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RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids

ABSTRACT: RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids

PROVIDER: PRJEB14077 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR1417400_1.fastq.gz	Fastqsanger.gz
	ERR1417400_2.fastq.gz	Fastqsanger.gz
	ERR1417401_1.fastq.gz	Fastqsanger.gz
	ERR1417401_2.fastq.gz	Fastqsanger.gz
	ERR1417402_1.fastq.gz	Fastqsanger.gz

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RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

Project description:This study has two components: (1) Human colon adenoma organoids (n=4 patients) were dissociated into single cells. Cells were incubated with a magnetic bead bound to an LGR5 antibody and run through a magnetic column. Magnet bound cells and flow through negative (FTN) cells were obtained. Magnet bound and FTN cells were incubated with an APC-check reagent (which binds to the magnetic bead on the LGR5 antibody) and DAPI, before being sorted by flow cytometry. 3 populations of live (DAPI-) cells were collected: FTN: Flow through negative. LGR5 negative by magnet and by flow cytometry SortedNeg: Magnet bound cells that were negative for LGR5 by flow cytometry SortedPos: Magnet bound cells that were positive for LGR5 by flow cytometry (2) Human colon organoids, as well as the tissue the organoid was derived from and adjcacent normal tissue (from n=19) were also profiled for known colorectal cancer associated mutations using the Qiagen Qiaseq Colorectal Cancer Panel, which provides targeted sequencing information for 71 genes.

2020-04-30 | E-MTAB-4698 | biostudies-arrayexpress

RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

Project description:RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

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The transcription factor GATA6 allows self-renewal of colon adenoma stem cells by repressing BMP gene expression.

Project description:Aberrant activation of WNT signaling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumor stem cell phenotype and identify the zinc-finger transcription factor GATA6 as key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells while it restricts BMP signaling to differentiated tumor cells. Genetic deletion of Gata6 in mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumor stem cells. In human tumors, GATA6 represses BMP4 gene expression through binding to a regulatory region that has been previously linked to increased susceptibility to develop CRC. Thus, GATA6 creates a permissive environment for tumor stem cell expansion by controlling the major signaling pathways that influence CRC initiation. Total RNA from biological replicates of VillinCreERT2Gata6+/+Apcfl/fl and VillinCreERT2Gata6fl/flApcfl/fl colon adenoma tumor organoids grown for one week in control media (see growth protocol).Total RNA was extracted using the TRIzolM-BM-. Plus RNA Purification Kit (Life Technologies).

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Gene expression changes in Apc-mutant mouse intestinal organoids 7 and 20 days after the induction of the Apc deletion

Project description:We isolated and selected intestinal adenoma organoids from Lgr5-EGFP-IRES-CreER; Apcflox/flox mice and added tamoxifen to induce the deletion of the Apc gene in the intestinal stem cells. Gene expressions on day7 and day20 after the addition of tamoxifen were compared, representing two stages with different colorectal cancer stem cell content. Total RNA obtained from Lgr5-EGFP-IRES-CreER; Apcflox/flox organoids were compared 7 days and 20 days after the addition of tamoxifen, cultured without the Wnt-agonist R-Spondin1.

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Analyzing the effect of Lef1 deletion in mouse intestinal organoids with single-cell RNA sequencing

Project description:We wanted to assess the role of Lef1 in ex vivo organoids using genetic mouse models of intestinal adenomas and scRNA-seq technology. Tumorigenesis was initiated by inducing Apc mutation in Lgr5+ stem cells. Intestinal cells of Lgr5-CreERT;Apc fl/fl (LApc) mouse and Lgr5-CreERT;Apc fl/fl; Lef1 fl/fl (LApcL) mouse were used to generate adenoma organoids. Organoids were cultured without growth factors for three passages and dissociated with Tryple express. We used WT mice as a control to distinguish adenoma cells. WT organoids were cultured with growth factors.

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Long-term in vitro expansion of epithelial organoids from human colon, adenoma, adenocarcinoma and Barrett's Esophagus

Project description:Background & Aims: We have recently established long-term culture conditions under which single crypts or stem cells derived from murine small intestine expand over long periods of time. Growing crypts undergo multiple crypt fission events, whilst simultaneously generating villus-like epithelial domains in which all differentiated cell types are present. We have now adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods: Based on the murine small intestinal culture system, we optimized the murine and human colon culture system. Results: Addition of Wnt3A to the growth factor cocktail allowed mouse colon crypts to expand indefinitely. Further addition of nicotinamide, a small molecule Alk inhibitor and a p38 inhibitor was essential for long-term human small intestine and colon culture. The culture system also allowed growth of murine Apcmin adenomas, human colorectal cancer and human esophageal metaplastic Barrett’s epithelium. Conclusion: The culture technology should be widely applicable as a research tool for infectious, inflammatory and neoplastic pathologies of the human gastrointestinal tract. Moreover, regenerative applications may become feasible with ex vivo expanded intestinal epithelia. Human organoids were grown embedded in Matrigel in HISC (Human intestinal stem cell culture) medium. Additionally, human small intestinal crypts and villi were isolated independently from a freshly operated sample. RNA was isolated using the RNeasy Micro kit (Qiagen). Samples were labled according to Agilent guidelines with Cy3, whereas human reference RNA (Stratagene) was labeled in Cy5. Feature Extraction Software was used to extract and normalize data.

2011-12-07 | E-GEOD-28907 | biostudies-arrayexpress

In vitro expansion of single adult Lgr5+ liver cells induced by Wnt-driven regeneration

Project description:The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist Rspo, the recently discovered ligand of Lgr5. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH-/- mice. These findings imply that previous findings on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous self-renewal. We first generated arrays from multiple wildtype tissues including muscle, white adipose tissues, brown adipose tissues, liver and pancreas. Then we generated arrays from liver derived cultures maintained in different conditions, and compared the expression profile with the corresponding parental tissues and other non-related tissues.

2013-01-28 | E-GEOD-32210 | biostudies-arrayexpress

Gene expression changes in Apc-mutant mouse intestinal organoids 7 and 20 days after the induction of the Apc deletion

2015-12-31 | GSE47569 | GEO

Cell-autonomous transformation of Lgr5-positive intestinal stem cells into gastric stem cells upon loss of transcription factor Cdx2

Project description:Stomach and intestinal adult epithelia harbor stem cells that are responsible for their continuous regeneration. Stomach and intestinal stem cells differ in their differentiation program and in the gene repertoire that they express. We show that single adult Lgr5-positive stem cells, isolated from 3D cultured small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into stomach-pyloric stem cells in the absence of this transcription factor. In order to obtain Cdx2null intestinal stem cells carrying the Lgr5-EGFP marker, 5-6 days old small intestinal organoids generated from Cdx2-/fl/Lgr5-EGFP-Ires-CreERT2 mice were incubated with 1 µM of 4-hydroxytamoxifen in intestinal culture medium for 16h to activate the Cre recombinase. Controls were 4-hydroxytamoxifen-untreated small intestinal (Control SI) and stomach (Control Sto) organoids issued from mice with the same genotype. The organoids were dissociated and sorted for EGFPhi. Cdx2null, Control SI and Control Sto clonal organoids were generated and expanded from Lgr5-EGFPhi single cells in stomach specific culture medium (ENRWfg) and RNA was isolated for RNA-Seq analysis. Cdx2+ Stomach (Sto) organoids were generated by infection of the wild type stomach organoids with lentiviral stock expressing Cdx2. They were cultured in stomach medium (ENRWfg) and RNA was isolated for RNA-Seq analysis

2014-12-05 | E-GEOD-62784 | biostudies-arrayexpress

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Project description:The goal of this study was to identify gene expression changes in response to loss of LGR5 in the LoVo colon cancer cell line.

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