Single Lgr5-GFP positive cells sorted from mouse
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE100831 | GEO | 2017/07/06
SECONDARY ACCESSION(S): PRJNA393195
REPOSITORIES: GEO
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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.
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Project description:The identification of Lgr5 as an intestinal stem cell marker has made it possible to isolate and study primary stem cells from small intestine and skins. Applying genome-wide resequencing of bisulfite-treated genomic DNA, we profiled the DNA methylation changes of FACS-sorted Lgr5+ve stem cells and their immediate undifferentiated daughter cells from small intestine and skin. In addition to this, we also analyzed terminally differentiated villus cells. We find that terminal differentiation of an adult solid-tissue stem cell does not require DNA methylation dynamics at transcriptional start sites, but is characterized by hypo-methylation of enhancer-like domains. 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 several FACS-sorted cell populations, one expressing GFP at high levels (GFPhi) and the other expressing GFP at low levels (GFPlow), both from small intestine and skin. We also isolated RNA from intestinal epithelial villus cells. Differentially labelled cRNA from GFPhi, GFPlow and villus cells from three different sorts (each combining three different mice) were hybridized on Affymetrix HT MG-430 PM arrays.
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