Injury-inducible stem cell potential of the intestinal enteroendocrine lineage: single-cell mRNA-seq profiling
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE99457 | GEO | 2017/07/10
SECONDARY ACCESSION(S): PRJNA388493
REPOSITORIES: GEO
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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 intestinal stem cells. Transcriptional differences between intestinal stem cells can be explored by the use of the Lgr5-eGFP-ires-CreERT2 knock-in mouse. In this mouse model GFP expression is driven from the Lgr5 locus, leading to highest GFP levels in the Lgr5 positive cells. Yet, due to the stability of the GFP protein, it is distributed upon cell division to the daughter cells. Here we arbitrarily sorted crypt cells based on GFP expression into five fractions. The fraction with the lowest GFP level is named 1+, the fraction with the highest level 5+. 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 five FACS sorted cell populations, with fraction 5+ expressing GFP at highest levels and 1+ expressing GFP at lowest levels. Differentially labelled cRNA from fractions 1+ to 4+ were hybridized against fraction 5+ on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in dye swap experiments, resulting in eight individual arrays.
2012-08-15 | E-GEOD-36497 | biostudies-arrayexpress
Project description:Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.
| S-EPMC5642297 | biostudies-literature
Project description:The identification of Lgr5 as an intestinal stem cell marker has made it possible to isolate and study primary intestinal stem cells. Transcriptional differences between intestinal stem cells can be explored by the use of the Lgr5-eGFP-ires-CreERT2 knock-in mouse. In this mouse model GFP expression is driven from the Lgr5 locus, leading to highest GFP levels in the Lgr5 positive cells. Yet, due to the stability of the GFP protein, it is distributed upon cell division to the daughter cells. Here we arbitrarily sorted crypt cells based on GFP expression into five fractions. The fraction with the lowest GFP level is named 1+, the fraction with the highest level 5+.
2012-08-15 | GSE36497 | GEO
Project description:We demonstrate that Bmi1-expressing cells represent the predominant epithelial ISC in early intestinal development and precede existence of the canonical Wnt-dependent Lgr5+ stem cell population. We reveal that early in development, Bmi1+ ISCs are highly proliferative, then transition to a slow-cycling state with the emergence of the Lgr5+ ISC. Combining single cell RNA-sequencing (scRNA-seq) with bulk population ATAC-seq (Assay for Transposase Accessible Chromatin sequencing) analyses, we identify that a non-canonical Wnt signaling pathway correlates with the temporal presence of the highly proliferative developmental Bmi1+ ISC population.
2022-09-09 | GSE212798 | GEO
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.
2013-04-25 | E-GEOD-46303 | biostudies-arrayexpress
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. Using the cell cycle specific expression og the mKi67 gene, we generated a novel Ki67-RFP knock-in allele which identifies dividing cells. Using Lgr5-GFP;Ki67-RFP mice, we isolated CBCs with distinct Wnt signaling levels and cell cycle features, and analyzed their global gene expression pattern using microarrays. We concluded that the cycling Lgr5hi stem cells exit the cell cycle in transition into the secretory lineage. Lgr5med Ki67low intermediate precursors reside in the zone of differentiation, resemble quiescent stem cells and generate the Dll1+ secretory precursors and the label retaining cells. Our findings support the cycling stem cell hypothesis and highlight the heterogeneity of early progenitors during lineage commitment. We used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter, and Ki67-TagRFP mice where the RFP is fused to the C-terminus of the endogenous Ki67 gene. RNA was isolated from several FACS sorted cell populations of combinations expressing different levels of GFP and RFP: GFP high RFP high (Lgr5hi Ki67hi), GFP high RFP low (Lgr5hi Ki67low), GFP medium RFP high (Lgr5med Ki67high) and GFP medium RFP low (Lgr5med Ki67low). Purified RNA was processed, hybridized, and scanned according to the manufacturerM-bM-^@M-^Ys protocol and were hybridized on Affymetrix Mouse Gene ST 1.1 arrays).
2014-09-08 | E-GEOD-52813 | 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, 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:A comparative bulk cell RNA-seq analysis of diverse intestinal stem cell populations was performed, for cells expressing the following markers: Lgr5-eGFPhi, Cd166+, Cd24lo, Grp78, upper side population (SP), Bmi1, mTert, Hopx, Dclk1, a lower side population (SP)
2017-07-10 | GSE99815 | GEO
Project description:We used unbiased whole genome bisulfite sequencing (WGBS) to identify DNA methylation changes in the intestinal stem cells (ISCs) or their progeny during the suckling period of mouse colon development. Lgr5-EGFP mice were used to identify ISC populations in the colons. WGBS were performed using EGFP labeled Lgr5+ ISCs and epithelial cell adhesion molecule (EpCAM) labeled epithelial cells isolated at the beginning and end of the suckling period (postnatal day 0-P0 and P21).
2015-08-25 | E-GEOD-58532 | biostudies-arrayexpress
Project description:Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into 3D epithelial organoids containing all cell types at nearnormal ratios. Culture conditions to generate the main cell types have been established previously, but signals inducing the various types of enteroendocrine cells (EECs) have remained elusive. Here we generate quiescent Lgr5+ stem cells in vitro by inhibition of the EGF-receptor (EGFR) and mitogen-associated protein kinase (MAPK) signaling pathways in organoids, a state that can be readily reversed. Quiescent Lgr5+ stem cells gain a distinct molecular signature, biased towards EEC differentiation. Indeed, combined inhibition of Wnt, Notch and MAPK pathways efficiently generates a diversity of EEC subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and cell fate choice, and provide an in vitro approach for the study of the elusive EECs.
2017-01-03 | GSE80636 | GEO