A Model for Human Liver Fibrosis

摘要 肝纤维化是一种病理状态，由细胞外基质(ECM)积聚引起 慢性肝损伤，是全球成人死亡的主要原因(每年约100万人)，原因是治疗不当 选择。为了解决这一局限性，我们开发了人类肝脏器官模型，使假设- 对治疗肝纤维化的新药候选进行驱动的、机械性的评估。其中一个型号是由 基因工程IPSC表达常染色体隐性遗传性多囊肾病常见致病突变 (ARPKD)。ARPKD类器官形成了肝纤维化的主要特征：它们积累了厚厚的胶原纤维； 并且有明显增加的胶原生成的肌成纤维细胞，其转录图谱类似于 存在于常见的(获得性)肝纤维化患者的肝组织中(病毒- 诱发肝硬变和晚期非酒精性脂肪性肝炎(NASH)。我们还开发了一种纳什有机体 纤维化模型；以及两种活细胞成像方法监测胶原纤维的出现和 胶原蛋白产生细胞。我们假设，由于在这个人类多血统中发展起来的纤维化， 肝脏类器官类似于先天性和获得性肝纤维化患者的肝脏器官，可用于 推进肝纤维化研究，发现和鉴定抗纤维化治疗方法。在目标1中，ARPKD和 NASH有机化合物被用来开发一个新的平台来评估10种药物的抗纤维化效果，这些药物 作用机制与肝纤维化有关，并确定具有增强抗肝纤维化作用的药物组合 功效。由于有九种药物是FDA批准的，但目前没有一种药物用于治疗肝纤维化，这些研究 可能具有重大的翻译重要性。在目标2中，这些模型评估了ECM的纤维化作用。 使用一种新的、完全化学定义的生物合成基质的线索。人们普遍认为ECM的变化将促进 纤维性重塑。一种新颖的合成化学方案使得能够调节关键机械(刚性， 粘弹性)和生物化学(细胞-粘附性配体特性)基质特性。ARPKD和NASH有机化合物 生长在合成基质中将使我们能够检查基质信号对纤维化的影响，这 分析包括单细胞RNA测序(scRNA-Seq)。在目标3中，确定常见的致病因素 在先天性和获得性肝纤维化中共有的，我们将我们的建模方法扩展到 Joubert综合征相关疾病(JSRD)的器官模型的建立和表征 在某些情况下导致肝纤维化的系统遗传性疾病，我们描述了一种独特的NASH有机化合物 模特。JSRD肝病不能在动物身上建立模型。JSRD和NASH有机化合物和等基因对照将 用scRNA-Seq、高维飞行时间质谱仪(CyTOF)和两个半靶点 代谢组学方法。JSRD有机化合物也将用于测试10种药物的抗纤维化效果(目标1)和 用于表征ECM对纤维化的影响(目标2)。此数据将提供有关共享的重要信息 不同病因的肝纤维化的调节机制。

Abstract Liver fibrosis is a pathological condition that results from extracellular matrix (ECM) accumulation in response to chronic liver injury and is a major global cause of death in adults (~1M per year) due to inadequate therapeutic options. To address this limitation, we have developed human hepatic organoid models that enable hypothesis- driven, mechanistic evaluation of novel drug candidates for treatment of liver fibrosis. One model is produced by engineering iPSC to express a common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). ARPKD organoids develop the key hallmarks of hepatic fibrosis: they accumulate thick collagen fibers; and have a marked increase in collagen-producing myofibroblasts whose transcriptomic profile is like those present in liver tissues obtained from patients with commonly occurring (acquired) forms of liver fibrosis (viral- induced cirrhosis and advanced non-alcoholic steatohepatitis, NASH). We also developed a NASH organoid fibrosis model; along with two live cell imaging methods for monitoring for the appearance of collagen fibers and collagen producing cells. We hypothesize that since the fibrosis that develops in this human, multi-lineage, hepatic organoid resembles that in patients with congenital and acquired forms of liver fibrosis, it can be used to advance liver fibrosis research and to discover and characterize anti-fibrotic therapies. In Aim 1, ARPKD and NASH organoids are used to develop a novel platform for assessing the anti-fibrotic efficacy of 10 agents whose mechanism of action is relevant to liver fibrosis, and to identify drug combinations with increased anti-fibrotic efficacy. Since nine are FDA-approved drugs, but none are currently used to treat liver fibrosis, these studies could have significant translational importance. In Aim 2, these models evaluate the fibrogenic effect of ECM cues using a novel, fully chemically defined, biosynthetic matrix. ECM changes are widely thought to promote fibrotic remodeling. A novel, synthetic chemistry scheme enables tuning of the key mechanical (stiffness, viscoelasticity) and biochemical (cell-adhesive ligand identity) matrix properties. ARPKD and NASH organoids grown in synthetic matrices will enable us to examine the effects that matrix cues have on fibrosis, and this analysis includes single cell RNA sequencing (scRNA-Seq). In Aim 3, to identify common pathogenetic drivers that are shared among congenital and acquired forms of liver fibrosis, we extend our modeling approach to generate and characterize organoid models for Joubert Syndrome Related Disorder (JSRD), which is a multi- system genetic disease that causes liver fibrosis in some cases, and we characterize a unique NASH organoid model. JSRD liver disease cannot be modeled in animals. JSRD and NASH organoids and isogenic controls will be analyzed using scRNA-Seq, high-dimensional time of flight mass cytometry (CyTOF) and two semi-targeted metabolomic methods. JSRD organoids will also be used to test the anti-fibrotic effects of 10 drugs (Aim 1) and for characterizing ECM effects on fibrosis (Aim 2). This data will provide important information about shared mechanisms that mediate liver fibrosis of different etiologies.