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

总结 非酒精性脂肪性肝病（NAFLD）是美国肝功能检查异常的最常见原因 它的进展形式，称为非酒精性脂肪性肝炎（NASH），将很快成为主要的适应症， 肝移植目前没有有效的药物来治疗NASH，没有生物标志物来确定 疾病进展或移植后复发的风险，并且没有高通量药物的有效平台 筛选虽然NASH与肥胖和糖尿病有关，但导致疾病的致病因素 进展为NASH/肝硬化知之甚少。与侵入性肝活检相比，外周血 单核细胞（PBMC）可以容易地从NASH和终末期NASH/肝硬化患者获得 需要肝脏移植并重新编程为诱导多能干细胞（iPSC）的患者。这些 iPSC然后可以分化成iPSC-肝细胞，iPSC-肝细胞是可以在培养基中培养的人肝样细胞。 体外生物工程系统适合于正常和慢性肝病肝硬度，使研究， 独立于使人体内分析复杂化的复合代谢和环境因素， 动物系统在本提案中，我们将利用iPSC-肝细胞平台来确定 细胞外基质（ECM）硬度和未折叠蛋白反应（UPR）细胞信号传导对肝脏脂质的影响 新陈代谢.我们将首先解开矩阵刚度和患者特异性之间的影响和相互作用 患者来源的iPSC中NASH的倾向，并分析对脂质代谢和脂质组学的影响（目的 1）。ER应激和未折叠蛋白反应（UPR）已被证明是重要的发病机制， 纳什因此，我们将使用iPSC-肝细胞来开发一个平台，用于确定UPR与肝细胞之间的相互作用。 与NASH相关的信号传导和脂质代谢（Aim 2）。iPSC-肝细胞将用ER应激处理 还原化合物，包括化学伴侣牛磺熊去氧胆酸（TUDC）或FXR/胆汁酸 激动剂，以及对细胞分化、基因表达和脂质代谢的影响将被研究。最后， iPSC-肝细胞将用于研究NASH在iPSC中的细胞信号传导和致病机制， 需要肝移植的快速进展性NASH/肝硬化患者。我们将开发iPSC- 肝细胞平台，使用iPSC-肝细胞鉴定负责NASH基质和UPR因子， NASH/肝硬化患者被列为肝移植（目的3）。此MPI提案利用了 两个机构的三名PI之间具有丰富的研究经验1）iPSC，生物工程基质， ECM生物学和移植手术，2）肝脏脂质代谢，细胞信号传导和移植肝脏学，以及 3)脂质组学和代谢组学。这些iPSC肝细胞平台的开发和优化将具有 对确定NASH的病理生理学，开发生物标志物以确定风险的重要意义 用于NASH进展以及用于药物开发和个性化药物筛选。

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.