Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche
By A. Ootani, Xingnan Li, E. Sangiorgi, Q. Ho, Hiroo Ueno, S. Toda, H. Sugihara, K. Fujimoto, Irving L. Weissman, M. Capecchi and 1 more. Nature Medicine, 2009
869 citations recorded by the source corpus.
The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the γ-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein–coupled receptor-5–positive (Lgr5+) and B lymphoma moloney murine leukemia virus insertion region homolog-1–positive (Bmi1+) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5+ cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
Subfields: Genetics, Hepatology, Oncology
View Paper: https://www.semanticscholar.org/paper/4b9d2c63c90ebe3302f9358dc92fea88b699681a
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Citations in the corpus
- The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations (2011, methodology): “FISH was performed on 6-μm paraffin-embedded Bmi1derived clonogenic spheroids by using Lgr5 digoxigenin-labeled probes as described (17), with simultaneous immunodetection of YFP using anti-GFP antibody (Aves).”
- Modeling Development and Disease with Organoids. (2016, background): “The structures were surrounded by myofibroblasts and were responsive to R-spondin and to Notch inhibition (see above) (Ootani et al., 2009).”
- Tracking adult stem cells (2011, background): “Alternatively, three-dimensional intestinal organ cultures have been initiated by using neonatal tissue; in this case, both the mesenchymal niche architecture and the multilineage epithelial lining could be preserved during long-term culture”
- Hypoxia-inducible factors in stem cells and cancer (2009, neutral): “Genetic, molecular and 3-Dimensional culture analyses [32] have identified a number of conserved signal transduction pathways that are provided by niche cells and promote stem cell maintenance.”
- A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. (2018, neutral): “In contrast to the simultaneously developed culture method by Ootani et al. (2009), our adult stem cell-based cultures lack mesenchymal cells and generally require the following niche factors: the mitogen epidermal growth factor (EGF), the Wnt-agonist R-spondin, the transforming growth factor beta…”
- R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling (2011, methodology): “We then turned our attention to the R-spondins as candidate ligands on the basis of their strong mitogenic effect, specifically on LGR5 cells (20, 24) and the lack of any unequivocally identified receptors.”
- Secreted and transmembrane wnt inhibitors and activators. (2013, background): “Moreover, because of its potent proliferative effect on the intestinal epithelium in vivo and in vitro, Rspo1 holds promise as therapeutic target for gastrointestinal diseases and regenerative medicine (Kim et al. 2005; Ootani et al. 2009; Sato et al. 2009).”
- Organoid modeling of the tumor immune microenvironment (2018, methodology): “Inserts containing a permeable, membranous bottom (PICM03050, Millicell-CM, Millipore) were inserted into tissue culture dishes as described (Li et al., 2014; Ootani et al., 2009).”
- CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. (2014, background): “Activation of Wnt signaling plays a key role in maintaining the stem cell pool in the gut and promoting self-renewal of CR-CSCs (de Lau et al., 2011; Leedham et al., 2013; Ootani et al., 2009; van Es et al., 2012).”