title: Investigating novel methods of enhancing in vitro models of drug induced liver injury
Creators: name: Heslop, James
Publisher: name: University of Liverpool Repository
Year: 2017

description: Drug induced liver injury (DILI) is a major cause of patient morbidity and mortality inferring considerable burdens onto healthcare and pharmaceutical sectors. As a consequence, substantial resources are directed towards triaging potentially dangerous new compounds at all stages of drug development. However, despite these efforts, hepatotoxic compounds remain the greatest cause of post-marketing drug withdrawal. One of the major factors preventing efficacious screening of new compounds is the lack of a truly representative in vitro model of hepatotoxicity. This thesis describes our efforts to utilise innovative and emerging techniques to further understand and develop in vitro models of hepatotoxicity. One such technique is the generation of hepatocyte-like cells from induced pluripotent stem cells (iPSCs). iPSC-derived hepatocyte-like cells offer a reproducible, physiologically-relevant, genotypically normal and population-representative model of hepatotoxicity; however, current differentiation protocols are not capable of producing hepatocyte-like cells beyond a relatively immature phenotype, limiting their use for toxicological studies. As part of the cellular reprogramming process the epigenome of the somatic cell undergoes dramatic changes; however, the studies have shown that this ‘resetting’ of the epigenome to a pluripotent state is an imperfect process, resulting in an altered differentiation propensity skewed towards the lineage of origin. We evaluated if using human hepatocytes as the starting cell type and utilising the inherent ‘epigenetic memory’ associated with iPSCs could enhance the maturity of hepatocyte-like cells. Despite a trend towards improvement in phenotype, no significant differences were found between isogenic hepatocyte-derived and fibroblast-derived iPSCs. The further development of hepatocyte-like cells is limited by the inability of current culture systems to adequately support the hepatic phenotype. Once placed into culture, primary human hepatocytes, the gold standard model of hepatotoxicity, quickly lose the metabolic qualities required for modelling drug induced liver injury. Thus, without a culture system which supports the hepatic phenotype, the differentiation of hepatocyte-like cells will remain sub-optimal. Using iTRAQ proteomics we attempted to identify the driving factors responsible for the process of hepatocyte dedifferentiation. Our results identified numerous novel factors, including HSF2, SMARCB1, ZEB1 and FOXO1 which may drive the selective loss of metabolic phenotype. The proteomic assessment of hepatocyte dedifferentiation also highlighted the loss of Nrf2-related proteins during culture. Further investigation of Nrf2 in hepatocytes revealed a potentially negative relationship between Nrf2 induction and the key metabolic enzyme, CYP3A4. Furthermore, Nrf2 gene and protein expression was shown to increase during hepatocyte-like cell differentiation. Taken together, these results suggest that Nrf2 may negatively regulate the hepatic phenotype, potentially preventing the establishment of a mature phenotype during hepatocyte-like cell differentiation. A mechanistic evaluation of Nrf2 during differentiation and dedifferentiation is therefore required to gain a fuller insight into the role it plays in the maintenance and acquisition of the hepatic phenotype. In summary, this thesis presents our contribution to the further understanding, development and enhancement of in vitro hepatotoxicity models, using innovative techniques to assess the impact of epigenetic memory on HLC differentiation, identifying novel influencing factors driving the loss of phenotype in hepatocyte culture systems and evaluating the influence of Nrf2 on the hepatic phenotype during differentiation and dedifferentiation.