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.

  • Protocol
  • Published:

Transplantation of intestinal organoids into a mouse model of colitis

Nature Protocols volume 17, pages 649–671 (2022)Cite this article

Subjects

Abstract

Intestinal organoids are fundamental in vitro tools that have enabled new research opportunities in intestinal stem cell research. Organoids can also be transplanted in vivo, which enables them to probe stem cell potential and be used for disease modeling and as a preclinical tool in regenerative medicine. Here we describe in detail how to orthotopically transplant epithelial organoids into the colon of recipient mice. In this assay, epithelial injury is initiated at the distal part of colon by the administration of dextran sulfate sodium, and organoids are infused into the luminal space via the anus. The infused organoids subsequently attach to the injured region and rebuild a donor-derived epithelium. The steps for cell infusion can be completed in 10 min. The assay has been applied successfully to organoids derived from both wild-type and genetically altered epithelial cells from adult colonic and small intestinal epithelium, as well as fetal small intestine. This is a versatile protocol, providing the technical basis for transplantation following alternative colonic injury models. It has been used previously for functional assays to probe cellular potential, and formed the basis for the first in-human clinical trial using colonic organoid transplantation therapy for intractable cases of ulcerative colitis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Outline of the DSS-grafting assay.
Fig. 2: Preparation of donor organoids.
Fig. 3: Step-by-step procedure for the organoid infusion.
Fig. 4: Tissue sampling, storage and sectioning.
Fig. 5: Images showing how to detect and process engraftments.
Fig. 6: Typical macroscopic results obtained using the protocol.
Fig. 7: Integration of organoids.
Fig. 8: Typical microscopic results obtained using the protocol.

Similar content being viewed by others

Data availability

All data generated or analyzed in this study are included in Supplementary Data with reference to the originally published paper.

References

  1. van der Flier, L. G. & Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).

    Article  PubMed  CAS  Google Scholar 

  2. Beumer, J. & Clevers, H. Cell fate specification and differentiation in the adult mammalian intestine. Nat. Rev. Mol. Cell Biol. 22, 39–53 (2021).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  4. Antfolk, M. & Jensen, K. B. A bioengineering perspective on modelling the intestinal epithelial physiology in vitro. Nat. Commun. 11, 6244 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Merenda, A., Fenderico, N. & Maurice, M. M. Wnt signaling in 3D: recent advances in the applications of intestinal organoids. Trends Cell Biol. 30, 60–73 (2020).

    Article  CAS  PubMed  Google Scholar 

  6. Sprangers, J., Zaalberg, I. C. & Maurice, M. M. Organoid-based modeling of intestinal development, regeneration, and repair. Cell Death Differ. 28, 95–107 (2021).

    Article  PubMed  Google Scholar 

  7. Date, S. & Sato, T. Mini-gut organoids: reconstitution of the stem cell niche. Annu. Rev. Cell Dev. Biol. 31, 269–289 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Nakamura, T. & Sato, T. Advancing intestinal organoid technology toward regenerative medicine. Cell Mol. Gastroenterol. Hepatol. 5, 51–60 (2018).

    Article  PubMed  Google Scholar 

  9. Clevers, H. et al. Tissue-engineering the intestine: the trials before the trials. Cell Stem Cell 24, 855–859 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Clevers, H. C. Organoids: avatars for personalized medicine. Keio J. Med. 68, 95 (2019).

    Article  PubMed  Google Scholar 

  11. Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat. Med. 18, 618–623 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Fordham, R. P. et al. Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13, 734–744 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yui, S. et al. YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell Stem Cell 22, 35–49e37 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Guiu, J. et al. Tracing the origin of adult intestinal stem cells. Nature 570, 107–111 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Thomas, E. D., Lochte, H. L. Jr., Lu, W. C. & Ferrebee, J. W. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N. Engl. J. Med. 257, 491–496 (1957).

    Article  CAS  PubMed  Google Scholar 

  17. Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Jensen, K. B., Driskell, R. R. & Watt, F. M. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat. Protoc. 5, 898–911 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Bergenheim, F. et al. Fluorescence-based tracing of transplanted intestinal epithelial cells using confocal laser endomicroscopy. Stem Cell Res. Ther. 10, 148 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Bergenheim, F. et al. A fully defined 3D matrix for ex vivo expansion of human colonic organoids from biopsy tissue. Biomaterials 262, 120248 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Morris, S. A. et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell 158, 889–902 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Miura, S. & Suzuki, A. Generation of mouse and human organoid-forming intestinal progenitor cells by direct lineage reprogramming. Cell Stem Cell 21, 456–471 e455 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. O’Rourke, K. P. et al. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nat. Biotechnol. 35, 577–582 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Ganesh, K. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat. Med. 25, 1607–1614 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Roper, J. et al. Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation. Nat. Protoc. 13, 217–234 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Roper, J. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat. Biotechnol. 35, 569–576 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cruz-Acuna, R. et al. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nat. Cell Biol. 19, 1326–1335 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cruz-Acuna, R. et al. PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery. Nat. Protoc. 13, 2102–2119 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Fumagalli, A. et al. Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids. Proc. Natl Acad. Sci. USA 114, E2357–E2364 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fumagalli, A. et al. A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression. Nat. Protoc. 13, 235–247 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Khalil, H. A. et al. Intestinal epithelial replacement by transplantation of cultured murine and human cells into the small intestine. PLoS One 14, e0216326 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. de Sousa e Melo, F. et al. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543, 676–680 (2017).

    Article  PubMed  CAS  Google Scholar 

  35. Fukuda, M. et al. Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon. Genes Dev. 28, 1752–1757 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sugimoto, S. et al. An organoid-based organ-repurposing approach to treat short bowel syndrome. Nature 592, 99–104 (2021).

    CAS  PubMed  Google Scholar 

  37. Sugimoto, S. et al. Reconstruction of the human colon epithelium in vivo. Cell Stem Cell 22, 171–176 e175 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Jee, J. H. et al. Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture. Stem Cells Int. 2019, 8472712 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Wirtz, S. et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat. Protoc. 12, 1295–1309 (2017).

    Article  CAS  PubMed  Google Scholar 

  40. Eichele, D. D. & Kharbanda, K. K. Dextran sodium sulfate colitis murine model: an indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis. World J. Gastroenterol. 23, 6016–6029 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Serra, D. et al. Self-organization and symmetry breaking in intestinal organoid development. Nature 569, 66–72 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  43. Liu, A. et al. Aging increases the severity of colitis and the related changes to the gut barrier and gut microbiota in humans and mice. J. Gerontol. A Biol. Sci. Med. Sci. 75, 1284–1292 (2020).

    Article  CAS  PubMed  Google Scholar 

  44. Perse, M. & Cerar, A. Dextran sodium sulphate colitis mouse model: traps and tricks. J. Biomed. Biotechnol. 2012, 718617 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Dieleman, L. A. et al. Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology 107, 1643–1652 (1994).

    Article  CAS  PubMed  Google Scholar 

  46. Mahler, M. et al. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am. J. Physiol. 274, G544–G551 (1998).

    CAS  PubMed  Google Scholar 

  47. Janda, C. Y. et al. Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling. Nature 545, 234–237 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by MEXT/JSPS KAKENHI (18K15743, 20H03657, 19H01050, 19H03634), Young Innovative Medical Science Unit (TMDU), Naoki Tsuchiya Research Grant, Japan Agency for Medical Research and Development (AMED) (20bm0704029h0003, 20bm0304001h0008, 20bk0104008h0003, 20bm0404055h0002), Marie Curie fellowship programme (625238 to S.Y.), the DFF mobilex programme (1333-00130B to S.Y.), European Union’s Horizon 2020 research and innovation programme (KBJ - ERCCoG682665) and the Novo Nordisk Foundation Center for Stem Cell Medicine, which is supported by Novo Nordisk Foundation grants (NNF17CC0027852 and NNF21CC0073729).

Author information

Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Satoshi Watanabe, Sakurako Kobayashi, Nobuhiko Ogasawara & Ryuichi Okamoto

  2. Department of Research and Development for Organoids, Juntendo University School of Medicine, Tokyo, Japan

    Tetsuya Nakamura

  3. Advanced Research Institute (IBD Lab), Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Mamoru Watanabe

  4. Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Kim B. Jensen

  5. Novo Nordisk Foundation Center for Stem Cell Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Kim B. Jensen

  6. Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Shiro Yui

Authors
  1. Satoshi Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sakurako Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nobuhiko Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ryuichi Okamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tetsuya Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mamoru Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kim B. Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shiro Yui
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, R.O., T.N., M.W., K.B.J. and S.Y.; methodology, S.W., S.K., N.O., T.N., M.W., K.B.J. and S.Y.; investigation, S.W., S.K., N.O., and S.Y.; writing—original draft, S.W., K.B.J., and S.Y.; writing—review and editing, all authors.; supervision, M.W.; funding acquisition, R.O., T.N., M.W., K.B.J. and S.Y.

Corresponding authors

Correspondence to Kim B. Jensen or Shiro Yui.

Ethics declarations

Competing interests

The authors declare competing interests. T.N. and M.W. are inventors on a patent related to organoid culture system and transplantation.

Peer review

Peer review information

Nature Protocols thanks Arianna Fumagalli, Jatin Roper and Eduardo Villablanca for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Yui, S. et al. Nat. Med. 18, 618–623 (2012): https://doi.org/10.1038/nm.2695

Yui, S. et al. Cell Stem Cell 22, 35–49.e7 (2018): https://doi.org/10.1016/j.stem.2017.11.001

Guiu, J. et al. Nature 570, 107–111 (2019): https://doi.org/10.1038/s41586-019-1212-5

Extended data

Extended Data Fig. 1 Detailed assessment of past cohorts that have undergone the protocol.

a, Comparison of engrafted areas (µm2) divided on tissue origin of organoid (colon or SI) and recipient model (RAG2-/- or C57BL/6J). Red line indicates mean of measurements in each sample set. b, Assessment of the impact of Wnt3a as a supplement in the medium used to culture small intestinal organoids on the engrafted area (µm2). Red lines indicate mean of measurements in each sample set. c, Impact of extracellular matrix used for culturing colonic organoids on the engraftment using RAG2-/- or C57BL/6J recipient animals. Red lines indicate mean of measurements in each sample set. d, Engraftment rates (%) of transplantation experiments conducted in RAG2-/- mice shown over time divided into early, middle and late. This illustrates the gradual increase in success rate as the assay becomes optimized. e, Transplantation efficiency (% positive engraftment) is independent of whether female and male are used as recipient animals (RAG2-/- or C57BL/6J). Red lines indicate mean of measurements in each sample set. f, Across operators the success rate (%) is very similar among 8 cohorts of recipients (C57BL/6J) mice. Red lines indicate mean of measurements in each sample set. The analysis represents additional analysis of the cohorts analyzed in Figs. 1c and 6e.

Extended Data Fig. 2 Additional examples of results obtained using the protocol.

a, Images represents colonic organoids derived from a GFP transgenic mice transplanted into a RAG2-/- mouse. A part is magnified in a’ with arrow heads indicating epithelial injury. Scale bar, 1 mm in a and 500 µm in a′. b, An example showing a RAG2-/- mouse without any engraftment and in the magnification in b′ it is evident that there is no obvious epithelial injury. Scale bar, 1 mm in b and 500 µm in b′. The samples presented in a) and b) are analyzed at 2 weeks after transplantation, and are obtained in cohorts that were not published previously. c, Schematic illustration of competition assay where the same number of tdTomato positive control organoids derived from Rosa26mT/mG reporter mice and GFP positive organoids derived from VillinCreER; YAP/TAZ dKO mice were combined and transplanted to the colon of a RAG2-/- mouse. Successful attachment of both organoids is shown in c′. Scale bar, 500 µm. c′ is an enlarged view of Fig. 6D from ref. 14.

Supplementary information

Supplementary Video 1

Anal infusion of a suspension of organoid fragments

Supplementary Video 2

Retraction of catheter and temporary closing of anus

Supplementary Data

Details of past cohorts, organoid sizes and areas of engraftment.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Watanabe, S., Kobayashi, S., Ogasawara, N. et al. Transplantation of intestinal organoids into a mouse model of colitis. Nat Protoc 17, 649–671 (2022). https://doi.org/10.1038/s41596-021-00658-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00658-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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