Expression data from mouse proximal intestinal epithelial Lgr5(hi) stem cells and differentiated villus cells (enterocytes from Atoh1 conditional knockout)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE41541 | GEO | 2012/10/13
SECONDARY ACCESSION(S): PRJNA177387
REPOSITORIES: GEO
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Project description:We used microarrays to detail the differentail gene expression between intestinal Lgr5(hi) stem cells and differentiated cells Assay the differential gene expression using total RNA from flow cytometry sorted Lgr5(hi) cells and EDTA isolated enterocytes from Atoh1 conditional knockout
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Project description:We previously identified Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal tumors. In this study, we have identified IL17RB as a cell surface marker distinctively expressed by Dclk1+ tuft-like tumor cells in mouse intestinal tumors. Using this tuft cell marker, we compared and analyzed the transcriptome of Lgr5-tuft marker-, Lgr5+tuft marker-, Lgr5-tuft marker+, and Lgr5+tuft marker+ tumor cells. These analyses revealed that tuft-like tumor cells in the intestinal tumors comprise two distinct subsets: highly differentiated tuft-like tumor cells (Lgr5-tuft marker+ cells) and tuft-like tumor cells with TCS potential (Lgr5+tuft marker+ cells).
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Project description:Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Validated markers of these early stem/progenitor populations are essential for deciphering their in vivo function and for evaluating their clinical potential for treating adult kidney disease. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5+ve cells via in vivo lineage tracing. The appearance and localization of Lgr5+ve cells coincided with that of the S-shaped body around E14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until P7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a novel progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential. Metanephric kidneys of timed pregnancies of ~14 days of gestation were harvested and cultured for ~10 days. Lgr5-EGFP-Ires-CreERT2-positive embryonic kidneys were identified by fluorescence microscopy and enzymatically digested to a single cell solution. GFPhi and GFPneg cells were sorted by flow cytometry (MoFlo; Dako). RNA, isolated using RNeasy (Qiagen), was analyzed using the Affymetrix platform.
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Project description:To further elucidate the role of the intestinal stem cell marker Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) in colorectal cancer (CRC), we exposed Lgr5-EGFP-IRES-Cre-ERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS) which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were characterized using gene expression profiling. In the AOM/DSS-induced mouse colon tumors Lgr5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal colon epithelial cells. To further elucidate the role of the intestinal stem cell marker Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) in colorectal cancer (CRC), we transduced SW480 CRC cells with lentiviral shRNA constructs to silence LGR5 expression. This resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells. Notch signaling was down-regulated upon LGR5 silencing. This was confirmed by immunohistochemistry against cleaved NOTCH1. Normal mouse colons and AOM/DSS-induced mouse colon tumors were flow-sorted into Lgr5 high and low cells before gene expression was measured. Fifteen independent experiments were performed using seven individual mice for normal colons and eight for tumors. Appropriate LGR5 status was confirmed by real-time qRT-PCR before measuring silencing induced gene expression. Three independent experiments were performed for each cell fraction using separately cultured cells for each experiment.
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Project description:Transcriptome profiling using Affymetrix GeneChip arrays was performed on sorted populations of Lgr5-expressing mouse ovarian surface epithelium. The ovary surface epithelium (OSE) undergoes ovulatory tear-and-remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has yet to be definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and Single Molecule Florescent-in-Situ-Hybridization (FISH) to document candidate Lgr5+ stem cells within the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in early neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and fimbria- mesovarian junction. Using conditional in vivo lineage tracing we identify embryonic and early neonate Lgr5+ populations as stem/progenitor cells contributing to the development of adult OSE and granulosa cell lineages, as well as the epithelia of the mesovarian and oviduct including its distal opening, fimbria. Long-term lineage tracing reveals that adult OSE-resident Lgr5+ populations contribute to epithelial homeostasis and OSE regenerative repair in vivo. Thus, Lgr5 is a marker of stem/progenitor cells of the ovary and tubal epithelia. Ovarian surface epithelium from pooled batches of Lgr5-eGFP-CreERT2 mice (n=8, per array) were sorted for cells expressing either high or low EGFP. Total RNA from three technical replicates per sorted population (Lgr5-high or Lgr5-low) was extracted with the RNeasy Micro Kit (QIAGEN), DNaseI-treated, and amplified with the Ovation Pico WTA V2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN). cDNA was biotinylated using the Encore Biotin Module (NuGEN Technologies). Biotiylated cDNA was hybridized to Affymetrix Mouse Genechip ST 2.0 expression arrays.
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Project description:Colorectal cancer tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is unknown. Using CRC patient derived organoids (PDOs), we found that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing scRNA-seq expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.
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