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BRB-seq data of murine intestinal organoids treated with 100ng/ml of Egf, Ereg or Nrg1 ligand for 24 h

ABSTRACT: Regeneration of the intestinal epithelium is a dynamic process requiring a transient Yap-dependent reprogramming of the epithelium into a fetal-like state. Adjacent stroma, including subepithelial fibroblasts, are believed to coordinate the process, but mechanisms are not well understood. Here, we identify Neuregulin 1 (NRG1) and Epiregulin (EREG) as fibroblast-derived EGF ligands in colon and intestine which are induced during regeneration and capable of supporting the growth of the intestinal epithelium. While EREG stimulated intestinal organoid growth similarly to EGF, NRG1 increased de novo crypt formation. The goal of this experiment was to observe the differences in gene expression profile of stromal-derived EGF ligands EREG and NRG1 on intestinal organoids compared to traditional used EGF. Organoids were derived from intestinal crypts of a WT mice C57BL/6J. To exclude the size differences observed in long term cultures of EREG and NRG1, we first cultured the organoids for three days in normal ENR media, removed exogenous EGF for 24h and added EGF, EREG or NRG1 to the culture medium for 24 hours prior to the RNA isolation. NRG1 treated organoids acquired a fetal/regenerative transcriptome including activation of the Yap pathway and induction of epithelial EGF ligand Ereg and Amphiregulin (Areg) expression, highlighting the dynamic orchestration of the EGF signaling during regeneration.

ORGANISM(S): Mus musculus

PROVIDER: GSE200704 | GEO | 2023/03/15

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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BRB-seq data of murine intestinal organoids treated with 100ng/ml of Egf, Ereg or Nrg1 ligand for 24 h

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