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Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing

A Corrigendum to this article was published on 01 April 2018

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Abstract

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

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Figure 1: A defined strategy for deriving colonic organoids from human embryonic stem cells (hESCs).
Figure 2: Global gene-expression analysis of hESC-derived COs or SIOs.
Figure 3: FAP colonic organoids show enhanced WNT activity and increased cell proliferation.
Figure 4: A platform for evaluating drug candidates that can rescue APC-mutation-induced hyperproliferation in human COs.

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Change history

  • 13 March 2018

    In the version of this article initially published, there were several instances of duplication of confocal microscopy images presented in Figures 1e, 3i and 4c,h,j. These errors occurred during the final preparation of the figures, when representative images were selected to illustrate the results obtained from multiple independent experiments. To ensure that these errors did not affect the paper’s conclusions, the authors verified all raw data used to quantify the results. The results presented in the original Figures 3j and 4d were not affected, but the quantification in Figure 4i was. This quantification was performed again, and the results supported the original conclusions of the paper. In addition, the western blot shown in Figure 3d came from a different replicate experiment than the one represented in Supplementary Figure 9g,h (which shows the raw data). The original blot has been replaced with the correct blot that matches the raw data originally provided. The conclusions of this experiment remain unchanged. All affected images have been now corrected in the HTML and PDF versions of the article.

References

  1. Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. McCracken, K.W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Dye, B.R. et al. In vitro generation of human pluripotent stem cell derived lung organoids. eLife 4, e05098 (2015).

    Article  PubMed Central  Google Scholar 

  6. Huang, L. et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nat. Med. 21, 1364–1371 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sherwood, R.I., Maehr, R., Mazzoni, E.O. & Melton, D.A. Wnt signaling specifies and patterns intestinal endoderm. Mech. Dev. 128, 387–400 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pitera, J.E., Smith, V.V., Thorogood, P. & Milla, P.J. Coordinated expression of 3′ hox genes during murine embryonal gut development: an enteric Hox code. Gastroenterology 117, 1339–1351 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Bekku, S. et al. Carbonic anhydrase I and II as a differentiation marker of human and rat colonic enterocytes. Res. Exp. Med. (Berl.) 198, 175–185 (1998).

    Article  CAS  Google Scholar 

  10. The Human Protein Atlas. CA4 (The Human Protein Atlas, n.d.); available at http://www.proteinatlas.org/ENSG00000167434–CA4/tissue/colon.

  11. Batlle, E. et al. Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251–263 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Bretscher, A. & Weber, K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc. Natl. Acad. Sci. USA 76, 2321–2325 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Allen, A., Hutton, D.A. & Pearson, J.P. The MUC2 gene product: a human intestinal mucin. Int. J. Biochem. Cell Biol. 30, 797–801 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. O'Connor, D.T., Burton, D. & Deftos, L.J. Chromogranin A: immunohistology reveals its universal occurrence in normal polypeptide hormone producing endocrine glands. Life Sci. 33, 1657–1663 (1983).

    Article  CAS  PubMed  Google Scholar 

  15. Roth, K.A., Kim, S. & Gordon, J.I. Immunocytochemical studies suggest two pathways for enteroendocrine cell differentiation in the colon. Am. J. Physiol. 263, G174–G180 (1992).

    CAS  PubMed  Google Scholar 

  16. Legay, C., Faudon, M., Héry, F. & Ternaux, J.P. 5-HT metabolism in the intestinal wall of the rat-I. The mucosa. Neurochem. Int. 5, 721–727 (1983).

    Article  CAS  PubMed  Google Scholar 

  17. Yan, K.S. et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc. Natl. Acad. Sci. USA 109, 466–471 (2012).

    Article  PubMed  Google Scholar 

  18. Takeda, N. et al. Interconversion between intestinal stem cell populations in distinct niches. Science 334, 1420–1424 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jenny, M. et al. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J. 21, 6338–6347 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Moll, R., Schiller, D.L. & Franke, W.W. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J. Cell Biol. 111, 567–580 (1990).

    Article  CAS  PubMed  Google Scholar 

  21. Green, M.D. et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol. 29, 267–272 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Beuling, E. et al. GATA4 mediates gene repression in the mature mouse small intestine through interactions with friend of GATA (FOG) cofactors. Dev. Biol. 322, 179–189 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Forster, R. et al. Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells. Stem Cell Reports 2, 838–852 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fujii, M. et al. A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell 18, 827–838 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sasikumar, R., Rejitha, J.R., Binumon, P.K. & Manoj, M. Role of heterozygous APC mutation in niche succession and initiation of colorectal cancer—a computational study. PLoS One 6, e22720 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Taketo, M.M. Apc gene knockout mice as a model for familial adenomatous polyposis. Prog. Exp. Tumor Res. 35, 109–119 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Yu, B., Lane, M.E., Pestell, R.G., Albanese, C. & Wadler, S. Downregulation of cyclin D1 alters cdk 4- and cdk 2-specific phosphorylation of retinoblastoma protein. Mol. Cell Biol. Res. Commun. 3, 352–359 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Voloshanenko, O. et al. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nat. Commun. 4, 2610 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Durno, C.A. Colonic polyps in children and adolescents. Can. J. Gastroenterol. 21, 233–239 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Faller, W.J. et al. mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517, 497–500 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Huang, S.M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Ignatenko, N.A. et al. Combination chemoprevention of intestinal carcinogenesis in a murine model of familial adenomatous polyposis. Nutr. Cancer 60 (Suppl. 1), 30–35 (2008).

    Article  PubMed  Google Scholar 

  36. Floquet, C., Rousset, J.P. & Bidou, L. Readthrough of premature termination codons in the adenomatous polyposis coli gene restores its biological activity in human cancer cells. PLoS One 6, e24125 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zilberberg, A., Lahav, L. & Rosin-Arbesfeld, R. Restoration of APC gene function in colorectal cancer cells by aminoglycoside- and macrolide-induced read-through of premature termination codons. Gut 59, 496–507 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Dow, L.E. et al. Apc restoration promotes cellular differentiation and establishes crypt homeostasis in colorectal cancer. Cell 161, 1539–1552 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Choy, J.Y. et al. A resource of ribosomal RNA-depleted RNA-seq data from different normal adult and fetal human tissues. Sci. Data 2, 150063 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Borras, E. et al. Genomic landscape of colorectal mucosa and adenomas. Cancer Prev. Res. (Phila.) 9, 417–427 (2016).

    Article  CAS  Google Scholar 

  41. Kanth, P. et al. Gene signature in sessile serrated polyps identifies colon cancer subtype. Cancer Prev. Res. (Phila.) 9, 456–465 (2016).

    Article  CAS  Google Scholar 

  42. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li, B. & Dewey, C.N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Greene, A.E., Charney, J., Nichols, W.W. & Coriell, L.L. Species identity of insect cell lines. In Vitro 7, 313–322 (1972).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

S.C. is funded by The New York Stem Cell Foundation (R-103) and NIH/NIDDK (1 DP2 DK098093-01, DP3DK111907-01). This study was supported in part by a shared facility contract from the New York Department of Health (NYSTEM, C029156; to T.E. and S.C.); a Tri-institutional Stem Cell Initiative grant (2013-001; to F. M. and S.C.); and a pilot grant from the Center for Advanced Digestion Care (CADC) at Weill Cornell Medical College (to S.C. and S.L.). S.C. is New York Stem Cell Foundation-Robertson Investigator. This work was also supported by grants R03CA176788 (US National Institutes of Health/National Cancer Institute), the MD Anderson Cancer Center Institutional Research Grant (IRG) Program (E.V.), and a gift from the Feinberg Family to E.V.; Cancer Prevention Educational Award (R25T CA057730, US National Institutes of Health/National Cancer Institute (to K.C.); Arnold O. Beckman postdoctoral fellowship to H.J.C. We thank H.E. Varmus (Weill Cornell Medical College) for his support and J. Jin at Weill Cornell Medical College for kindly providing human aortic smooth muscle cells. We are grateful for the technical support and advice provided by H.S. Ralph in the Cell Screening Core Facility, J. McCormick in the Flow Cytometry Facility and L. Cohen-Gould in the Electron Microscopy Facility at Weill Cornell Medical College.

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Contributions

S.C., T.E., S.L., and F.R.M. designed the project; M.C., S.-Y.T., S.A., T.S., N.H.P.H.J.C., M.W., and M.G. performed experiments; E.V., K.C., T. Z., and J.Z.X. performed the bioinformatics analysis; M.C., E.V., K.C., and S.C. analyzed data; and M.C., E.V., F.R.M., S.L., T.E., and S.C. wrote the manuscript.

Corresponding authors

Correspondence to Todd Evans or Shuibing Chen.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9 and Supplementary Tables 1–3 (PDF 42809 kb)

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Crespo, M., Vilar, E., Tsai, SY. et al. Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing. Nat Med 23, 878–884 (2017). https://doi.org/10.1038/nm.4355

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