Endocrine Abstracts

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a disease spectrum ranging from intrahepatic lipid accumulation to fibrosis, cirrhosis, and hepatocellular carcinoma. Despite the severity, there are no efficacious therapies, mainly due to the complexity of disease and the lack of predictive pre-clinical models. We hypothesised that iPSC-derived hepatocytes (Opti-HEP) offer a suitable platform for the development of in vitro MASLD models recapitulating all aspects of the disease phenotype. Wild-type iPSCs and iPSCs harbouring the MASLD risk-associated PNPLA3-I148M variant were differentiated to Opti-HEP. Differentiation status was determined by qPCR, immunocytochemistry, western blotting, colorimetric assays, and ELISA. Wild-type and disease Opti-HEP were treated with a mixture of fatty acids, glucose, or a combination of both for 7 days. Differences in lipid accumulation, insulin sensitivity, hepatic inflammation, and CYP450 expression were determined by qPCR, western blotting, and BODIPY staining. Wild-type and PNPLA3-I148M Opti-HEP demonstrated mature hepatocyte phenotype, as evidenced by liver maturity marker expression (albumin, alpha-1-antitrypsin, HNF4A), urea secretion, gluconeogenesis, and CYP450 activity. Fatty acid and glucose treatment of wild-type Opti-HEP significantly increased lipid accumulation compared to vehicle-treated cells, and this effect was further exacerbated in PNPLA3-I148M cells. High glucose treatment impaired AKT phosphorylation (Ser473) in wild-type and PNPLA3-I148M Opti-HEP suggestive of insulin resistance, whilst fatty acid treatment resulted in ~20% reduction in Opti-HEP viability alongside increases in pro-inflammatory cytokine IL-1β, IL-6, IL-8, and TNFα expression. Endorsing these findings, fatty acid and glucose treatment significantly decreased drug metabolising genes expression (CYP3A4, CYP2B6, CYP2C9), suggesting impaired drug metabolism activity. We have successfully produced iPSC-derived hepatocytes with comparable functionality to that observed in primary human hepatocytes. By applying dietary and genetic interventions associated with MASLD development, we have generated a novel model that recapitulates the MASLD phenotype in vitro, offering an efficient pre-clinical platform for the large-scale efficacy screening of novel therapeutics against the disease.