SUMMARYNon-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver function tests in the USand its progressive form termed non-alcoholic steatohepatitis (NASH) will soon be the leading indication forliver transplantation. There are currently no effective medications to treat NASH no biomarkers to determinedisease progression or risk of post-transplant recurrence and no effective platforms for high-throughput drugscreening. Although NASH is related to obesity and diabetes the pathogenic factors that cause diseaseprogression to NASH/Cirrhosis are poorly understood. Compared to an invasive liver biopsy peripheral bloodmononuclear cells (PBMCs) can be easily obtained from patients with NASH and end-stage NASH/Cirrhosispatients requiring liver transplantation and re-programmed into induced pluripotent stem cells (iPSCs). TheseiPSCs may then be differentiated into iPSC-hepatocytes which are human liver-like cells that can be cultured inex vivo bioengineered systems tailored to normal and cirrhotic liver stiffness enabling an investigation that isindependent of the compounding metabolic and environmental factors that complicate analysis within human oranimal systems. In this proposal we will utilize an iPSC-hepatocyte platform to determine the effects ofextracellular matrix (ECM) stiffness and unfolded protein response (UPR) cell signaling on hepatic lipidmetabolism. We will initially unwind the impact and interplay between matrix stiffness and patient-specificpropensity for NASH in patient-derived iPSCs and analyze the impact on lipid metabolism and lipidomics (Aim1). ER stress and the unfolded protein response (UPR) has been shown to be important in the pathogenesis ofNASH. Thus we will use iPSC-hepatocytes to develop a platform for determining the interaction between UPRsignaling and lipid metabolism relevant to NASH (Aim 2). iPSC-hepatocytes will be treated with ER stressreducing compounds including the chemical chaperone tauroursodeoxycholic acid (TUDC) or FXR/bile acidagonists and the effects on cell differentiation gene expression and lipid metabolism will investigated. FinallyiPSC-hepatocytes will be used to study the cell signaling and pathogenic mechanisms of NASH in iPSCs frompatients with rapidly progressive NASH/Cirrhosis that require liver transplantation. We will develop an iPSC-hepatocyte platform identifying matrix and UPR factors responsible for NASH using iPSC-hepatocytes fromNASH/Cirrhosis patients listed for liver transplantation (Aim 3). This MPI proposal leverages the collaborationbetween three PIs at two institutions with extensive experience investigating 1) iPSCs bioengineering matricesECM biology and transplant surgery 2) hepatic lipid metabolism cell signaling and transplant hepatology and3) lipidomics and metabolomics. The development and optimization of these iPSC-hepatocyte platforms will haveimportant implications for determining the pathophysiology of NASH developing biomarkers to determine riskfor NASH progression and for use in drug development and personalized drug screening.