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Active Lgr5 Positive Stem Cells Drive Self-Renewal in the Pyloric Stomach and Efficiently Build Pyloric Units In-vitro

ABSTRACT: The Wnt target gene Lgr5 marks cycling stem cells in the small intestine, colon and hair-follicle. In the adult stomach, Lgr5 expression is confined to 3-4 proliferating cells at the gland base throughout the pylorus and limited numbers of corpus-type glands adjacent to the esophagus and squamous forestomach. In-vivo lineage tracing reveals that the pyloric Lgr5+ve cells rapidly generate all the major cell-types present in the gastric epithelium and maintain this multipotent stem cell activity over at least 20 months. In-vitro, single adult Lgr5+ve cells efficiently generate gastric units closely resembling mature pyloric glands. We conclude that Lgr5 is marking an active stem cell population at the base of the pyloric glands contributing to the long-term renewal of the adult gastric epithelium. The transcriptome of the adult Lgr5+ve stem cells harbors multiple Wnt target genes, implying a role for Wnt signaling in regulating pyloric stem cell function in-vivo. Conditional deletion of APC in these Lgr5+ve stem cells rapidly initiates tumor formation, supporting a potential role for aberrant Wnt signaling activity in gastric cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE17485 | GEO | 2010/02/01

SECONDARY ACCESSION(S): PRJNA118757

REPOSITORIES: GEO

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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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Active Lgr5 Positive Stem Cells Drive Self-Renewal in the Pyloric Stomach and Efficiently Build Pyloric Units In-vitro

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