Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium

ABSTRACT: Immortal spheroids were generated from fetal mouse intestine using the culture system developed to culture organoids from adult intestinal epithelium. Spheroids are made of a monostratified polarized epithelium displaying a poorly differentiated intestinal phenotype. The proportion of spheroids generated from intestinal explants progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Spheroid cells show indefinite self-renewing properties but exhibit a transcriptome strikingly different from that of adult intestinal stem cells reminiscent of incompletely caudalized progenitors. The receptor Lgr4, but not Lgr5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the E14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, thus qualifying them as a new type of intestinal stem-like cells. Two-channel microarray experiments were performed from spheroid/organoid pairs isolated each from a given embryo. Following initial seeding of small intestine from a given embryo/mouse (at E16, E18 or P0), spheroids and organoids were selectively picked up for each animal and replated for 3 passages to reach sample homogeneity. Hybridization was performed on the 4 independent pairs with dye-swap.

ORGANISM(S): Mus musculus

SUBMITTER: Frederick Libert

PROVIDER: E-GEOD-49803 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

Mustata Roxana C RC Vasile Gabriela G Fernandez-Vallone Valeria V Strollo Sandra S Lefort Anne A Libert Frédérick F Monteyne Daniel D Pérez-Morga David D Vassart Gilbert G Garcia Marie-Isabelle MI

Cell reports 20131017 2

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome ...[more]

PMID: 24139799

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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.

Dataset Information

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium

Publications

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

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