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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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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