Proteome-wide analyses of human hepatocytes during differentiation and dedifferentiation

Authors

  • Cliff Rowe,

    1. Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, AV Hill Building, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
    2. Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Building, Liverpool, UK
    3. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
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  • Dave T. Gerrard,

    1. Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, AV Hill Building, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
    2. Bioinformatics, Faculty of Life Sciences, Michael Smith Building, Manchester, UK
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  • Roz Jenkins,

    1. Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Building, Liverpool, UK
    2. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
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  • Andrew Berry,

    1. Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, AV Hill Building, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
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  • Kesta Durkin,

    1. Human Genetics Division, University of Southampton, Southampton, UK
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  • Lars Sundstrom,

    1. SARTRE, School of Clinical Sciences, University of Bristol, Bristol, UK
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  • Chris E. Goldring,

    1. Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Building, Liverpool, UK
    2. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
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  • B. Kevin Park,

    1. Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Building, Liverpool, UK
    2. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
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  • Neil R. Kitteringham,

    1. Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Building, Liverpool, UK
    2. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
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  • Karen Piper Hanley,

    1. Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, AV Hill Building, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
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  • Neil A. Hanley

    Corresponding author
    1. Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, AV Hill Building, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
    2. MRC Centre for Drug Safety Science, University of Liverpool and University of Manchester, UK
    3. Department of Endocrinology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK
    • Address reprint requests to: Professor Neil Hanley, AV Hill Building, University of Manchester, Oxford Road, Manchester, M13 9PT, UK. E-mail: Neil.Hanley@manchester.ac.uk; fax: +44 (0)161 275 5958.

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  • The copyright line on this article was changed on July 30, 2014, after original online publication.

  • Potential conflict of interest: Nothing to report.

Abstract

Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise, with differentiated hepatocyte-like cells currently considered to be “fetal-like” in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. Conclusion: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein “toolkit” for determining human hepatocyte maturity in cultured cells. (Hepatology 2013;58:799–809)

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