Cell Stem Cell
Volume 20, Issue 2, 2 February 2017, Pages 177-190.e4
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Article
Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells

https://doi.org/10.1016/j.stem.2016.11.001Get rights and content
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Highlights

  • EGFR inhibition halts DNA replication and proliferation of Lgr5+ ISCs through MEK

  • Lgr5+ ISCs reactivated from quiescence retain multilineage differentiation potential

  • Combined EGFR/Wnt/Notch inhibition produces enteroendocrine cells with high purity

  • RNA sequencing shows regional identity and heterogeneity in hormone-producing EECs

Summary

Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence in vitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment.

Keywords

organoids
quiescence
intestinal stem cells
EGFR signaling
enteroendocrine cell
Lgr5

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