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Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.

TitleMechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.
Publication TypeJournal Article
Year of Publication2017
AuthorsHeslop JA, Rowe C, Walsh J, Sison-Young R, Jenkins R, Kamalian L, Kia R, Hay DC, Jones RP, Malik HZ, Fenwick S, Chadwick AE, Mills J, Kitteringham NR, Goldring CEP, B Park K
JournalArch Toxicol
Volume91
Issue1
Pagination439-452
Date Published2017 Jan
ISSN1432-0738
KeywordsCell Dedifferentiation, Cells, Cultured, Electron Transport Complex I, Gene Expression Profiling, Gene Expression Regulation, Developmental, Hepatocytes, Humans, Kinetics, Mitochondria, Liver, Pharmacology, Protein Stability, Proteome, Reproducibility of Results, Rotenone, Toxicology, Uncoupling Agents
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.

DOI10.1007/s00204-016-1694-y
Alternate JournalArch. Toxicol.
PubMed ID27039104
PubMed Central IDPMC5225178
Publication institute
CRM