High Yap and Mll1 promote a persistent regenerative cell state induced by Notch signaling and loss of p53
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Mus musculus
SUBMITTER: Ramon Oliveira Vidal
PROVIDER: E-MTAB-6588 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We isolated and selected intestinal adenoma organoids from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox mice and added tamoxifen to induce the deletion of the Apc and Prox1 genes in the intestinal epitheliul ex vivo. Microarray experiments were carried out 7 days after the addition of tamoxifen. Total RNA obtained from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox organoids were compared 7 days after the addition of tamoxifen and 5 days after the selection for Apc-mutant organoids in the absence of the Wnt-agonist R-Spondin1.
2014-08-15 | E-GEOD-47568 | 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.
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2014-04-18 | E-GEOD-35093 | biostudies-arrayexpress
Project description:The purpose of this study was to characterise iPSC-derived human intestinal epithelial organoids (iPSCo) by comparing these cultures with primary purified intestinal epithelial cells (IEC). Intestinal epithelial organoid (IEO) cultures were derived from at least three different lines of iPSCs, RNA was extracted and gene expression was profiled using RNA-sequencing. We compared these profiles with datasets we have previously derived from purified IEC from mature terminal ileum (TI) and sigmoid colon (SC) as well as human fetal proximal gut (FPG) and fetal distal gut (FDG).
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Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.
2015-05-07 | E-GEOD-65253 | biostudies-arrayexpress
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.
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Project description:Bats are the most important natural reservoirs for a variety of emerging viruses that cause several illnesses in humans and other mammals. Increased viral shedding by bats is thought to be linked to an increased ability of many bat species to tolerate viral infection. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is thought to have originated in bats, since viruses with high sequence similarity have been detected in bat feces. However, there is no robust in vitro model for assessing the SARS-CoV-2 infection in the bat GI tract. Here, we established gastrointestinal organoid cultures from Jamaican fruit bats (JFB, Artibeus jamaicensis), which replicated the characteristic morphology of the gastrointestinal epithelium and showed tissue specific gene expression patterns and cell differentiation. To analyze whether JFB intestinal epithelial cells are susceptible to SARS-CoV-2, we performed in vitro infection experiments. Increased SARS-CoV-2 RNA was found in both cell lysates and supernatants from the infected organoids after 48 h, and sgRNA also was detected, indicating that the JFB intestinal epithelium supports limited viral replication. However, no infectious virus was released into the culture media, and no cytopathic effects were observed. Gene expression studies revealed a significant induction of type I interferon and inflammatory cytokine genes in response to active SARS-CoV-2 virus but not to TLR agonist treatment. Untargeted analysis of the organoid proteome using data-independent acquisition mass spectrometry (DIA-MS) revealed a significant increase in proteins and pathways associated with inflammatory signaling, cell turnover and repair, and SARS-CoV-2 infection. Collectively, our data suggest that primary intestinal epithelial cells from JFBs are largely resistant to SARS-CoV-2 infection and cell damage, likely because they are able to mount a strong antiviral interferon and regenerative response upon infection.
2024-01-26 | PXD036016 | Pride