Advertisement

Use of Biliary Organoids in Cholestasis Research

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1981)

Abstract

Cholangiocytes play a crucial role in the pathophysiology of cholestasis. However, research on human cholangiocytes has been restricted by challenges in long-term propagation and large-scale expansion of primary biliary epithelium. The advent of organoid technology has overcome this limitation allowing long-term culture of a variety of epithelia from multiple organs. Here, we describe two methods for growing human cholangiocytes in organoid format. The first applies to the generation of intrahepatic bile ducts using human induced pluripotent stem cells using a protocol of differentiation that recapitulates physiological bile duct development. The second method allows the propagation of primary biliary epithelium from the extrahepatic ducts or gallbladder. Both protocols result in large numbers of cholangiocyte organoids expressing biliary markers and maintaining key cholangiocyte functions.

Key words

Cholangiocytes Human pluripotent stem cells Extrahepatic biliary epithelium Gallbladder Common bile duct Organoids Cholestasis Endoscopic retrograde cholangiopancreatography Liver biopsy 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was financially supported by grants by the Addenbrooke’s Charitable Trust (ACT) (F.S.), Academy of Medical Sciences (F.S.), NIHR (F.S.), and the Rosetrees Trust Interdisciplinary project grant “Generation and transplantation of a bioengineered human bile duct” (F.S. and L.V.). L.V. lab is funded by the ERC proof of concept grant Relieve-Chol, by the ERC advanced grant New-Chol, the Cambridge University Hospitals National Institute for Health Research Biomedical Research Center and the core support grant from the Wellcome Trust and Medical Research Council to the Wellcome-Medical Research Council Cambridge Stem Cell Institute.

References

  1. 1.
    Glaser S, Francis H, DeMorrow S et al (2006) Heterogeneity of the intrahepatic biliary epithelium. World J Gastroenterol 12:3523–3536CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lazaridis KN, Strazzabosco M, Larusso NF (2004) The cholangiopathies: disorders of biliary epithelia. Gastroenterology 127:1565–1577CrossRefGoogle Scholar
  3. 3.
    Lazaridis KN, LaRusso NF (2015) The cholangiopathies. Mayo Clin Proc 90:791–800CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sampaziotis F, de Brito MC, Madrigal P et al (2015) Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat Biotechnol 33:845–852CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459:262–265CrossRefPubMedGoogle Scholar
  6. 6.
    Sampaziotis F, de Brito MC, Geti I et al (2017) Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells. Nat Protoc 12:814–827CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sampaziotis F, Justin AW, Tysoe OC et al (2017) Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids. Nat Med 23:954–963CrossRefPubMedGoogle Scholar
  8. 8.
    Tanimizu N, Miyajima A, Mostov KE (2007) Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture. Mol Biol Cell 18:1472–1479CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dianat N, Dubois-Pot-Schneider H, Steichen C et al (2014) Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells. Hepatology 60:700–714CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ogawa M, Ogawa S, Bear CE et al (2015) Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat Biotechnol 33:853–861CrossRefPubMedGoogle Scholar
  11. 11.
    Esteller A (2008) Physiology of bile secretion. World J Gastroenterol 14:5641–5649CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Marinelli RA, Tietz PS, Pham LD et al (1999) Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. Am J Physiol 276:G280–G286PubMedGoogle Scholar
  13. 13.
    Boyer JL (2013) Bile formation and secretion. Compr Physiol 3:1035–1078PubMedPubMedCentralGoogle Scholar
  14. 14.
    Xia X, Francis H, Glaser S et al (2006) Bile acid interactions with cholangiocytes. World J Gastroenterol 12:3553–3563CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Strazzabosco M (1997) Transport systems in cholangiocytes: their role in bile formation and cholestasis. Yale J Biol Med 70:427–434PubMedPubMedCentralGoogle Scholar
  16. 16.
    Si-Tayeb K, Lemaigre FP, Duncan SA et al (2010) Organogenesis and development of the liver. Dev Cell 18:175–189CrossRefPubMedGoogle Scholar
  17. 17.
    Antoniou A, Raynaud P, Cordi S et al (2009) Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology 136:2325–2333CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zong Y, Panikkar A, Xu J et al (2009) Notch signaling controls liver development by regulating biliary differentiation. Development 136:1727–1739CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Strazzabosco M, Fabris L (2012) Development of the bile ducts: essentials for the clinical hepatologist. J Hepatol 56:1159–1170CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Wellcome Trust and MRC Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative MedicineUniversity of CambridgeCambridgeUK
  2. 2.Department of MedicineUniversity of CambridgeCambridgeUK
  3. 3.Department of SurgeryUniversity of CambridgeCambridgeUK

Personalised recommendations

Cite protocol

Buy options