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A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5⁺ intestinal stem cell maintenance.

ABSTRACT: Lgr5⁺ intestinal stem cells (ISCs) drive the fast renewal of intestinal epithelium. Several signaling pathways have been shown to regulate ISC fates. However, it is unclear what are the essential signals to sustain the ISC self-renewal. Here we show that coordination between Wnt and BMP signaling activity is necessary and sufficient to maintain Lgr5⁺ ISCs self-renewal. The key function of R-spondin1 is to achieve a high activity of Wnt signaling in the organoid culture. Using the GSK3 inhibitor CHIR-99021 and the BMP type I receptor inhibitor LDN-193189, we can maintain Lgr5⁺ ISCs without growth factors in vitro. Our results define the basic signaling pathways sustaining Lgr5⁺ ISCs and set up a convenient and economical culture system for their in vitro expansion. This work also set up an example for growth factor-free culture of other adult stem cells.

SUBMITTER: Li Y

PROVIDER: S-EPMC6120946 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5+ intestinal stem cell maintenance.

Li Yehua Y Liu Yuan Y Liu Bofeng B Wang Jilian J Wei Siting S Qi Zhen Z Wang Shan S Fu Wei W Chen Ye-Guang YG

Cell discovery 20180904

Lgr5+ intestinal stem cells (ISCs) drive the fast renewal of intestinal epithelium. Several signaling pathways have been shown to regulate ISC fates. However, it is unclear what are the essential signals to sustain the ISC self-renewal. Here we show that coordination between Wnt and BMP signaling activity is necessary and sufficient to maintain Lgr5+ ISCs self-renewal. The key function of R-spondin1 is to achieve a high activity of Wnt signaling in the organoid culture. Usi ...[more]

PMID: 30181900

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Project description:The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Dataset Information

A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5⁺ intestinal stem cell maintenance.

Publications

A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5<sup>+</sup> intestinal stem cell maintenance.

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