Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in developed countries. An in vitro NAFLD model would permit mechanistic studies and enable high-throughput therapeutic screening. While hepatic cancer-derived cell lines are a convenient, renewable resource, their genomic, epigenomic and functional alterations mean their utility in NAFLD modelling is unclear. Additionally, the epigenetic mark 5-hydroxymethylcytosine (5hmC), a cell lineage identifier, is rapidly lost during cell culture, alongside expression of the Ten-eleven-translocation (TET) methylcytosine dioxygenase enzymes, restricting meaningful epigenetic analysis. Hepatocyte-like cells (HLCs) derived from human embryonic stem cells can provide a non-neoplastic, renewable model for liver research. Here, we have developed a model of NAFLD using HLCs exposed to lactate, pyruvate and octanoic acid (LPO) that bear all the hallmarks, including 5hmC profiles, of liver functionality. We exposed HLCs to LPO for 48 h to induce lipid accumulation. We characterized the transcriptome using RNA-seq, the metabolome using ultra-performance liquid chromatography-mass spectrometry and the epigenome using 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) sequencing. LPO exposure induced an NAFLD phenotype in HLCs with transcriptional and metabolomic dysregulation consistent with those present in human NAFLD. HLCs maintain expression of the TET enzymes and have a liver-like epigenome. LPO exposure-induced 5hmC enrichment at lipid synthesis and transport genes. HLCs treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and additionally retain TET expression and 5hmC. This in vitro model of NAFLD will be useful for future mechanistic and therapeutic studies.This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Highlights

  • Non-alcoholic fatty liver disease (NAFLD) affects around 25 –33% of the population and up to 75% of obese individuals in developed countries [1,2]

  • Hepatocyte-like cells (HLCs) treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and retain TET expression and 5hmC

  • NAFLD encompasses a spectrum of liver disease and while simple steatosis is considered relatively benign, it can progress to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and approximately 25% of those with cirrhosis will develop hepatocellular carcinoma (HCC) [3,4]

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Summary

Introduction

Non-alcoholic fatty liver disease (NAFLD) affects around 25 –33% of the population and up to 75% of obese individuals in developed countries [1,2]. Exposure of HLCs to a nutrient cocktail of lactate, pyruvate and octanoic acid produces a robust NAFLD-like phenotype in vitro We believe this model will be of importance for further mechanistic studies of NAFLD on defined genetic backgrounds. The automated high-throughput system for HLC differentiation, staining and objective image analysis is depicted in figure 1a; for detailed methods, see the electronic supplementary material. (g) qPCR of mRNA levels of genes relevant to human NAFLD in control (Con, blue) versus 48 h LPO-treated (red) HLCs. Data are expressed relative to the mean of control genes PPIA/B2M and were analysed by one-way ANOVA with Bonferroni correction. 5hmC DNA immunoprecipitation (hmeDIP) was performed and libraries sequenced on an Ion Torrent semiconductor sequencer using the Ion PITM Hi-QTM Sequencing Kit and an Ion PITM Chip Kit v3 (Thermo Fisher Scientific, Paisley, UK) to a depth of approximately 30 million reads (see the electronic supplementary material). Data point and bar graphs are shown as mean + s.e.m

Results
Discussion
27. Thomson JP et al 2015 DNA immunoprecipitation
28. Cameron K et al 2015 Recombinant laminins drive
47. Nestor CE et al 2012 Tissue type is a major modifier
48. Szulwach KE et al 2011 5-hmC-mediated epigenetic
Findings
32. Satapati S et al 2012 Elevated TCA cycle function in
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