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Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

Nature Medicine volume 15, pages 701–706 (2009)Cite this article

Abstract

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.

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Figure 1: Long-term intestinal culture.
Figure 2: Intestinal cultures from juvenile and adult mice.
Figure 3: Wnt signaling regulates proliferation of cultured intestinal epithelium.
Figure 4: Notch and Neurogenin-3 regulate intestinal cell fate in vitro.
Figure 5: Putative intestinal stem cell populations with or without R-spondin1 treatment in culture.

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Acknowledgements

We are grateful to members of the Kuo laboratory, C. Chartier, R. Nusse, L. Chia, M. Lee and M. Pech for helpful discussions. We are indebted to M. Amieva for video recording, J. Yuan, P. Chu, H. Ideguchi and S. Nakahara for technical assistance, M. Kay (Stanford University) for adenovirus expressing Ngn3, P. Soriano (Mount Sinai School of Medicine) for Rosa26-1 vector, J. Chen (Stanford University) for Wnt reporter cells, R. Nusse (Stanford University) for Wnt3a L cells, and M. Selsted (University of California–Irvine) and E. Sibley (Stanford University) for antibodies specific for cryptidin and intestinal hydrolases, respectively. A.O. was a California Institute for Regenerative Medicine Scholar. X.L. was supported by a Dean's Fellowship from Stanford University, and H.U. was supported by the Floren Family Fund. This work was supported by US National Institutes of Health grant R01 DK069989-01, California Institute for Regenerative Medicine RS1-00243-1 and the Broad Medical Research Foundation (C.J.K.), US National Institutes of Health grant R01 CA86065-06 and the Smith Family Fund (I.L.W.), the Ichiro Kanehara Foundation, Kato Memorial Trust for Nambyo Research and Nagao Memorial Trust (A.O.), and Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research No. 20592023 (S.T.). We acknowledge the generous support of the Stanford University Digestive Disease Center (US National Institutes of Health grant P30 DK56339; H.U. and C.J.K.).

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Authors and Affiliations

  1. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California, USA

    Akifumi Ootani, Xingnan Li, Quoc T Ho & Calvin J Kuo

  2. Howard Hughes Medical Institute and Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Eugenio Sangiorgi & Mario R Capecchi

  3. Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA

    Hiroo Ueno & Irving L Weissman

  4. Department of Pathology, Saga Medical School, Saga, Japan

    Shuji Toda & Hajime Sugihara

  5. Department of Internal Medicine, Saga Medical School, Saga, Japan

    Kazuma Fujimoto

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  1. Akifumi Ootani
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Correspondence to Akifumi Ootani or Calvin J Kuo.

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Competing interests

A.O. and C.J.K. have a provisional patent on the culture method described in the article. I.L.W. has over $10,000 in Amgen stock and is a director of both Cellerone, Inc. and StemCells, Inc.

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Ootani, A., Li, X., Sangiorgi, E. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15, 701–706 (2009). https://doi.org/10.1038/nm.1951

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