Article | Published:

Long-term culture and expansion of primary human hepatocytes

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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Idiosyncratic drug hepatotoxicity. Nat. Rev. Drug Discov. 4, 489–499 (2005).

  2. 2.

    et al. Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc. Natl. Acad. Sci. USA 106, 15714–15719 (2009).

  3. 3.

    & Microscale culture of human liver cells for drug development. Nat. Biotechnol. 26, 120–126 (2008).

  4. 4.

    , & in Tissue Engineering II vol. 103 (eds. Lee, K. & Kaplan, D.) 309–329 (Springer, 2007).

  5. 5.

    , & Growth stimulation of adult rat hepatocytes in a primary culture by soluble factor(s) secreted from nonparenchymal liver cell. Cell Struct. Funct. 14, 217–229 (1989).

  6. 6.

    , & Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells. Exp. Cell Res. 172, 228–242 (1987).

  7. 7.

    et al. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat. Chem. Biol. 9, 514–520 (2013).

  8. 8.

    et al. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation. Cell Biol. Toxicol. 13, 375–386 (1997).

  9. 9.

    et al. Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy. Cell Transplant. 17, 1083–1094 (2008).

  10. 10.

    et al. CYP3A4 catalytic activity is induced in confluent Huh7 hepatoma cells. Drug Metab. Dispos. 38, 995–1002 (2010).

  11. 11.

    , & Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties. Drug Metab. Dispos. 31, 1035–1042 (2003).

  12. 12.

    et al. Primary hepatocytes outperform Hep G2 cells as the source of biotransformation functions in a bioartificial liver. Ann. Surg. 220, 59–67 (1994).

  13. 13.

    et al. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 168, 66–73 (2007).

  14. 14.

    et al. Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells–derived and fetal hepatocytes. Hepatology 62, 265–278 (2015).

  15. 15.

    et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).

  16. 16.

    et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297–305 (2010).

  17. 17.

    et al. Accurate prediction of drug-induced liver injury using stem cell-derived populations. Stem Cells Transl. Med. 3, 141–148 (2014).

  18. 18.

    , , , & Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126, 1147–1156 (2004).

  19. 19.

    et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab. Dispos. 38, 516–525 (2010).

  20. 20.

    et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 28, 69–87 (2012).

  21. 21.

    et al. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int. 25, 389–402 (2005).

  22. 22.

    et al. Generation of proliferating human hepatocytes using Upcyte technology: characterisation and applications in induction and cytotoxicity assays. Xenobiotica 42, 939–956 (2012).

  23. 23.

    & Human papillomavirus oncoproteins: pathways to transformation. Nat. Rev. Cancer 10, 550–560 (2010).

  24. 24.

    Molecular mechanism of carcinogenesis by human papillomavirus-16. J. Dermatol. 27, 73–86 (2000).

  25. 25.

    et al. Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes. J. Hepatol. 46, 26–36 (2007).

  26. 26.

    et al. Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy. Cell Transplant. 17, 1083–1094 (2008).

  27. 27.

    et al. Oncostatin M-induced effects on EMT in human proximal tubular cells: differential role of ERK signaling. Am. J. Physiol. Renal Physiol. 293, F1714–F1726 (2007).

  28. 28.

    , , , & Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice. Hepatology 39, 635–644 (2004).

  29. 29.

    et al. Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration. J. Biol. Chem. 283, 9886–9895 (2008).

  30. 30.

    et al. Response to IL-6 of HPV-18 cervical carcinoma cell lines. Virology 258, 344–354 (1999).

  31. 31.

    , , , & Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat. Rev. Cancer 14, 736–746 (2014).

  32. 32.

    et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467, 707–710 (2010).

  33. 33.

    et al. Frequent aneuploidy among normal human hepatocytes. Gastroenterology 142, 25–28 (2012).

  34. 34.

    & Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol. In Vitro 21, 1581–1591 (2007).

  35. 35.

    et al. Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc. Natl. Acad. Sci. USA 107, 3141–3145 (2010).

  36. 36.

    et al. Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes. Hepatology 54, 406–417 (2011).

  37. 37.

    et al. Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J. Virol. 81, 4591–4603 (2007).

  38. 38.

    et al. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102, 9294–9299 (2005).

  39. 39.

    et al. Role for TBC1D20 and Rab1 in hepatitis C virus replication via interaction with lipid droplet-bound nonstructural protein 5A. J. Virol. 86, 6491–6502 (2012).

  40. 40.

    et al. The lipid droplet is an important organelle for hepatitis C virus production. Nat. Cell Biol. 9, 1089–1097 (2007).

  41. 41.

    The current status of primary hepatocyte culture. Int. J. Exp. Pathol. 79, 393–409 (1998).

  42. 42.

    , , & Recent advances in human hepatocyte culture systems. Biochem. Biophys. Res. Commun. 274, 1–3 (2000).

  43. 43.

    et al. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation. Dev. Cell 23, 681–690 (2012).

  44. 44.

    et al. C/EBP-β/LAP controls down-regulation of albumin gene transcription during liver regeneration. J. Biol. Chem. 271, 22262–22270 (1996).

  45. 45.

    , , , & The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63, 4417–4421 (1989).

  46. 46.

    , & Measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function. Methods Enzymol. 206, 454–462 (1991).

  47. 47.

    et al. Xenobiotic metabolism by cultured primary porcine hepatocytes. Tissue Eng. 6, 467–479 (2000).

  48. 48.

    , , & Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab. Dispos. 32, 699–706 (2004).

  49. 49.

    et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100, 197–207 (2000).

  50. 50.

    et al. Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells. Nat. Commun. 5, 4825 (2014).

  51. 51.

    et al. Quantitative differences in phase I and II metabolism between rat precision-cut liver slices and isolated hepatocytes. Drug Metab. Dispos. 23, 1274–1279 (1995).

  52. 52.

    et al. The identity of glutathione-S-transferase B with ligandin, a major binding protein of liver. Proc. Natl. Acad. Sci. USA 71, 3879–3882 (1974).

Download references


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


  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


  1. Search for Gahl Levy in:

  2. Search for David Bomze in:

  3. Search for Stefan Heinz in:

  4. Search for Sarada Devi Ramachandran in:

  5. Search for Astrid Noerenberg in:

  6. Search for Merav Cohen in:

  7. Search for Oren Shibolet in:

  8. Search for Ella Sklan in:

  9. Search for Joris Braspenning in:

  10. Search for Yaakov Nahmias in:


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.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1–2

About this article

Publication history





Further reading