Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA168973 | ENA |
REPOSITORIES: ENA
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Project description:PeyerM-bM-^@M-^Ys Patches consist of domains of specialized intestinal epithelium overlying Gut-Associated Lymphoid Tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, so-called M cells. We have recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor Spi-B, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In Spi-B-/- mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. In intestinal organoid (M-bM-^@M-^Xmini-gutM-bM-^@M-^Y) culture, we show that stimulation with RankL induces SpiB expression within 24hrs and subsequently of other M cell markers. We conclude that RankL-induced expression of Spi-B is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells. Small intestinal organoids were derived from wildtype (WT) mice. Recombinant mouse RankL (BioLegend) was added to the organoid culture medium in concentrations of 50-200ng/ml and fresh medium was added at day 2 and day 5. At the indicated time points, organoids were harvested for RNA isolation and microarray analysis to look for gene expression changes in reponse to RanKL.
2012-06-18 | E-GEOD-38785 | biostudies-arrayexpress
Project description:Peyer’s Patches consist of domains of specialized intestinal epithelium overlying Gut-Associated Lymphoid Tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, so-called M cells. We have recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor Spi-B, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In Spi-B-/- mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. In intestinal organoid (‘mini-gut’) culture, we show that stimulation with RankL induces SpiB expression within 24hrs and subsequently of other M cell markers. We conclude that RankL-induced expression of Spi-B is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.
2012-06-19 | GSE38785 | GEO
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
Project description:The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. Conversely, forced expression of Cdx2 in gastric organoids results in their intestinalization. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2. Small intestinal crypts and stomach glands were isolated from Cdx2-/fl / Lgr5-EGFP-CreERT2 mice and cultured for a week in order to generate small intestinal (SI) and stomach (Sto) in vitro organoids. The Lgr5-CreERT2 enzyme activity has been induced by overnight 4-hydroxytamoxifen induction. Tamoxifen treated and untreated Lgr5-EGFPhi SI and Sto stem cells were FACS sorted and seeded back into ENRWfg (Sto med) culture conditions in order to generate Cdx2-/fl small intestinal (Control SI), Cdx2null small intestinal (Cdx2null SI) and Cdx2-/fl stomach (Control Sto) clonal organoids. Cdx2-/fl SI organoids and Cdx2-/fl Sto organoids have been also cultured in ENR (SI med) to induce differentiation. After some passages of clonal organoid expansion, RNA was isolated from Control SI, Cdx2null SI and Control Sto Lgr5-EGFPhi FACS sorted stem cell populations and from smal intestinal and stomach organoids cultured in different conditions and hybridized on Affymetrix Mouse Gene ST 1.1 arrays.
2014-12-04 | E-GEOD-51751 | biostudies-arrayexpress
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.
2021-12-10 | GSE179853 | GEO
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.
2014-12-05 | GSE62784 | GEO
Project description:Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) which expand 5-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality We generated arrays from whole pancreas, islet, acinar and duct (Sox9+ sorted) cells and pancreas derived cultures maintained in our defined medium. For the clustering analysis we substracted the pancreas array to all arrays. Genes 2 fold differentially expressed in the ductal array were used for the analsyis. For the genes enriched in organoid cultures compared to pancreas we substracted the organoid culture arrays to the pancreas array. Genes >2-fold differentially expressed were used for the analysis.
2013-09-17 | E-GEOD-50103 | biostudies-arrayexpress
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.
2015-12-31 | E-GEOD-47569 | biostudies-arrayexpress
Project description:Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) which expand 5-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality
2013-09-17 | GSE50103 | GEO
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