Ex-vivo bioengineered technology to unravel dysfunction due to non-alcoholic steatohepatitis (NASH)

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SUMMARY Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver function tests in the US and its progressive form, termed non-alcoholic steatohepatitis (NASH), will soon be the leading indication for liver transplantation. There are currently no effective medications to treat NASH, no biomarkers to determine disease progression or risk of post-transplant recurrence and no effective platforms for high-throughput drug screening. Although NASH is related to obesity and diabetes, the pathogenic factors that cause disease progression to NASH/Cirrhosis are poorly understood. Compared to an invasive liver biopsy, peripheral blood mononuclear cells (PBMCs) can be easily obtained from patients with NASH and end-stage NASH/Cirrhosis patients requiring liver transplantation and re-programmed into induced pluripotent stem cells (iPSCs). These iPSCs may then be differentiated into iPSC-hepatocytes, which are human liver-like cells that can be cultured in ex vivo bioengineered systems tailored to normal and cirrhotic liver stiffness, enabling an investigation that is independent of the compounding metabolic and environmental factors that complicate analysis within human or animal systems. In this proposal, we will utilize an iPSC-hepatocyte platform to determine the effects of extracellular matrix (ECM) stiffness and unfolded protein response (UPR) cell signaling on hepatic lipid metabolism. We will initially unwind the impact and interplay between matrix stiffness and patient-specific propensity for NASH in patient-derived iPSCs and analyze the impact on lipid metabolism and lipidomics (Aim 1). ER stress and the unfolded protein response (UPR) has been shown to be important in the pathogenesis of NASH. Thus, we will use iPSC-hepatocytes to develop a platform for determining the interaction between UPR signaling and lipid metabolism relevant to NASH (Aim 2). iPSC-hepatocytes will be treated with ER stress reducing compounds including the chemical chaperone tauroursodeoxycholic acid (TUDC) or FXR/bile acid agonists, and the effects on cell differentiation, gene expression and lipid metabolism will investigated. Finally, iPSC-hepatocytes will be used to study the cell signaling and pathogenic mechanisms of NASH in iPSCs from patients 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 from NASH/Cirrhosis patients listed for liver transplantation (Aim 3). This MPI proposal leverages the collaboration between three PIs at two institutions with extensive experience investigating 1) iPSCs, bioengineering matrices, ECM biology, and transplant surgery, 2) hepatic lipid metabolism, cell signaling and transplant hepatology, and 3) lipidomics and metabolomics. The development and optimization of these iPSC-hepatocyte platforms will have important implications for determining the pathophysiology of NASH, developing biomarkers to determine risk for NASH progression and for use in drug development and personalized drug screening.