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

项目摘要 纤维化肝病是一个日益严重的公共卫生问题，严重影响全球人口。纤维化 肝病通常是持续性损伤的结果;慢性病毒性肝炎B和C以及NAFLD是 最普遍的原因。虽然B型和C型肝炎的抗病毒治疗方案降低了病毒性肝硬化的负担， 其他原因如NAFLD越来越普遍，现在影响发达国家14-27%的人。 尽管疾病负担很高，但目前还没有批准的治疗方法。相反，缺乏共识 最佳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理解存在差距。同样地， 动物模型的实用性和临床相关性同样存在争议。目前，许多型号 使用，但每一个都只概括了人类病理生理学的孤立方面。的需求尚未得到满足 概括纤维化肝病的关键要素以实现机械解剖的人类相关系统。 在成功的抗病毒治疗后，一些但不是所有患者的肝纤维化可测量的改善 治疗加强了在分子水平上更好地理解纤维化的需要，以帮助发展 预防纤维化或促进退化的新疗法。对于NAFLD，尽管识别出越来越多的 与易感性相关的遗传变异和生活方式依赖性风险因素的数量， 它们单独或协同地促进疾病进展的作用在很大程度上仍不清楚。 这项提案的主要研究目标是利用一种独特的可再生和可遗传操纵的 人多能干细胞（hPSC）衍生的多细胞培养系统以解决上述缺口。在 在这个多细胞系统中，我们共培养hPSC衍生的肝细胞、肝星状细胞（HSC）和 在健康和疾病中，以概括肝脏生理学的复杂性的方式对巨噬细胞进行研究。 在多细胞培养物中模拟肝炎病毒感染和NAFLD，我们发现HCV感染和NAFLD均与HCV感染有关。 脂毒性环境诱导炎症信号和星状细胞活化。脂毒性环境也引发了其他 NAFLD临床表型的特征。消除HCV逆转的纤维化样表型并用 如在临床中观察到的，奥贝胆酸显示出NAFLD样特征的改善。 在三个目标，使用细胞和分子的方法，我们利用这部小说的独特功能， 平台，以解决任何现有的离体人类相关技术无法充分回答的问题 系统，包括细胞因子在HSC活化中的作用（Aim 1），活化后逆转的机制 (Aim 2）和遗传和生活方式相关的危险因素在NAFLD中的作用（目的3）。了解 星状细胞活化和纤维化发展的分子机制对于开发诊断是至关重要的 并设计新的治疗方法来阻止纤维化或促进退化。此外，阐明机制 遗传变异和风险因素如何导致肝病进展可能提供了制定 针对特定患者亚组优化的靶向抗纤维化干预。

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.