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Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell

Nature Medicine volume 18, pages 618–623 (2012)Cite this article

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Abstract

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5+ cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP+ colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5+ colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

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Figure 1: Long-term, serum-free culture of colonic epithelial cells.
Figure 2: Lgr5+ stem cells are enriched in cultured organoids.
Figure 3: Transplantation of cultured cells improves acute colitis.
Figure 4: Donor-derived cells regenerate functional colonic epithelium.
Figure 5: Single Lgr5+ stem-cell–derived cultured cells serve as long-lived, multipotential stem cells in vivo.

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References

  1. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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 

  5. Radtke, F. & Clevers, H. Self-renewal and cancer of the gut: two sides of a coin. Science 307, 1904–1909 (2005).

    Article  CAS  Google Scholar 

  6. 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 

  7. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Avansino, J.R., Chen, D.C., Woolman, J.D., Hoagland, V.D. & Stelzner, M. Engraftment of mucosal stem cells into murine jejunum is dependent on optimal dose of cells. J. Surg. Res. 132, 74–79 (2006).

    Article  CAS  Google Scholar 

  10. Tait, I.S., Evans, G.S., Flint, N. & Campbell, F.C. Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation. Am. J. Surg. 167, 67–72 (1994).

    Article  CAS  Google Scholar 

  11. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  Google Scholar 

  12. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).

    Article  CAS  Google Scholar 

  13. Booth, C., Patel, S., Bennion, G.R. & Potten, C.S. The isolation and culture of adult mouse colonic epithelium. Epithelial Cell Biol. 4, 76–86 (1995).

    CAS  PubMed  Google Scholar 

  14. Whitehead, R.H., Demmler, K., Rockman, S.P. & Watson, N.K. Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology 117, 858–865 (1999).

    Article  CAS  Google Scholar 

  15. Kanayama, M. et al. Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G230–G239 (2007).

    Article  CAS  Google Scholar 

  16. Tahara, Y. et al. Hepatocyte growth factor facilitates colonic mucosal repair in experimental ulcerative colitis in rats. J. Pharmacol. Exp. Ther. 307, 146–151 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Wei, Q. et al. R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem. 282, 15903–15911 (2007).

    Article  CAS  Google Scholar 

  19. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).

    Article  CAS  Google Scholar 

  20. Gerbe, F. et al. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. J. Cell Biol. 192, 767–780 (2011).

    Article  CAS  Google Scholar 

  21. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  22. Haramis, A.P. et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004).

    Article  CAS  Google Scholar 

  23. 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 

  24. 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 

  25. van Es, J.H., de Geest, N., van de Born, M., Clevers, H. & Hassan, B.A. Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors. Nat. Commun. 1, 18 (2010).

    Article  Google Scholar 

  26. Wong, G.T. et al. Chronic treatment with the γ-secretase inhibitor LY-411,575 inhibits β-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J. Biol. Chem. 279, 12876–12882 (2004).

    Article  CAS  Google Scholar 

  27. Okamoto, R. et al. Requirement of Notch activation during regeneration of the intestinal epithelia. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G23–G35 (2009).

    Article  CAS  Google Scholar 

  28. Wirtz, S., Neufert, C., Weigmann, B. & Neurath, M.F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2, 541–546 (2007).

    Article  CAS  Google Scholar 

  29. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    Article  CAS  Google Scholar 

  30. Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  Google Scholar 

  31. Binnerts, M.E. et al. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc. Natl. Acad. Sci. USA 104, 14700–14705 (2007).

    Article  CAS  Google Scholar 

  32. Hayflick, L. & Moorhead, P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–621 (1961).

    Article  CAS  Google Scholar 

  33. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).

    Article  CAS  Google Scholar 

  34. Carmon, K.S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl. Acad. Sci. USA 108, 11452–11457 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Okabe (Osaka University) for EGFP transgenic mice and Y. Kato, J. Inazawa, I. Sekiya (TMDU), H. Snippert and R. Vries (Hubrecht Institute) for technical assistance. This study was supported by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, by the Health and Labour Sciences Research Grants for Research on Intractable Diseases from Ministry of Health, Labour and Welfare of Japan, and by a grant from the European Research Council and from the Dutch Cancer Foundation.

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Author notes

  1. Toshiro Sato

    Present address: Present address: Department of Gastroenterology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.,

  2. Shiro Yui and Tetsuya Nakamura: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

    Shiro Yui, Yasuhiro Nemoto, Tomohiro Mizutani, Xiu Zheng, Takashi Nagaishi, Kiichiro Tsuchiya & Mamoru Watanabe

  2. Department of Advanced Therapeutics for Gastrointestinal Diseases, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

    Tetsuya Nakamura & Ryuichi Okamoto

  3. Hubrecht Institute and University Medical Centre, Utrecht, The Netherlands

    Toshiro Sato & Hans Clevers

  4. Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

    Shizuko Ichinose

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  1. Shiro Yui
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Contributions

T. Nakamura, H.C. and M.W. designed the study. S.Y., T. Nakamura and T.S. performed experiments and analyzed data. T. Nakamura, T.S. and H.C. wrote the paper. Y.N., T. Nagaishi and K.T. assisted in transplantation experiments. T.M., X.Z. and K.T. gave support in gene analysis. R.O. helped with the immunohistochemistry. S.I. advised on the electron microscopy. H.C. and M.W. gave conceptual advice and supervised the project.

Corresponding authors

Correspondence to Hans Clevers or Mamoru Watanabe.

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Competing interests

H.C. and T.S. hold patents from the Hubrecht Institute, The Netherlands for the intestinal stem cells culture system.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Methods and Supplementary Table 1 (PDF 5382 kb)

Supplementary Video 1

A representative colonic crypt forming a cystic structure (MOV 6656 kb)

Supplementary Video 2

A colonic organoid grown from a single cell (MOV 17180 kb)

Supplementary Video 3

A dynamic expansion of Lgr5+ stem cells in growing organoids (MOV 28188 kb)

Supplementary Video 4

Another example of a growing organoid showing preferential expansion of Lgr5+ cells (MOV 28669 kb)

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Yui, S., Nakamura, T., Sato, T. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat Med 18, 618–623 (2012). https://doi.org/10.1038/nm.2695

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