Improving differentiation and maturation of hESCs-derived hepatocytes for biomedical applications

Abstract

The potential for generating hepatocytes from hPSCs holds immense promise for in vitro disease modelling and drug development. However, achieving hepatocytes that faithfully replicate in vivo hepatocytes has proven challenging, influenced by various factors within the cellular microenvironment (Dziedzicka et al., 2021; M. H. Kim et al., 2021). Among these factors, cell seeding density (Dziedzicka et al., 2021) and extracellular matrices play pivotal roles in shaping liver development and hepatocyte functions. Nevertheless, their precise functions in hepatocyte differentiation and maturation during hPSC differentiation remain elusive. This project focuses on refining the differentiation of hESCs into HLCs with an emphasis on enhancing maturation, functionality, and reproducibility for biomedical applications such as disease modelling and drug testing. Prior HLC differentiation experiments in our laboratory relied on splitting ratios, resulting in inconsistent outcomes. To address reproducibility, I investigated the impact of cell seeding density on DE formation, examining L-SD, M-SD, and H-SD seeding densities. The findings highlighted M-SD as the better condition, marked by elevated SOX17 and GATA6 expression and improved DE differentiation. Furthermore, this study delved into the influence of ECMs, specifically COL and MG, on the structural and functional properties of hESCs-HLCs. Results indicated that COL-based conditions enhanced albumin secretion and urea production, resembling portal hepatocyte functions, while MG boosted CYP3A4 activity and glycogen storage, mirroring pericentral hepatocyte characteristics. Moreover, the research unveiled the differential regulation of Wnt target genes (LEF1 and AXIN2) by MG and COL, suggesting their potential impact on hepatocyte zonation through modulation of the Wnt signalling pathway—a known factor in establishing hepatocyte zonation (Benhamouche et al., 2006; Burke et al., 2009; Ma et al., 2020). In conclusion, this study underscores the significance of cell seeding density and specific ECM compositions in generating and maturing hESCs-HLCs, offering insights into creating zone-specific liver disease models and pharmaceutical applications.