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

非酒精性脂肪性肝病（NAFLD）已成为西方国家最常见的肝脏疾病，影响了数百万患者并对卫生保健系统提出了挑战。NAFLD与肥胖密切相关，其特征是肝细胞中的脂质积累，并与发展为非酒精性脂肪性肝炎（NASH）的风险相关，NASH在脂肪变性的基础上伴有炎症，可导致肝硬化和癌症。研究表明，遗传多态性可增加NAFLD和nash的风险。最常见的遗传风险因素之一是基因TM6SF2 （c.499C>T; rs58542926）的单核苷酸多态性，该多态性可导致E167K氨基酸改变。临床研究证实，该变异可改变肝脏脂质代谢，并与NAFLD和NASH相关。对小鼠和细胞系的研究表明，TM6SF2参与肝细胞中甘油三酯和胆固醇的运输。然而，目前尚不清楚E167K变异如何在分子和功能水平上影响TM6SF2。我们提出回答这个问题，以促进我们对NAFLD/NASH发病机制的理解，并揭示新的治疗靶点。为了确定我们的结果的稳健性和可翻译性，我们将使用完全遗传特征的人类诱导多能干细胞（iPSCs）来模拟和研究TM6SF2 E167K风险因素。为此，我们从NAFLD/NASH患者中生成了携带TM6SF2 E167K变体的iPSC系，在这些系中对其进行校正，并将其引入常用的野生型系。为了能够研究脂肪变性和炎症进展，我们将从iPSCs中生成肝细胞以及促炎性和抗炎性巨噬细胞。此外，我们将在基于生物芯片的肝窦启发3D系统中研究这些细胞，该系统比传统细胞培养更忠实地复制人类肝脏生理和病理生理。重要的是，我们已经使用CRISPR技术生成了必要的iPSC系，建立了将其定向分化为肝细胞和巨噬细胞的稳健方案，并生产了肝脏生物芯片。因此，我们可以专注于利用这些细胞和生物芯片建模和研究NALD/NASH的病理机制。通过从相同的iPSC细胞系中产生肝细胞和巨噬细胞，我们希望排除由不同遗传背景引起的偏差。该项目的成功完成将揭示TM6SF2 E167K变异的分子和功能后果，并建立NALD/NASH病理机制建模系统，包括遗传风险因素的具体影响。此外，该项目将为独立研究者的职业生涯提供一个平台，例如通过进一步开发生物芯片，包括ipsc衍生的肝星状细胞，从而允许研究脂肪变性和炎症下游的纤维化，包括筛选作用于NAFLD/NASH这些关键步骤的药物。

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.