Phd Student The Chinese University of Hong Kong (CUHK), Hong Kong
Abstract: Human liver organoids (HLOs) derived from human pluripotent stem cells (hPSCs) hold a promise for modeling liver development and disease. However, HLOs generated using current protocols lack physiological complexity and cellular diversity, with hepatocytes exhibiting a fetal-like phenotype, limiting their utility for advanced applications. This study aimed to investigate into the underlying mechanism to enhance the functionality of HLOs. In this study, we optimized multiple conditions to generate HLOs from H9 human embryonic stem cells (hESCs). RNA sequencing (RNA-seq) and immunostaining were used to characterize the lineage composition of the HLOs, while functional assays evaluated key liver-specific functions, such as urea synthesis and albumin production. To further explore the mechanisms underlying hepatocyte maturation, HepaRG cells were co-cultured with HUVECs (human umbilical vein endothelial cells) and HHSECs (human hepatic sinusoidal endothelial cells). We successfully generated improved HLOs from H9 hESCs. RNA-seq analysis revealed the expression of hepatocyte markers alongside non-parenchymal cell markers. Immunostaining confirmed the presence of hepatocyte-specific HNF4A and stellate cell-specific vimentin in distinct regions of the organoids. Notably, the mutually exclusive expression patterns of HNF4A and CK19 (cholangiocyte marker) highlighted the spatial organization of these cells within the organoids. The HLOs expressed key genes essential for liver function, including those involved in cholesterol and fat metabolism, drug detoxification, and hepatic-specific transcriptional regulation. Furthermore, co-culturing HepaRG cells with HUVECs and HHSECs significantly enhanced hepatocyte proliferation and maturation compared to monoculture, supporting the hypothesis that multi-lineage composition enhances HLO functionality. In summary, this study optimized multiple factors to generate HLOs with improved lineage composition and functionality. Future work will focus on characterizing the responses of these organoids to liver injury factors and further investigating the roles of specific cell types, particularly endothelial cells, in promoting hepatocyte maturation.
