Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Long-term culture and expansion of primary human hepatocytes

Nature Biotechnology volume 33, pages 1264–1271 (2015)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Induction of reversible proliferation in E6- and E7-transduced primary human hepatocytes.
Figure 2: Fetal and mature hepatocyte markers in E6/E7low hepatocytes.
Figure 3: Differentiated hepatocytes show functional polarization, gene expression and CYP450 activity similar to those of primary cells.
Figure 4: Differentiated hepatocytes predict toxicological outcomes.
Figure 5: Transduced hepatocytes of different ethnic backgrounds differentially respond to HCVcc infection.

Similar content being viewed by others

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Kidambi, S. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Khetani, S.R. & Bhatia, S.N. Microscale culture of human liver cells for drug development. Nat. Biotechnol. 26, 120–126 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Nahmias, Y., Berthiaume, F. & Yarmush, M. in Tissue Engineering II vol. 103 (eds. Lee, K. & Kaplan, D.) 309–329 (Springer, 2007).

  5. Yamamoto, N., Imazato, K. & Masumoto, A. 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).

    Article  CAS  PubMed  Google Scholar 

  6. Shimaoka, S., Nakamura, T. & Ichihara, A. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Tsuruga, Y. 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).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Guillouzo, A. 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).

    Article  CAS  PubMed  Google Scholar 

  14. Avior, Y. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Parent, R., Marion, M.-J., Furio, L., Trépo, C. & Petit, M.-A. Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126, 1147–1156 (2004).

    Article  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  20. Gerets, H.H. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. Moody, C.A. & Laimins, L.A. Human papillomavirus oncoproteins: pathways to transformation. Nat. Rev. Cancer 10, 550–560 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Tsuruga, Y. 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).

    Article  PubMed  Google Scholar 

  27. Pollack, V. 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).

    Article  CAS  PubMed  Google Scholar 

  28. Nakamura, K., Nonaka, H., Saito, H., Tanaka, M. & Miyajima, A. Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice. Hepatology 39, 635–644 (2004).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  31. Yu, H., Lee, H., Herrmann, A., Buettner, R. & Jove, R. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat. Rev. Cancer 14, 736–746 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  34. Westerink, W.M. & Schoonen, W.G. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ndongo-Thiam, N. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Nevo-Yassaf, I. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Runge, D., Michalopoulos, G.K., Strom, S.C. & Runge, D.M. Recent advances in human hepatocyte culture systems. Biochem. Biophys. Res. Commun. 274, 1–3 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  45. Münger, K., Phelps, W.C., Bubb, V., Howley, P.M. & Schlegel, R. 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).

    PubMed  PubMed Central  Google Scholar 

  46. Arlotto, M.P., Trant, J.M. & Estabrook, R.W. Measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function. Methods Enzymol. 206, 454–462 (1991).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  48. Donato, M.T., Jimenez, N., Castell, J.V. & Gomez-Lechon, M.J. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  51. Ekins, S. 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).

    CAS  PubMed  Google Scholar 

  52. Habig, W.H. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and 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. Department of Gastroenterology, Liver Unit, 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

Authors
  1. Gahl Levy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Bomze
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefan Heinz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sarada Devi Ramachandran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Astrid Noerenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Merav Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Oren Shibolet
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ella Sklan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joris Braspenning
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yaakov Nahmias
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Yaakov Nahmias.

Ethics declarations

Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Characterization of genetically induced proliferation in human hepatocytes.

(A) Western blot analysis for p53 expression in HeLa (negative control) and MDA-MB-231 cell lines (positive control), source cryopreserved human hepatocytes as well as the resulting E6 and E7 transduced hepatocytes (E6/E7LOW). E6/E7LOW hepatocytes retained p53 expression in the presence or absence of OSM, similar to their source hepatocytes and the MDA-MB-231 control, while HeLa cells transformed by infection do not. (B) OSM signaling activates the HPV noncoding upstream regulatory region (URR) in HepG2 cells, suggesting a viral feedback loop (Fig. 1A). To test whether OSM treatment up-regulates E6 and E7 in transduced hepatocytes lacking the URR, we compared the expression of E6 and E7 in transduced hepatocytes and HepG2 cells in the presence or absence of OSM. OSM stimulation of E6/E7LOW hepatocytes does not increase E6 and E7 expression (p=0.19, n=3), while HepG2 cells containing dormant URR show up-regulation of E6 and E7, with a 2-fold induction (p=0.008, n=3). (C) Growth curves of transduced hepatocytes exposed to 10 ng/mL HGF, 20 ng/mL EGF, or 10 ng/mL OSM. OSM stimulation resulted in rapid expansion, with 34±2 hours doubling time. EGF and HGF stimulation was not different from control. (D) Growth curves of transduced hepatocytes exposed to EGF or HGF during OSM induced proliferation. Both EGF and HGF increased proliferation over OSM alone, resulting in 32±1 hour doubling time, which was not significantly different from OSM alone over the given time frame. (E) OSM-induced expansion of E6/E7LOW and E6/E7HIGH hepatocytes promotes the acquisition of a fibroblastoid phenotype (Fig. 1E). To demonstrate the cells are hepatocytes that underwent EMT we carried out a qRT-PCR analysis showing the cells express epithelial specific EpCAM and are negative to mesenchyme intermediate filament vimentin. Cells also expressed low levels of albumin and AFP, marking a hepatocyte origin. Gene expression analysis presented in log scale. Values are normalized to human fibroblasts. (F) To evaluate whether transduced hepatocytes express the fetal marker CYP3A7 we compared expression levels by qRT-PCR of proliferating (E6/E7LOW+OSM) and differentiated (E6/E7LOW) hepatocytes, primary hepatocytes, HepG2, and fetal hepatocytes. Differentiated transduced hepatocytes express CYP3A7 at 32% of primary hepatocyte levels, and three orders of magnitude lower than fetal hepatocytes, showing transduced hepatocytes do not up-regulate fetal CYP3A7 (n=3). (G) BrdU labeling of OSM-stimulated hepatocytes shows 23% of the cells are proliferating at each time point. However, only 10±5% of the cells were positive for albumin (top right). In contrast, over 95±5% of differentiated cells were positive for albumin during OSM-deprivation induced differentiation when BrdU incorporation was <1% (bottom right). We note that BrdU incorporation is carried out in 1 hour while cell doubling time ranges from 33 to 49 hours, suggesting only 23% of the population replicates at each given moment. Importantly, the bi-modal albumin expression pattern mimics previously reported loss of albumin expression during liver regeneration. (H) Western blot analysis of STAT3 phosphorylation in differentiated hepatocytes (E6/E7low) and proliferating hepatocytes (E6/E7low + OSM) treated with Stattic STTC).

Supplementary Figure 2 CYP450 activity in genetically induced primary human hepatocytes.

CYP450 activity of differentiated hepatocytes (donor 653) compared with HepG2 cells and cryopreserved primary human hepatocytes. Permutation test shows that CYP450 activity profile of differentiated hepatocytes is not significantly different from the profile of primary cells (p=0.44, n=5), while HepG2 activity profile was significantly higher (p=0.04, n=5).

Supplementary Figure 3 Variability between different hepatocyte donors in CYP450 expression and drug toxicity.

(A) Quantitative gene expression analysis of primary human hepatocytes and HepG2 cells compared with differentiated hepatocyte from donors 653, 422A, and 10. All three lines show comparable levels of gene expression. (B) Graph comparing the TC50 of 18 compounds (Supplementary Table 1) in differentiated hepatocytes from donors 653, 151, 10, and 422A against TC50 values for primary human hepatocytes. Toxicity was measured using the MTS assay. All donors showed an R2 correlation of 0.99 (n=3). Values presented in Log scale.

Supplementary Figure 4 Toxicity curves for differentiated and proliferating hepatocytes treated with known hepatotoxic and control compounds (donor 653).

(A) Dose dependent toxicity curves comparing day four differentiated hepatocytes (red diamonds) and proliferating hepatocytes (blue squares) treated with increasing doses of toxic and control compounds for 24 hours (n=3). To evaluate the potential of these E6/E7LOW hepatocytes to predict human response we tested 9 known hepatotoxic drugs and 3 control compounds of similar structure. We show that differentiated hepatocytes show toxic response to all 9 hepatotoxic drugs, matching the toxicological end-point in all cases including apoptosis, steatosis, and cholestasis. (B) Table comparing TC50 values obtained for proliferating E6/E7LOW hepatocytes, differentiated hepatocytes and cryopreserved primary human hepatocytes.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–2 (PDF 1163 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Levy, G., Bomze, D., Heinz, S. et al. Long-term culture and expansion of primary human hepatocytes. Nat Biotechnol 33, 1264–1271 (2015). https://doi.org/10.1038/nbt.3377

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research