Intestinal epithelium are generated by intestinal stem cells, which are recognized morphologically as slender columnar cells at the base of the crypt. Stem cells produce transit-amplifying (TA) cells, which divide a number of times and the daughter cells differentiate into absorptive enterocytes as well as secretory-lineages. Intestinal stem cells highly express Lgr5 which is decreased in TA cells. Here, we show that the zinc transported SLC39A7/ZIP7 is essential for the proliferation of TA cells and maintenance of intestinal stem cells. Lgr5Med TA cells derived from Zip7-deficient mice upregulated the expression of unfold protein responses-related genes including pro-apoptotic genes, indicating of induction of ER stress in these cells. The same effect was seen in Lgr5Hi stem cells derived from Zip7-deficient mice. We conclude that ZIP7 is fundamental to the maintenance of crypt homeostasis by resolving ER stress. Small intestinal crypts were isolated form tamoxifen-treated control (Zip7flox/+, Villin-CreERT2, Lgr5-EGFP-ires-CreERT2) and tamoxifen-treated Zip7â??IEC (Zip7flox/flox, Villin-CreERT2, Lgr5-EGFP-ires-CreERT2) mice. We FACS purified intestinal crypt cells according to their Lgr5 expression levels. RNA was isolated from four FACS sorted cell populations: Lgr5Hi cells and Lgr5Med cells derived from control mice, Lgr5Hi cells and Lgr5Med cells derived from Zip7â??IEC mice. Isolated RNA was analyzed using the Affymetrix platform.
2016-08-19 | E-GEOD-72190 | biostudies-arrayexpress