Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Li, L. & Xie, T. Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol. 21, 605–631 (2005).

    Article  CAS  Google Scholar 

  2. Barker, N., van de Wetering, M. & Clevers, H. The intestinal stem cell. Genes Dev. 22, 1856–1864 (2008).

    Article  CAS  Google Scholar 

  3. Potten, C.S., Booth, C. & Pritchard, D.M. The intestinal epithelial stem cell: the mucosal governor. Int. J. Exp. Pathol. 78, 219–243 (1997).

    Article  CAS  Google Scholar 

  4. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    Article  CAS  Google Scholar 

  5. Sangiorgi, E. & Capecchi, M.R. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40, 915–920 (2008).

    Article  CAS  Google Scholar 

  6. Scoville, D.H., Sato, T., He, X.C. & Li, L. Current view: intestinal stem cells and signaling. Gastroenterology 134, 849–864 (2008).

    Article  CAS  Google Scholar 

  7. Cheng, H. & Leblond, C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141, 537–561 (1974).

    Article  CAS  Google Scholar 

  8. Scadden, D.T. The stem-cell niche as an entity of action. Nature 441, 1075–1079 (2006).

    Article  CAS  Google Scholar 

  9. Crosnier, C., Stamataki, D. & Lewis, J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat. Rev. Genet. 7, 349–359 (2006).

    Article  CAS  Google Scholar 

  10. Williams, E.D., Lowes, A.P., Williams, D. & Williams, G.T. A stem cell niche theory of intestinal crypt maintenance based on a study of somatic mutation in colonic mucosa. Am. J. Pathol. 141, 773–776 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Pinto, D., Gregorieff, A., Begthel, H. & Clevers, H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709–1713 (2003).

    Article  CAS  Google Scholar 

  12. Kuhnert, F. et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl. Acad. Sci. USA 101, 266–271 (2004).

    Article  CAS  Google Scholar 

  13. van Es, J.H. et al. Notch/γ-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959–963 (2005).

    Article  CAS  Google Scholar 

  14. Fre, S. et al. Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964–968 (2005).

    Article  CAS  Google Scholar 

  15. Pageot, L.P. et al. Human cell models to study small intestinal functions: recapitulation of the crypt-villus axis. Microsc. Res. Tech. 49, 394–406 (2000).

    Article  CAS  Google Scholar 

  16. Kaeffer, B. Mammalian intestinal epithelial cells in primary culture: a mini-review. In Vitro Cell. Dev. Biol. Anim. 38, 123–134 (2002).

    Article  Google Scholar 

  17. Bjerknes, M. & Cheng, H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006).

    Article  CAS  Google Scholar 

  18. Frisch, S.M. & Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124, 619–626 (1994).

    Article  CAS  Google Scholar 

  19. Sträter, J. et al. Rapid onset of apoptosis in vitro follows disruption of β1-integrin/matrix interactions in human colonic crypt cells. Gastroenterology 110, 1776–1784 (1996).

    Article  Google Scholar 

  20. Booth, C., O'Shea, J.A. & Potten, C.S. Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium. Exp. Cell Res. 249, 359–366 (1999).

    Article  CAS  Google Scholar 

  21. Perreault, N. & Beaulieu, J.F. Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp. Cell Res. 245, 34–42 (1998).

    Article  CAS  Google Scholar 

  22. Abud, H.E., Watson, N. & Heath, J.K. Growth of intestinal epithelium in organ culture is dependent on EGF signalling. Exp. Cell Res. 303, 252–262 (2005).

    Article  CAS  Google Scholar 

  23. Blanpain, C., Horsley, V. & Fuchs, E. Epithelial stem cells: turning over new leaves. Cell 128, 445–458 (2007).

    Article  CAS  Google Scholar 

  24. Kim, K.A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005).

    Article  CAS  Google Scholar 

  25. Milano, J. et al. Modulation of notch processing by γ-secretase inhibitors causes intestinal goblet cell metaplasia and induction of genes known to specify gut secretory lineage differentiation. Toxicol. Sci. 82, 341–358 (2004).

    Article  CAS  Google Scholar 

  26. Schonhoff, S.E., Giel-Moloney, M. & Leiter, A.B. Neurogenin 3–expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Dev. Biol. 270, 443–454 (2004).

    Article  CAS  Google Scholar 

  27. López-Díaz, L. et al. Intestinal neurogenin 3 directs differentiation of a bipotential secretory progenitor to endocrine cell rather than goblet cell fate. Dev. Biol. 309, 298–305 (2007).

    Article  Google Scholar 

  28. Wang, J. et al. Mutant neurogenin-3 in congenital malabsorptive diarrhea. N. Engl. J. Med. 355, 270–280 (2006).

    Article  CAS  Google Scholar 

  29. Schmidt, G.H., Winton, D.J. & Ponder, B.A. Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine. Development 103, 785–790 (1988).

    CAS  PubMed  Google Scholar 

  30. Ueno, H. & Weissman, I.L. Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. Dev. Cell 11, 519–533 (2006).

    Article  CAS  Google Scholar 

  31. Griffith, L.G. & Swartz, M.A. Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 7, 211–224 (2006).

    Article  CAS  Google Scholar 

  32. Yamada, K.M. & Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell 130, 601–610 (2007).

    Article  CAS  Google Scholar 

  33. Bryant, D.M. & Mostov, K.E. From cells to organs: building polarized tissue. Nat. Rev. Mol. Cell Biol. 9, 887–901 (2008).

    Article  CAS  Google Scholar 

  34. Nelson, C.M. & Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 22, 287–309 (2006).

    Article  CAS  Google Scholar 

  35. Pampaloni, F., Reynaud, E.G. & Stelzer, E.H. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839–845 (2007).

    Article  CAS  Google Scholar 

  36. Bingmann, D. & Kolde, G. PO2-profiles in hippocampal slices of the guinea pig. Exp. Brain Res. 48, 89–96 (1982).

    Article  CAS  Google Scholar 

  37. Pruniéras, M., Regnier, M. & Woodley, D. Methods for cultivation of keratinocytes with an air-liquid interface. J. Invest. Dermatol. 81, 28s–33s (1983).

    Article  Google Scholar 

  38. Toda, S. et al. A new organotypic culture of thyroid tissue maintains three-dimensional follicles with C cells for a long term. Biochem. Biophys. Res. Commun. 294, 906–911 (2002).

    Article  CAS  Google Scholar 

  39. Ootani, A., Toda, S., Fujimoto, K. & Sugihara, H. Foveolar differentiation of mouse gastric mucosa in vitro. Am. J. Pathol. 162, 1905–1912 (2003).

    Article  Google Scholar 

  40. Amemori, S. et al. Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G923–G929 (2007).

    Article  CAS  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Akifumi Ootani or Calvin J Kuo.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–8 and Supplementary Methods (PDF 1144 kb)

Supplementary Video 1 (MOV 4751 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1951

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing