Drug induced liver injury (DILI) is currently the leading cause of acute liver failure in the western world and is also cited as the primary reason for the post market withdrawal of new drugs. Given the clinical implications of DILI and the associated costs to the industry, there is a need to improve the preclinical assessment of new therapeutics. For in vitro toxicity screening, primary human hepatocytes are still considered the gold standard due to their functional metabolic profile. Despite their predictive utility, primary hepatocytes rapidly dedifferentiate in culture, are limited by donor availability, and are phenotypically variable. As an alternative, it has been proposed that stem cell derived hepatocyte-like cells (HLCs) could be adopted for this purpose. HLCs offer several advantages over primary cells as they are derived from a renewable cell source, have a defined genetic background and display a consistent phenotype. Although HLCs hold great promise, they lack the functional maturity of adult primary cells and are believed to be more characteristic of foetal hepatocytes. In particular, many of the enzymes responsible for the metabolism of xenobiotics are either absent or expressed at low levels in these cultures. In many cases, adverse drug reactions result from the generation of toxic metabolites. Of particular relevance to this process is the activity of phase I enzymes belonging to the cytochrome p450 (CYP450) monooxygenase family. Several studies have indicated that CYP450 expression and activity in HLCs is severely limited. To overcome this functional deficit and improve the predictive value of HLCs, we have investigated the developmental expression of CYP450 genes and have adapted a differentiation protocol accordingly. Based on observations in mice we specifically examined signalling pathways associated with the postnatal period, with a focus on the foetal / neonatal transition and the suckling / weaning transition. Using an in vitro embryonic mouse liver model of hepatic differentiation, we have demonstrated that cAMP signalling enhances the expression of Cyp2e1 and Cyp3a11. Alongside this we have also shown that cAMP and the ketone body b-hydroxybutyrate (BHBA) synergistically enhance the metabolic activity of Cyp3a11/13 in murine explants. Furthermore, in combination, these factors significantly improved the maturational status of HLCs derived from H9 human embryonic stem cells (hESCs), with notable advancements in CYP2E1 and CYP3A4/5 dependent metabolism. When cultured for an extended period in these conditions, CYP3A4/5 activity continued to improve, and we were also able to detect activity of the adult marker CYP2A6. Preliminary evidence also indicates that CYP2B6 activity is upregulated in HLC cultures treated with triiodothyronine (T3), 9-cis retinoic acid, the PPARa agonist WY-14643 and to much a greater extent by b-estradiol (E2). Collectively, the findings in this thesis describe a set of conditions that can be used to enhance the metabolic capacity of HLC cultures.
|Date of Award||1 Apr 2020|
|Supervisor||David Tosh (Supervisor) & Stephen Ward (Supervisor)|
- Drug Metabolism
- Stem Cells
- Cytochrome p450