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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Potten, C.S., Booth, C. & Pritchard, D.M. The intestinal epithelial stem cell: the mucosal governor. Int. J. Exp. Pathol. 78, 219–243 (1997).
Bjerknes, M. & Cheng, H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006).
Barker, N., van de Wetering, M. & Clevers, H. The intestinal stem cell. Genes Dev. 22, 1856–1864 (2008).
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).
Radtke, F. & Clevers, H. Self-renewal and cancer of the gut: two sides of a coin. Science 307, 1904–1909 (2005).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
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).
Sangiorgi, E. & Capecchi, M.R. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40, 915–920 (2008).
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).
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).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).
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).
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).
Kanayama, M. et al. Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G230–G239 (2007).
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).
Kim, K.A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005).
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).
Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).
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).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Haramis, A.P. et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004).
Fre, S. et al. Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964–968 (2005).
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).
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).
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).
Okamoto, R. et al. Requirement of Notch activation during regeneration of the intestinal epithelia. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G23–G35 (2009).
Wirtz, S., Neufert, C., Weigmann, B. & Neurath, M.F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2, 541–546 (2007).
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).
Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Binnerts, M.E. et al. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc. Natl. Acad. Sci. USA 104, 14700–14705 (2007).
Hayflick, L. & Moorhead, P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–621 (1961).
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
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).
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.
H.C. and T.S. hold patents from the Hubrecht Institute, The Netherlands for the intestinal stem cells culture system.
Supplementary Figures 1–7, Supplementary Methods and Supplementary Table 1 (PDF 5382 kb)
A representative colonic crypt forming a cystic structure (MOV 6656 kb)
A colonic organoid grown from a single cell (MOV 17180 kb)
A dynamic expansion of Lgr5+ stem cells in growing organoids (MOV 28188 kb)
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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