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

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.

Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation

“…Hepatocyte-only systems can be successfully be used to address hepatic metabolism. Whole genome analysis of primary human hepatocytes (PHHs) and two commonly used cell lines HepaRG and HepG2, has demonstrated that genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 when compared to both PHHs and liver samples [ 2 , 3 ]. Furthermore, HepaRG maintained in 3D-spheroids showed albumin production levels and CYP activity more similar to the liver when compared with 2D-monolayers [ 4 ].…”

Beyond gene expression

“…In the case of biomarkers, currently, there are ~20 biomarkers (Table 1) in research, and out of these only α-fetoprotein (alpha-fetoprotein or AFP) has a clinical application; even though it is ineffective for detecting early lesions [1,[6][7][8]. Of the other markers used in research, none have reached the standard level of clinical practice so far [6,9].…”

“…With the invention of Sanger sequencing (also known as the first generation of sequencing) in 1977, the landscape of the DNA sequencing changed forever [1][2][3].…”

“…The liver plays a central role in metabolizing exogenous substances. After oral uptake xenobiotics pass through the digestive tract and enter the liver via the portal vein, where metabolism by phase I and II enzymes take place [1]. Xenobiotics or their metabolites may damage the liver with fatal consequences for the individual [2].…”

“…This crowded environment of the cell results in continuous interaction of all these biomolecules. Many interactions are nonspecific[1, 2]; however, others form the basis for a variety of biological processes including, but not limited to, cell cycle regulation, differentiation, protein folding, translation, transcription, post-translational modifications, gene expression, enzyme inhibition, and antibody-antigen interactions[3, 4]. These bound molecules that possess important functions are termed protein complexes.…”