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

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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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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Authors

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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