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

在基于人类 iPSC 的微生理系统中模拟脂肪肝疾病的发生和进展

• 批准号: 442552105
• 负责人: Dr. Marko Gröger
• 金额: --
• 依托单位: Division of Transplant Surgery
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/442552105?language=en
• 关键词: Modeling; fatty; liver; disease; development

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.

非酒精性脂肪性肝病（NAFLD）已成为西方国家最常见的肝病，影响着数百万患者，并对医疗保健系统构成挑战。NAFLD与肥胖密切相关，其特征是肝细胞中的脂质积聚，并与发生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，并导致肝硬化和癌症。研究发现遗传多态性增加了NAFLD和NASH的风险。最常见的遗传风险因素之一是基因TM 6SF 2（c.499C>T; rs 58542926）中的单核苷酸多态性，该多态性导致E167 K氨基酸改变。临床研究证实，这种变异改变了肝脏脂质代谢，并与NAFLD和NASH相关。在小鼠和细胞系中的研究表明，TM 6SF 2参与从肝细胞转运甘油三酯和胆固醇。然而，目前尚不清楚E167 K变体如何在分子和功能水平上影响TM 6SF 2。我们建议回答这个问题，以促进我们对NAFLD/NASH发病机制的理解，并揭示新疗法的靶点。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表征的人诱导多能干细胞（iPSC）来建模和研究TM 6SF 2 E167 K风险因素。为此，我们已经从携带TM 6SF 2 E167 K变体的NAFLD/NASH患者产生了iPSC系，在这些系中对其进行了校正，并将其引入到常用的野生型系中。为了能够研究脂肪变性和炎症进展，我们将从iPSC中产生肝细胞以及促炎和抗炎巨噬细胞。此外，我们将在基于生物芯片的肝窦状隙启发的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学和病理生理学。重要的是，我们已经使用CRISPR技术产生了必要的iPSC系，建立了强大的方案，使其定向分化为肝细胞和巨噬细胞，并生产了肝脏生物芯片。因此，我们可以专注于使用这些细胞和生物芯片来建模和研究NALD/NASH病理机制。通过从相同的iPSC系产生肝细胞和巨噬细胞，我们期望排除由不同遗传背景引起的偏差。拟议项目的成功完成将揭示TM 6SF 2 E167 K变体的分子和功能后果，并建立一个NALD/NASH病理机制建模系统，包括遗传风险因素的特定影响。此外，该项目还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以包括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。