Transplantation of intestinal organoids into a mouse model of colitis

Satoshi Watanabe, Sakurako Kobayashi, Nobuhiko Ogasawara, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe, Kim B. Jensen, Shiro Yui

Published: 2022-02-01 DOI: 10.1038/s41596-021-00658-3

Extended

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

a , Comparison of engrafted areas (µm 2 ) 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 (µm 2 ). 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 .

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References

Intestinal organoids are fundamental invitro tools that have enabled new research opportunities in intestinal stem cell research. Organoids can also be transplanted invivo, which enables them to probe stem cell potential and be used for disease modeling
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References
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS

van der Flier, L. G. & Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).
Beumer, J. & Clevers, H. Cell fate specification and differentiation in the adult mammalian intestine. Nat. Rev. Mol. Cell Biol. 22, 39–53 (2021).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Antfolk, M. & Jensen, K. B. A bioengineering perspective on modelling the intestinal epithelial physiology in vitro. Nat. Commun. 11, 6244 (2020).
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).
Sprangers, J., Zaalberg, I. C. & Maurice, M. M. Organoid-based modeling of intestinal development, regeneration, and repair. Cell Death Differ. 28, 95–107 (2021).
Date, S. & Sato, T. Mini-gut organoids: reconstitution of the stem cell niche. Annu. Rev. Cell Dev. Biol. 31, 269–289 (2015).
Nakamura, T. & Sato, T. Advancing intestinal organoid technology toward regenerative medicine. Cell Mol. Gastroenterol. Hepatol. 5, 51–60 (2018).
Clevers, H. et al. Tissue-engineering the intestine: the trials before the trials. Cell Stem Cell 24, 855–859 (2019).
Clevers, H. C. Organoids: avatars for personalized medicine. Keio J. Med. 68, 95 (2019).
Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018).
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).
Fordham, R. P. et al. Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13, 734–744 (2013).
Yui, S. et al. YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell Stem Cell 22, 35–49e37 (2018).
Guiu, J. et al. Tracing the origin of adult intestinal stem cells. Nature 570, 107–111 (2019).
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).
Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).
Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006).
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).
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).
Bergenheim, F. et al. Fluorescence-based tracing of transplanted intestinal epithelial cells using confocal laser endomicroscopy. Stem Cell Res. Ther. 10, 148 (2019).
Bergenheim, F. et al. A fully defined 3D matrix for ex vivo expansion of human colonic organoids from biopsy tissue. Biomaterials 262, 120248 (2020).
Morris, S. A. et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell 158, 889–902 (2014).
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).
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).
Ganesh, K. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat. Med. 25, 1607–1614 (2019).
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).
Roper, J. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat. Biotechnol. 35, 569–576 (2017).
Cruz-Acuna, R. et al. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nat. Cell Biol. 19, 1326–1335 (2017).
Cruz-Acuna, R. et al. PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery. Nat. Protoc. 13, 2102–2119 (2018).
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).
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).
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).
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).
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).
Sugimoto, S. et al. An organoid-based organ-repurposing approach to treat short bowel syndrome. Nature 592, 99–104 (2021).
Sugimoto, S. et al. Reconstruction of the human colon epithelium in vivo. Cell Stem Cell 22, 171–176 e175 (2018).
Jee, J. H. et al. Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture. Stem Cells Int. 2019, 8472712 (2019).
Wirtz, S. et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat. Protoc. 12, 1295–1309 (2017).
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).
Serra, D. et al. Self-organization and symmetry breaking in intestinal organoid development. Nature 569, 66–72 (2019).
Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).
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).
Perse, M. & Cerar, A. Dextran sodium sulphate colitis mouse model: traps and tricks. J. Biomed. Biotechnol. 2012, 718617 (2012).
Dieleman, L. A. et al. Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology 107, 1643–1652 (1994).
Mahler, M. et al. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am. J. Physiol. 274, G544–G551 (1998).
Janda, C. Y. et al. Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling. Nature 545, 234–237 (2017).

Transplantation of intestinal organoids into a mouse model of colitis

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