Modeling fatty liver disease development and progression in a human iPSC-based microphysiological system

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Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in Western countries, impacting millions of patients and challenging the health care system. NAFLD is closely related to obesity, characterized by lipid accumulation in hepatocytes and associated with a risk for developing non-alcoholic steatohepatitis (NASH), which is accompanied by inflammation on top of steatosis and causes liver cirrhosis as well as cancer. Studies revealed genetic polymorphisms that increase the risk for developing NAFLD and NASH.0 One of the most common genetic risk factors is a single-nucleotide polymorphism in the gene TM6SF2 (c.499C>T; rs58542926) that causes an E167K amino acid change. Clinical studies established that this variant alters hepatic lipid metabolism and is associated with both NAFLD and NASH. Studies in mice and cell lines showed that TM6SF2 is involved in the transport of triglycerides and cholesterol from hepatocytes. However, it is currently not known how the E167K variant affects TM6SF2 at the molecular and functional level. We propose to answer this question to advance our understanding of NAFLD/NASH pathogenesis and reveal targets for new therapies. To ascertain robustness and translatability of our results, we will use fully genetically characterized human induced pluripotent stem cells (iPSCs) to model and investigate the TM6SF2 E167K risk factor. For this, we have generated iPSC lines from NAFLD/NASH patients carrying the TM6SF2 E167K variant, corrected it in these lines and introduced it into a commonly used wildtype line. To be able to study steatosis and progression to inflammation, we will generate hepatocytes as well as pro- and anti-inflammatory macrophages from the iPSCs. In addition, we will study these cells in a biochip-based liver sinusoid-inspired 3D system that more faithfully replicates human liver physiology and pathophysiology than conventional cell cultures. Importantly, we have generated the necessary iPSC lines using CRISPR technology, established robust protocols for their directed differentiation into hepatocytes as well as macrophages and produced the liver biochip. Therefore, we can focus on modeling and investigating NALD/NASH pathomechanisms using these cells and the biochip. By generating hepatocytes and macrophages from the same iPSC line, we expect to exclude biases caused by different genetic backgrounds. Successful completion of the proposed project will reveal the molecular and functional consequences of the TM6SF2 E167K variant and establish a system for modeling NALD/NASH pathomechanisms, including specific effects of genetic risk factors. In addition, this project will provide a platform for launching a career as an independent investigator, for example by further developing the biochip to include iPSC-derived hepatic stellate cells, thereby allowing studies of fibrosis downstream of steatosis and inflammation, including screens for drugs acting on these key steps of NAFLD/NASH.