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Long-term culture and expansion of primary human hepatocytes

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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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.

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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).

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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.

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Correspondence to Yaakov Nahmias.

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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.

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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

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