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Long-term culture and expansion of primary human hepatocytes

Nature Biotechnology volume 33, pages 12641271 (2015) | Download Citation

Abstract

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 1013 to 1016 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development.

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Acknowledgements

The authors wish to thank D. Kitsberg, E. Flashner and T. Golan-Lev for technical support. We also wish to thank M. Vinken, V. Rogiers, N. Benvenisty and S. Bhatia for their comments and suggestions. This work was funded by the Förderprogram Biotechnologie Baden-Würtenberg (project 720.830-4-03; S.H., S.D.R., A.N. and J.B.), European Research Council Starting Grant TMIHCV (project 242699; G.L., D.B., M.C. and Y.N.), and HeMiBio: a jointly funded consortium by the European Commission and Cosmetics Europe, as part of the SEURAT-1 cluster (project HEALTH-F5-2010-266777).

Author information

Affiliations

  1. Alexander Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.

    • Gahl Levy
    • , David Bomze
    • , Merav Cohen
    •  & Yaakov Nahmias
  2. Upcyte Technologies GmbH, Hamburg, Germany.

    • Stefan Heinz
    •  & Astrid Noerenberg
  3. Medicyte GmbH, Heidelberg, Germany.

    • Sarada Devi Ramachandran
    •  & Joris Braspenning
  4. Liver Unit, Department of Gastroenterology, Tel Aviv Medical Center and Tel Aviv University, Tel Aviv, Israel.

    • Oren Shibolet
  5. Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

    • Ella Sklan
  6. Department of Cell and Developmental Biology, The Hebrew University of Jerusalem, Jerusalem, Israel.

    • Merav Cohen
    •  & Yaakov Nahmias
  7. Fraunhofer Translational Center, Würzburg, Germany

    • Joris Braspenning

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Contributions

G.L., S.H., S.D.R., A.N. and Y.N. designed and performed experiments and analyzed data; D.B., M.C., E.S. and O.S. provided materials, technical support and conceptual advice; J.B. and Y.N., administered experiments and wrote the paper.

Competing interests

Y.N., G.L., A.N., S.H. and J.B. submitted a patent application on the method described in this work.

Corresponding author

Correspondence to Yaakov Nahmias.

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DOI

https://doi.org/10.1038/nbt.3377

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