Development of optimised human iPSC-derived hepatocytes with improved liver function for in vitro metabolic disease modelling and toxicity studies

Background & AimsLiver disease is a rising cause of mortality worldwide. Primary human hepatocytes (PHH) and hepatocellular cancer cells are currently used in drug development, however, they come with limitations, including limited supply, rapid loss of function, and tumorigenic origin. In addition, current iPSC differentiation protocols lead to the generation of hepatocyte-like cells with compromised liver-related features. We hypothesised that optimisation of iPSC differentiation protocols can lead to the generation of hepatocyte-like cells with improved metabolic functionality for disease modelling and toxicity screening studies. MethodsHealthy human iPSCs were differentiated to hepatocyte-like cells (Opti-HEP) using a novel 3-step differentiation protocol. Hepatocyte functionality was assessed, including liver maturity marker expression, urea synthesis, de novo gluconeogenesis, and CYP450 expression, activity, and induction. Suitability of Opti-HEP to predict drug-induced liver injury (DILI) was evaluated by cell viability assays. CRISPR/Cas9 gene editing was employed to generate in vitro inherited metabolic disease models. ResultsOpti-HEP expressed similar liver maturity marker levels to those seen in primary human hepatocytes (PHH), in addition to functional urea and gluconeogenesis pathways. CYP450 expression and activity were comparable between Opti-HEP and PHH, with both cell types showing similar levels of CYP3A4 induction upon 1,25-hydroxy-vitamin D3 treatment. Opti-HEP accurately predicted DILI, following treatment with 7 drugs of known DILI liability. CRISPR-derived Opti-HEP harbouring mutations for inherited metabolic disorders (Ornithine Transcarbamylase Deficiency, Progressive Familial Intrahepatic Cholestasis Type 2, Citrullinemia Type 1) recapitulated key pathophysiological disease features, including reduced protein expression, impaired urea secretion, and bile acid transport. ConclusionsWe demonstrate the generation of optimised iPSC-derived hepatocytes with enhanced liver functionality that is comparable to PHH. These data alongside the expansion capacity and amenability of these cells highlight the opportunities this model can offer in the space of disease modelling and large-scale drug efficacy and hepatotoxicity screening.