Embryonic or Induced Pluripotent Stem Cell-Derived Hepatocellular Systems for HCV Culture

  • Xianfang Wu
  • Viet Loan Dao Thi
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


Human-induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) constitute a powerful tool for modeling hepatotropic pathogen infections in cell culture. Meanwhile, CRISPR-Cas9 technology enables precise editing of stem cell genomes to generate patient-specific disease models and thus development of personalized experimental systems. Here we present a detailed stepwise protocol for the differentiation of stem cells to hepatocyte-like cells for HCV studies in cell culture. We also outline the use of an inducible iCRISPR platform for the rapid and efficient modification of host factors of interest to better understand their function during HCV infection.

Key words

Human embryonic/induced pluripotent stem cells Hepatocyte-like cells iCRISPR Precision medicine 


  1. 1.
    Goossens N, Hoshida Y (2015) Personalized management of hepatocellular carcinoma based on molecular information: future prospects. Clin Liver Dis 5:132–135CrossRefGoogle Scholar
  2. 2.
    Conti F, Buonfiglioli F, Scuteri A, Crespi C, Bolondi L, Caraceni P et al (2016) Early occurrence and recurrence of hepatocellular carcinoma in HCV-related cirrhosis treated with direct-acting antivirals. J Hepatol 65:727–733CrossRefGoogle Scholar
  3. 3.
    Reig M, Marino Z, Perello C, Inarrairaegui M, Ribeiro A, Lens S et al (2016) Unexpected high rate of early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy. J Hepatol 65:719–726CrossRefGoogle Scholar
  4. 4.
    Asselah T, Bieche I, Sabbagh A, Bedossa P, Moreau R, Valla D et al (2009) Gene expression and hepatitis C virus infection. Gut 58:846–858CrossRefGoogle Scholar
  5. 5.
    Heim MH (2013) 25 years of interferon-based treatment of chronic hepatitis C: an epoch coming to an end. Nat Rev Immunol 13:535–542CrossRefGoogle Scholar
  6. 6.
    Patin E, Kutalik Z, Guergnon J, Bibert S, Nalpas B, Jouanguy E et al (2012) Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection. Gastroenterology 143:1244–1252CrossRefGoogle Scholar
  7. 7.
    Wu X, Robotham JM, Lee E, Dalton S, Kneteman NM, Gilbert DM et al (2012) Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation. PLoS Pathog 8:e1002617CrossRefGoogle Scholar
  8. 8.
    Schwartz RE, Trehan K, Andrus L, Sheahan TP, Ploss A, Duncan SA et al (2012) Modeling hepatitis C virus infection using human induced pluripotent stem cells. Proc Natl Acad Sci U S A 109:2544–2548CrossRefGoogle Scholar
  9. 9.
    Shlomai A, Schwartz RE, Ramanan V, Bhatta A, de Jong YP, Bhatia SN et al (2014) Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems. Proc Natl Acad Sci U S A 111:12193–12198CrossRefGoogle Scholar
  10. 10.
    Carpentier A, Tesfaye A, Chu V, Nimgaonkar I, Zhang F, Lee SB et al (2014) Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model. J Clin Invest 124:4953–4964CrossRefGoogle Scholar
  11. 11.
    Paganelli M, Dallmeier K, Nyabi O, Scheers I, Kabamba B, Neyts J et al (2013) Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection. Hepatology 57:59–69CrossRefGoogle Scholar
  12. 12.
    Roelandt P, Obeid S, Paeshuyse J, Vanhove J, Van Lommel A, Nahmias Y et al (2012) Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus. J Hepatol 57:246–251CrossRefGoogle Scholar
  13. 13.
    Yoshida T, Takayama K, Kondoh M, Sakurai F, Tani H, Sakamoto N et al (2011) Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection. Biochem Biophys Res Commun 416:119–124CrossRefGoogle Scholar
  14. 14.
    Kaneko S, Kakinuma S, Asahina Y, Kamiya A, Miyoshi M, Tsunoda T et al (2016) Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus. Sci Rep 6:29358CrossRefGoogle Scholar
  15. 15.
    Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular SystemsGoogle Scholar
  16. 16.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872CrossRefGoogle Scholar
  17. 17.
    Gonzalez F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV et al (2014) An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15:215–226CrossRefGoogle Scholar
  18. 18.
    Lindenbach BD, Meuleman P, Ploss A, Vanwolleghem T, Syder AJ, McKeating JA et al (2006) Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci U S A 103:3805–3809CrossRefGoogle Scholar
  19. 19.
    Jones CT, Catanese MT, Law LM, Khetani SR, Syder AJ, Ploss A et al (2010) Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nat Biotechnol 28:167–171CrossRefGoogle Scholar
  20. 20.
    Zhou X, Sun P, Lucendo-Villarin B, Angus AG, Szkolnicka D, Cameron K et al (2014) Modulating innate immunity improves hepatitis C virus infection and replication in stem cell-derived hepatocytes. Stem Cell Reports 3:204–214CrossRefGoogle Scholar
  21. 21.
    Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L et al (2011) EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat Med 17:589–595CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Laboratory of Virology and Infectious Diseases, Center for the Study of Hepatitis CThe Rockefeller UniversityNew YorkUSA
  2. 2.Center for Integrative Infectious Disease ResearchUniversity Hospital HeidelbergHeidelbergGermany
  3. 3.Laboratory of Virology and Infectious DiseasesCenter for the Study of Hepatitis C, The Rockefeller UniversityNew YorkUSA

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