ABSTRACT Background and aims Non-alcoholic fatty liver disease (NAFLD) is a major health care challenge and new therapies are urgently needed. However, the mechanisms underlying disease remain to be understood. Indeed, studying NAFLD remains challenging due to the lack of model systems recapitulating the different aspects of the human pathology. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity to address this limitation since they can be differentiated into large quantity of liver cells. Here, we took advantage of hiPSCs to develop a multi-cellular platform mimicking the complex interplays involved in NAFLD progression. Methods hiPSCs-derived hepatocyte like cells (HLCs), cholangiocytes, stellate cells, and macrophages were co-cultured in a collagen-based 3D system to reproduce the liver microenvironment. Fatty acid treatments led to a NAFLD phenotype involving cell-cell interactions which were investigated by transcriptomic and functional analyses. Results Hepatic cells were grown up to 4weeks in 3D, retaining key functions and markers. Importantly, co-cultured cells spontaneously reorganised into physiologically relevant connections: HLCs arranged around biliary structures, which established contacts with stellate cells, while macrophages organised around HLCs. Fatty acid treatments induced steatosis and lipotoxicity in HLCs. Furthermore, fat-laden HLCs prompted a non-parenchymal cells response altering tissue architecture. Conclusions Our multicellular platform provides a new approach to model interactions between human hepatic cells during NAFLD progression. Such approach has the potential to investigate the sequential events driving chronic liver diseases, including hepatocellular injury, inflammation and fibrosis. Furthermore, our system provides a unique and urgently needed tool to investigate the molecular mechanisms associated with NAFLD and ultimately to validate new targets for therapeutics development. List of abbreviations COs, cholangiocytes organoids; FFA, free fatty acids; hiPSCs, human induced pluripotent stem cells; HLCs, hepatocyte like cells; HSCs, hepatic stellate cells; M0, hiPSCs-derived macrophages; NAFLD, non-alcoholic fatty liver disease; NPCs, non-parenchymal cells; OA, oleic acid; PA, palmitic acid.

Novel 3D Approach to Model Non-Alcoholic Fatty Liver Disease using human Pluripotent Stem Cells