Advertisement

Wnt Signaling pp 141-159 | Cite as

The Generation of Organoids for Studying Wnt Signaling

  • Jarno Drost
  • Benedetta Artegiani
  • Hans CleversEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1481)

Abstract

We established an in vitro culture model in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. Moreover, organoids can easily be genetically modified using different genome modification strategies, including viral delivery of transgenes and CRISPR/Cas9 technology. These combined characteristics make them a useful in vitro model system to study many biological processes including the contribution of cellular signaling pathways to tissue homeostasis and disease. Here we describe our current laboratory protocols to establish human intestinal organoids and how to genetically modify both mouse and human intestinal organoids to study cellular signaling pathways, specifically Wnt signaling. Moreover, we provide a detailed protocol for lentiviral transduction and CRISPR/Cas9-mediated genome modification of organoid cultures.

Key words

Organoids Intestine Wnt signaling Lentivirus CRISPR/Cas9 

Notes

Acknowledgements

We thank Johan H. van Es for critical reading of the manuscript. We are grateful for support from the following: The Netherlands Organisation for Scientific Research (NWO-ZonMw) VENI grant to J.D. (91614138); FEBS long term fellowship to B.A.

References

  1. 1.
    Clevers H, Nusse R (2012) Wnt/β-catenin signaling and disease. Cell 149:1192–1205CrossRefPubMedGoogle Scholar
  2. 2.
    Moser AR, Mattes EM, Dove WF, Lindstrom MJ, Haag JD, Gould MN (1993) ApcMin, a mutation in the Apc gene, predisposes to mammary carcinomas and focal alveolar hyperplasias. Proc Natl Acad Sci U S A 90:8977–8981CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Moser AR, Pitot HC, Dove WF (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247:322–324CrossRefPubMedGoogle Scholar
  4. 4.
    Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cell build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265CrossRefPubMedGoogle Scholar
  5. 5.
    Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, Clevers H (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141:1762–1772CrossRefPubMedGoogle Scholar
  6. 6.
    Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T, Stange DE, Begthel H, van den Born M, Danenberg E, van den Brink S, Korving J, Abo A, Peters PJ, Wright N, Poulsom R, Clevers H (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6:25–36CrossRefPubMedGoogle Scholar
  7. 7.
    Jung P, Sato T, Merlos-Suárez A, Barriga FM, Iglesias M, Rossell D, Auer H, Gallardo M, Blasco MA, Sancho E, Clevers H, Batlle E (2011) Isolation and in vitro expansion of human colonic stem cells. Nat Med 17:1225–1227CrossRefPubMedGoogle Scholar
  8. 8.
    Huch M, Bonfanti P, Boj SF, Sato T, Loomans CJ, van de Wetering M, Sojoodi M, Li VS, Schuijers J, Gracanin A, Ringnalda F, Begthel H, Hamer K, Mulder J, van Es JH, de Koning E, Vries RG, Heimberg H, Clevers H (2013) Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J 32:2708–2721CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H (2013) In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494:247–250CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Karthaus WR, Iaquinta PJ, Drost J, Gracanin A, van Boxtel R, Wongvipat J, Dowling CM, Gao D, Begthel H, Sachs N, Vries RG, Cuppen E, Chen Y, Sawyers CL, Clevers HC (2014) Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159:163–175CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Drost J et al (2016) Organoid culture systems for prostate epithelial and cancer tissue. Nat Protocols 11:347–358Google Scholar
  12. 12.
    Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H (2011) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9:81–83CrossRefPubMedGoogle Scholar
  13. 13.
    Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent CK, Nieuwenhuis EE, Beekman JM, Clevers H (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13:653–658CrossRefPubMedGoogle Scholar
  14. 14.
    Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, Sachs N, Overmeer RM, Offerhaus GJ, Begthel H, Korving J, van de Wetering M, Schwank G, Logtenberg M, Cuppen E, Snippert HJ, Medema JP, Kops GJ, Clevers H (2015) Sequential cancer mutations in cultured human intestinal stem cells. Nature 521:43–47CrossRefPubMedGoogle Scholar
  15. 15.
    Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T (2015) Modelling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 21:256–262PubMedGoogle Scholar
  16. 16.
    Sato T, Clevers H (2013) Primary mouse small intestinal epithelial cell cultures. Methods Mol Biol 945:319–328CrossRefPubMedGoogle Scholar
  17. 17.
    Van Es JH, Clevers H (2015) Generation and analysis of mouse intestinal tumors and organoids harboring APC and K-Ras mutations. Methods Mol Biol 1267:125–144CrossRefPubMedGoogle Scholar
  18. 18.
    Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170CrossRefPubMedGoogle Scholar
  19. 19.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefPubMedGoogle Scholar
  20. 20.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, Kuo CJ (2009) Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15:701–718CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jarno Drost
    • 1
  • Benedetta Artegiani
    • 1
  • Hans Clevers
    • 1
    Email author
  1. 1.Hubrecht Institute-KNAW and University Medical Center UtrechtUtrechtThe Netherlands

Personalised recommendations