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

Patrício Godoy ,Nils Blüthgen,Rolf Gebhardt,Raymond Reif,Umesh Chaudhari,Regina Stöber,Reinhard Guthke,Cristina Cadenas,Jan G Hengstler,Uta Dahmen,Steven Dooley,René P Zahedi,Ahmed Ghallab,Georg Damm,Karolina Edlund,Olaf Dirsch,Rosemarie Marchan,Gisela Campos,Kesavan Meganathan,Agapios Sachinidis,Agata Widera,Daniel Seehofer,Christoph Meyer,Larissa Pütter,Wolfgang E Thasler,Kathrin Gianmoena,Ute Hofmann,Seddik Hammad,Thomas S Weiss,Jens M Kelm ,Andreas K Nüssler ,Wolfgang Schmidt‐Heck

doi:10.1007/s00204-016-1761-4

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1761-4) contains supplementary material, which is available to authorized users.

Highlights

Hepatocyte in vitro systems represent a well-accepted tool in many fields of basic and applied research such as pharmacology and toxicology, tissue engineering and clinical hepatocyte transplantation (Godoy et al 2013)
We report that alterations in cultivated human and mouse hepatocytes resemble those in inflammatory liver diseases, and that similar transcriptional networks and transcription factors are responsible for the identified changes
To understand the structure of gene expression alterations in hepatocytes, we compared genome-wide expression changes that occur during cultivation to changes in liver diseases and cell lines and stem cell-derived hepatocyte-like cells. a Principal component analysis representing the 1000 genes with highest variance in cultivated mouse hepatocytes, the AML12 cell line at 3 and 24 h in culture and human-induced pluripotent stem cell-derived hepatocyte-like cells

Summary

Introduction

Hepatocyte in vitro systems represent a well-accepted tool in many fields of basic and applied research such as pharmacology and toxicology, tissue engineering and clinical hepatocyte transplantation (Godoy et al 2013) Despite of their widespread use, research with primary hepatocytes remains challenging (Godoy et al 2013). Isolating hepatocytes from their physiological environment in the liver causes alterations in cell physiology and major gene expression alterations (Godoy et al 2013; Zellmer et al 2010) It has never been studied whether these changes only represent in vitro artifacts or whether they resemble disease-relevant processes. Such a situation has since long been acknowledged for liver fibrosis, where cultivated stellate cells undergo similar activation mechanisms as in the fibrotic liver (De Minicis et al 2007). The resulting transcriptomic network directory offers a blueprint for interventions for improving the in vitro systems and for interfering with disease-relevant processes

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Conclusion