title: Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.
abstract: The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Heslop
authors.affiliation: CN Bio, Centre for Innovation and Enterprise, Oxford University Begbroke Science Park, Begbroke, Oxfordshire, OX5 1PF, UK.
authors.forename: Cliff
authors.initials: C
authors.lastname: Rowe
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Walsh
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Rowena
authors.initials: R
authors.lastname: Sison-Young
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Roz
authors.initials: R
authors.lastname: Jenkins
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Laleh
authors.initials: L
authors.lastname: Kamalian
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Kia
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: David
authors.initials: D
authors.lastname: Hay
authors.affiliation: University Hospital Aintree, Longmoor Lane, Liverpool, L9 7AL, UK.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Jones
authors.affiliation: University Hospital Aintree, Longmoor Lane, Liverpool, L9 7AL, UK.
authors.forename: Hassan Z
authors.initials: HZ
authors.lastname: Malik
authors.affiliation: University Hospital Aintree, Longmoor Lane, Liverpool, L9 7AL, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Fenwick
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Chadwick
authors.affiliation: AstraZeneca, Personalised Healthcare and Biomarkers, Alderley Park, Cheshire, SK10 4TG, UK.
authors.forename: John
authors.initials: J
authors.lastname: Mills
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Kitteringham
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK. Chrissy@liv.ac.uk.
authors.forename: Chris E P
authors.initials: CE
authors.lastname: Goldring
authors.affiliation: Division of Molecular and Clinical Pharmacology, The Institute of Translational Medicine, MRC Centre for Drug Safety Science, The University of Liverpool, Liverpool, L69 3GE, UK.
authors.forename: B
authors.initials: B
authors.lastname: Kevin Park
mesh_terms: D054337:Cell Dedifferentiation / Q000187:drug effects; D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010600:Pharmacology / Q000379:methods*; D055550:Protein Stability / Q000187:drug effects; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012402:Rotenone / Q000494:pharmacology; D014116:Toxicology / Q000379:methods*; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D020543:Proteome; D014475:Uncoupling Agents; D012402:Rotenone; D042967:Electron Transport Complex I
keywords: Cytochrome P450s; Donor-variation; Human hepatocytes; Mass spectrometry; Mitochondria; iTRAQ