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Transplantation of intestinal organoids into a mouse model of colitis

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.

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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.

Data availability

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

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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

Authors

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.

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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.

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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

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