Comparing in vitro human liver models to in vivo human liver using RNA-Seq

Rajinder K Gupta ,Marcel Van Herwijnen,Jos Kleinjans,Richard Maclennan,Sylvia Escher,Yannick Schrooders,Leroy Elenschneider,Jan G Hengstler,Duncan Hauser,Patrick Guye,Laia Tolosa,Bas Ter Braak,Wolfgang Moritz,Paul Walker,Wiebke Albrecht,Bob Van De Water,José V Castell ,Ahmed Ghallab,Tanja Hansen,Tim Brecklinghaus,Tine Tricot,Cathérine Verfaillie ,Marcel Leist,Florian Caiment

doi:10.1007/s00204-020-02937-6

Abstract

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Highlights

The liver plays a central role in metabolizing exogenous substances
An overview of the analyzed samples, human liver tissue specimens in vivo and in vitro liver cell models is given in Table 1, detailed information on samples and protocols is available in Suppl. methods 1a-g, and details on samples selected after each filtration step are provided in Suppl
Different in vitro liver cell models have been developed for studying the effects of toxic compounds in humans

Summary

Introduction

Extended author information available on the last page of the article metabolism by phase I and II enzymes take place (Cribb et al 2005). Xenobiotics or their metabolites may damage the liver with fatal consequences for the individual (Moeller et al 2012). It is important to identify compounds that cause hepatotoxic effects to avoid exposure to humans. While the use of animal models has proven to be of great importance in biological research (Dey et al 2010; Ericsson et al 2013; Hau 2008; Simmons 2008), it remains challenging to translate the results to humans.

Methods

Results

Conclusion