A renewable and genetically tractable human stem cell-derived multicellular platform for the study of fibrotic liver diseases

项目摘要 纤维性肝病是一个日益严重的公共卫生问题，严重影响着全球人口。纤维性 肝脏疾病通常是持续侮辱的结果；慢性乙型和丙型病毒性肝炎以及非酒精性脂肪肝是 最普遍的原因。虽然针对乙肝和丙型肝炎的抗病毒疗法减轻了病毒性肝炎的负担， 像NAFLD这样的其他原因越来越普遍，现在发达国家有14%-27%的人受到影响。 尽管疾病负担很高，但目前还没有得到批准的治疗方法。相反，缺乏共识 关于最优药物靶点或策略的研究反映了我们对疾病驱动因素的机械性理解上的差距。同样， 动物模型的实用性和临床相关性同样存在争议。目前有多款车型 使用过，但每一个都只概括了人类病理生理学的孤立方面。有一种未得到满足的需求 与人类相关的系统，概括了纤维性肝病的关键因素，使机械解剖成为可能。 部分但不是所有患者在成功抗病毒治疗后肝纤维化的可测量改善 治疗加强了在分子水平上更好地了解纤维化形成的必要性，以帮助发展 预防纤维化或鼓励病情恶化的新疗法。对于非酒精性脂肪肝，尽管发现 与易感性相关的遗传变异和依赖生活方式的风险因素的数量，通过 它们单独或协同作用对疾病进展的贡献在很大程度上仍不清楚。 这项提议的主要研究目标是开发一种独特的可再生和可遗传操纵的 人类多能干细胞(HPSC)衍生的多细胞培养系统，以解决上述空白。在……里面 在这个多细胞系统中，我们将hPSC来源的肝细胞、肝星状细胞(HSCs)和 巨噬细胞以一种概括了健康和疾病中肝脏生理的复杂性的方式。 在多细胞培养中模拟肝炎病毒感染和NAFLD，我们发现丙型肝炎病毒感染和NAFLD 脂毒环境诱导炎症信号和星状细胞激活。一种脂毒环境也引发了其他 非酒精性脂肪肝的临床表型特征。丙型肝炎病毒逆转肝纤维化样表型的消除和治疗 临床观察显示，乙酰胆酸对NAFLD样特征有改善作用。 跨越三个目标，使用细胞和分子方法，我们利用了这部小说的独特特征 解决任何现有的与体外人类相关的问题无法充分回答的问题的平台 系统，包括细胞因子在肝星状细胞激活中的作用(目标1)，激活后逆转的机制 (目标2)以及遗传和生活方式相关风险因素在非酒精性脂肪肝中的作用(目标3)。了解 星状细胞激活和纤维化发展的分子机制对发展诊断至关重要 以及设计新的治疗方法来阻止纤维化或促进消退。此外，还阐明了其作用机制 基因变异和风险因素是如何导致肝病进展的可能提供了一个机会 针对特定患者亚组优化的有针对性的抗纤维化干预。

Project Summary Fibrotic liver disease is a growing public health concern significantly impacting the global population. Fibrotic liver disease is often the result of sustained insults; chronic viral hepatitis B and C as well as NAFLD are the most prevalent causes. While antiviral regimens for hepatitis B and C have decreased the viral cirrhosis burden, other causes like NAFLD are increasingly prevalent, now affecting 14-27% of individuals in developed countries. Despite this high disease burden, there are currently no approved therapies. Rather, the lack of consensus on optimal drug targets or strategies reflects a gap in our mechanistic understanding of disease drivers. Similarly, the utility and clinical relevance of animal models is equally controversial. A multitude of models are currently used, but each recapitulates only isolated aspects of human pathophysiology. There is an unmet need for human-relevant systems that recapitulate key elements of fibrotic liver disease to enable mechanistic dissection. The measurable improvement in liver fibrosis in some but not all patients following successful antiviral treatment reinforces the need for better understanding of fibrogenesis at a molecular level to aid development of new treatments to prevent fibrosis or encourage regression. For NAFLD, despite identification of increasing numbers of susceptibility-associated genetic variants and lifestyle-dependent risk factors, the mechanisms by which they individually or synergistically contribute to disease progression remain largely unclear. The primary research goal of this proposal is to exploit a unique renewable and genetically manipulatable human pluripotent stem cell (hPSC)-derived multicellular culture system to address the aforementioned gaps. In this multicellular system, we coculture hPSC-derived hepatocytes, hepatic stellate cells (HSCs), and macrophages in a manner that recapitulates the complexity of liver physiology in both health and disease. Modeling hepatitis virus infection and NAFLD in the multicellular cultures, we found that both HCV infection and a lipotoxic milieu induced inflammatory signals and stellate cell activation. A lipotoxic milieu also triggered other features of NAFLD clinical phenotypes. Eliminating HCV reversed fibrosis-like phenotypes and treating with obeticholic acid showed improvement in NAFLD-like features, as observed in the clinic. Across three aims, using cell and molecular approaches, we capitalize on the unique features of this novel platform to address questions that cannot be adequately answered with any existing ex vivo human-relevant system, including the roles of cytokines in HSC activation (Aim 1), the mechanisms of reversion after activation (Aim 2) and the role of genetics and lifestyle-associated risk factors in NAFLD (Aim 3). Understanding the molecular mechanisms of stellate cell activation and fibrosis development are critical for developing diagnostics and designing new treatments to block fibrosis or promote regression. In addition, elucidating the mechanisms of how genetic variants and risk factors contribute to liver disease progression may offer the opportunity to craft targeted antifibrotic interventions optimized for particular patient subgroups.