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Arch Toxicol. 2016 Oct;90(10):2513-29. doi: 10.1007/s00204-016-1761-4. Epub 2016 Jun 23.

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

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IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany.
Facultad de Ciencias Biológicas, Departamento de Fisiología, Universidad de Concepción, Concepción, Chile.
IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany.
Leibniz Institute for Natural Product Research and Infection Biology eV-Hans-Knöll Institute, Jena, Germany.
Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany.
Department of Forensic and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.
BG Trauma Center, Siegfrid Weller Insitut, Eberhard Karls University Tübingen, Tübingen, Germany.
Center for Liver Cell Research, Department of General, Visceral, Transplantation, Vascular and Thorax Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany.
Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, Berlin, Germany.
Center for Liver Cell Research, University Children Hospital (KUNO), Regensburg University Hospital, Regensburg, Germany.
Institute of Pathology, Friedrich-Schiller-University of Jena, Jena, Germany.
Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Friedrich-Schiller-University of Jena, Jena, Germany.
Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany.
Institute of Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA.
InSphero AG, Wagistrasse 27, 8952, Schlieren, Switzerland.
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, University of Tuebingen, Auerbachstrasse 112, 70376, Stuttgart, Germany.
Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
Institute of Pathology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany.


It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.


Bioinformatics; Differentiation; Gene arrays; Inflammation; Metabolism

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