(549c) An in Vitro hpsc 3D MODEL of Embryological LIVER Morphogenesis for Advanced Hepatic Tissue Construction

We aimed to design an in vitro model of embryonic liver morphogenesis to mimic the progenitor cell intercommunication regulating liver growth. Liver morphogenesis initiates from the coordinated interactions of the liver progenitor cells that invade the septum transversum mesenchyme (STM) and rapidly proliferate to generate complex tissue. To elucidate the site-specific cellular crosstalk that governs this step we developed a hepatic organoid outgrowth assay to determine how endothelium/hepatic progenitor cells organize to form primitive sinusoidal structures. Initially to model embryonic liver tissue, in vitro derived hepatic endodermal (HTE) cells, were replated into ultralow attachment (ULA) round bottom 384 well plates (Corning) to form compact spheroids (n=10). To determine the effect of collective cell migration on nascent sinusoid formation, organoids were embedded into matrigel droplets in an outgrowth assay (n=10). These migratory organoids demonstrated radial, uniform, cord-like projections into the matrix, similar morphologically to primitive hepatic sinusoids that form during liver development between E9 to E10 (mouse). These structures displayed enhanced gene expression consistent with migration, including epithelial-to mesenchymal transition (EMT) markers and early liver differentiation genes more than controls. Immunolocalization of organoids demonstrated liver and biliary markers such as albumin, CK18, Sox9 and HNF6. Endothelium progenitor cells stained positive for VEGFR2 and VWF and co-aligned with hepatic progenitor cells in a radial fashion. The morphogenetic model presented here represents a novel approach to modeling embryonic liver growth, nascent sinusoidal development, and tissue organization, a rate-limiting step during liver development. Further studies will investigate the genomic and biophysical cues that regulate such multicellular events. These studies will improve models to create 3D liver tissue in vitro for various liver regenerative medicine applications, including modeling human development, liver disease, and tissue regeneration.