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pp 1-13 | Cite as

Derivation of Intestinal Organoids from Human Induced Pluripotent Stem Cells for Use as an Infection System

  • Jessica L. ForbesterEmail author
  • Nicholas Hannan
  • Ludovic Vallier
  • Gordon Dougan
Protocol
Part of the Methods in Molecular Biology book series

Abstract

Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs).

Keywords

Organoids hIPSCs Intestinal epithelium Differentiation Microinjection Host–pathogen interactions 

Notes

Acknowledgments

This work was supported by the Medical Research Council and the Wellcome Trust. We thank David Goulding for providing TEM images.

References

  1. 1.
    Fatehullah A, Tan SH, Barker N (2016) Organoids as an in vitro model of human development and disease. Nat Cell Biol 18(3):246–254CrossRefGoogle Scholar
  2. 2.
    Finkbeiner SR, Zeng XL, Utama B, Atmar RL, Shroyer NF, Estes MK (2012) Stem cell-derived human intestinal organoids as an infection model for rotaviruses. MBio 3(4), e00159-12, doi:ARTN e00159.10.1128/mBio.00159-12CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Zhang YG, Wu S, Xia Y, Sun J (2014) Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions. Physiol Rep 2(9), e12147, doi:10.14814/phy2.12147CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Leslie JL, Huang S, Opp JS, Nagy MS, Kobayashi M, Young VB, Spence JR (2014) Persistence and toxin production by Clostridium difficile within human intestinal organoids results in disruption of epithelial paracellular barrier function. Infect Immun 83:138–145. doi: 10.1128/IAI.02561-14 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Date S, Sato T (2015) Mini-gut organoids: reconstitution of the stem cell niche. Annu Rev Cell Dev Biol 31:269–289. doi: 10.1146/annurev-cellbio-100814-125218 CrossRefPubMedGoogle Scholar
  6. 6.
    Jung P, Sato T, Merlos-Suarez 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(10):1225–1227. doi: 10.1038/nm.2470 CrossRefPubMedGoogle Scholar
  7. 7.
    Forbester JL, Goulding D, Vallier L, Hannan N, Hale C, Pickard D, Mukhopadhyay S, Dougan G (2015) Interaction of Salmonella enterica Serovar Typhimurium with intestinal organoids derived from human induced pluripotent stem cells. Infect Immun 83(7):2926–2934. doi: 10.1128/IAI.00161-15 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hannan NR, Fordham RP, Syed YA, Moignard V, Berry A, Bautista R, Hanley NA, Jensen KB, Vallier L (2013) Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Reports 1(4):293–306. doi: 10.1016/j.stemcr.2013.09.003 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    McKernan R, Watt FM (2013) What is the point of large-scale collections of human induced pluripotent stem cells? Nat Biotechnol 31(10):875–877. doi: 10.1038/nbt.2710 CrossRefPubMedGoogle Scholar
  10. 10.
    Birket MJ, Ribeiro MC, Verkerk AO, Ward D, Leitoguinho AR, den Hartogh SC, Orlova VV, Devalla HD, Schwach V, Bellin M, Passier R, Mummery CL (2015) Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nat Biotechnol 33(9):970–979. doi: 10.1038/nbt.3271 CrossRefGoogle Scholar
  11. 11.
    Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379. doi: 10.1038/nature12517 ADSCrossRefPubMedGoogle Scholar
  12. 12.
    Hale C, Yeung A, Goulding D, Pickard D, Alasoo K, Powrie F, Dougan G, Mukhopadhyay S (2015) Induced pluripotent stem cell derived macrophages as a cellular system to study Salmonella and other pathogens. PLoS One 10(5):e0124307, doi:ARTN e0124307. 10.1371/journal.pone.0124307CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jessica L. Forbester
    • 1
    Email author
  • Nicholas Hannan
    • 2
  • Ludovic Vallier
    • 1
    • 3
  • Gordon Dougan
    • 1
  1. 1.Wellcome Trust Sanger InstituteHinxton, CambridgeUK
  2. 2.University of NottinghamNottinghamUK
  3. 3.Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, Department of SurgeryUniversity of CambridgeCambridgeUK

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