Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma

  1. Elisa De Crignis
  2. Tanvir Hossain
  3. Shahla Romal
  4. Fabrizia Carofiglio
  5. Panagiotis Moulos
  6. Mir Mubashir Khalid
  7. Shringar Rao
  8. Ameneh Bazrafshan
  9. Monique MA Verstegen
  10. Farzin Pourfarzad
  11. Christina Koutsothanassis
  12. Helmuth Gehart
  13. Tsung Wai Kan
  14. Robert-Jan Palstra
  15. Charles Boucher
  16. Jan MN IJzermans
  17. Meritxell Huch
  18. Sylvia F Boj
  19. Robert Vries
  20. Hans Clevers
  21. Luc JW van der Laan
  22. Pantelis Hatzis
  23. Tokameh Mahmoudi  Is a corresponding author
  1. Erasmus MC, Netherlands
  2. Biomedical Sciences Research Center 'Alexander Fleming', Greece
  3. Erasmus University Medical Center, Netherlands
  4. Foundation Hubrecht Organoid Technology (HUB), Netherlands
  5. HybridStat Predictive Analytics, Greece
  6. University Medical Centre Utrecht, Netherlands
  7. University of Cambridge, United Kingdom

Abstract

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

Data availability

Sequencing data that support the findings of this study have been deposited in GEO with the accession code GSE 126798.

The following data sets were generated

Article and author information

Author details

  1. Elisa De Crignis

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  2. Tanvir Hossain

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  3. Shahla Romal

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6404-0015
  4. Fabrizia Carofiglio

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  5. Panagiotis Moulos

    Biomedical Sciences Research Center, Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece
    Competing interests
    The authors declare that no competing interests exist.
  6. Mir Mubashir Khalid

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1862-6795
  7. Shringar Rao

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  8. Ameneh Bazrafshan

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  9. Monique MA Verstegen

    Department of Surgery, Erasmus University Medical Center, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  10. Farzin Pourfarzad

    Foundation Hubrecht Organoid Technology (HUB), Foundation Hubrecht Organoid Technology (HUB), Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  11. Christina Koutsothanassis

    HybridStat Predictive Analytics, HybridStat Predictive Analytics, Vari, Greece
    Competing interests
    The authors declare that no competing interests exist.
  12. Helmuth Gehart

    Hubrecht Institute-KNAW, University Medical Centre Utrecht, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  13. Tsung Wai Kan

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  14. Robert-Jan Palstra

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  15. Charles Boucher

    Viroscience, Erasmus MC, 3015 CN Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  16. Jan MN IJzermans

    Department of Surgery, Erasmus University Medical Center, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  17. Meritxell Huch

    Wellcome Trust/Cancer Research UK, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  18. Sylvia F Boj

    Foundation Hubrecht Organoid Technology (HUB), Foundation Hubrecht Organoid Technology (HUB), Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  19. Robert Vries

    Foundation Hubrecht Organoid Technology (HUB), Foundation Hubrecht Organoid Technology (HUB), Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  20. Hans Clevers

    Hubrecht Institute-KNAW, University Medical Centre Utrecht, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  21. Luc JW van der Laan

    Department of Surgery, Erasmus University Medical Center, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  22. Pantelis Hatzis

    Biomedical Sciences Research Center, Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece
    Competing interests
    The authors declare that no competing interests exist.
  23. Tokameh Mahmoudi

    Biochemistry, Erasmus MC, 3015 CN Rotterdam, Netherlands
    For correspondence
    t.mahmoudi@erasmusmc.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2060-9353

Funding

European Research Council ERC (STG 337116)

  • Tokameh Mahmoudi

Dutch Aids Fonds (201614)

  • Tokameh Mahmoudi

Bristol Meyers Squibb (AI424-543)

  • Elisa De Crignis

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: The Medical Ethical Council of the Erasmus Medical Center approved the use of this material for research purposes (reference number: MEC-2014-060, Department of Surgery, Erasmus MC), and informed consent was provided from all patients.

Reviewing Editor

  1. Melanie Ott, University of California, San Francisco, United States

Publication history

  1. Preprint posted: March 5, 2019 (view preprint)
  2. Received: August 25, 2020
  3. Accepted: July 29, 2021
  4. Accepted Manuscript published: July 30, 2021 (version 1)
  5. Version of Record published: August 24, 2021 (version 2)

Copyright

© 2021, De Crignis et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Elisa De Crignis
  2. Tanvir Hossain
  3. Shahla Romal
  4. Fabrizia Carofiglio
  5. Panagiotis Moulos
  6. Mir Mubashir Khalid
  7. Shringar Rao
  8. Ameneh Bazrafshan
  9. Monique MA Verstegen
  10. Farzin Pourfarzad
  11. Christina Koutsothanassis
  12. Helmuth Gehart
  13. Tsung Wai Kan
  14. Robert-Jan Palstra
  15. Charles Boucher
  16. Jan MN IJzermans
  17. Meritxell Huch
  18. Sylvia F Boj
  19. Robert Vries
  20. Hans Clevers
  21. Luc JW van der Laan
  22. Pantelis Hatzis
  23. Tokameh Mahmoudi
(2021)
Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma
eLife 10:e60747.
https://doi.org/10.7554/eLife.60747

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