Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Zoltan Wiener

PROVIDER: E-GEOD-47569 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

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