Marcus J. Lyall

State of the art simulations of aortic haemodynamics feature full fluid-structure interaction (FSI) and coupled 0D boundary conditions. Such analyses require not only significant computational resource but also weeks to months of run time, which compromises the effectiveness of their translation to a clinical workflow. This article employs three computational fluid methodologies, of varying levels of complexity with coupled 0D boundary conditions, to simulate the haemodynamics within a patient-specific aorta. The most comprehensive model is a full FSI simulation. The simplest is a rigid walled incompressible fluid simulation while an alternative middle-ground approach employs a compressible fluid, tuned to elicit a response analogous to the compliance of the aortic wall. The results demonstrate that, in the context of certain clinical questions, the simpler analysis methods may capture the important characteristics of the flow field.

Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations that occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, selected CGIs underwent hypo-hydroxymethylation prior to hypermethylation, while retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprograming of DNA methylation at silent CGI during progression. Cancer Res; 76(10); 3097-108. ©2016 AACR.

Non-alcoholic fatty liver disease (NAFLD) is now the commonest cause of liver disease in developed countries affecting 25-33% of the general population and up to 75% of those with obesity. Recent data suggest that alterations in DNA methylation may be related to NAFLD pathogenesis and progression and we have previously shown that dynamic changes in the cell lineage identifier 5-hydroxymethylcytosine (5hmC) may be important in the pathogenesis of liver disease. We used a model of diet-induced obesity, maintaining male mice on a high-fat diet (HFD) to generate hepatic steatosis. We profiled hepatic gene expression, global and locus-specific 5hmC and additionally investigated the effects of weight loss on the phenotype. HFD led to increased weight gain, fasting hyperglycaemia, glucose intolerance, insulin resistance and hepatic periportal macrovesicular steatosis. Diet-induced hepatic steatosis associated with reversible 5hmC changes at a discrete number of functionally important genes. We propose that 5hmC profiles are a useful signature of gene transcription and a marker of cell state in NAFLD and suggest that 5hmC profiles hold potential as a biomarker of abnormal liver physiology.

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’.