A Scalable 3D Human Liver Co-culture Platform for Hepatitis B Virus Infection

用于乙型肝炎病毒感染的可扩展 3D 人类肝脏共培养平台

• 批准号: 9814819
• 负责人: Salman R Khetani
• 金额: $ 24.1万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-11 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9814819
• 关键词: 3-Dimensional; Adult; Antibodies; Area; Binding; Biological Models; Cancer Etiology; Cancerous; Cell Culture Techniques; Cell Line; Cells; Chronic; Chronic Hepatitis B; Circular DNA; Cirrhosis; Clinical; Coculture Techniques; Collagen; Collagen Fiber; Disease; Disease Progression; Drug Screening; Endothelial Cells; Engineering; Enzymes; Episome; Europe; Extracellular Matrix; Extracellular Matrix Proteins; Fibrosis; Future; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genetic Transcription; Gold; HBV Animal Model; Hepatic Stellate Cell; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Surface Antigens; Hepatitis B Virus; Hepatitis B e Antigens; Hepatocyte; Human; Immune; Immune Response Genes; In Vitro; Infection; Inflammation; Inflammatory; Innate Immune Response; Kinetics; Kupffer Cells; Liver; Measures; Modeling; Myofibroblast; Outcome; Pan Genus; Pathogenesis; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Phenotype; Physiological; Polyethylene Glycols; Primary carcinoma of the liver cells; Protocols documentation; RNA; Resources; Serum; Source; Stem cells; Testing; Viral Pathogenesis; Virus; Virus Diseases; cell immortalization; cell type; chemokine; clinically relevant; cytokine; drug development; drug metabolism; drug testing; enzyme activity; high reward; high risk; immortalized cell; in vivo; induced pluripotent stem cell; inhibitor/antagonist; liver function; mortality; new therapeutic target; novel; patient population; pre-clinical; screening; three dimensional cell culture; viral DNA

Abstract / Project Summary Hepatitis B virus (HBV) infects ~240 million people chronically and can progress to cirrhosis and hepatocellular carcinoma (HCC), the third leading cause of cancer mortality. Current drug therapies to treat HBV cannot eliminate the persistent covalently closed circular (ccc)DNA, which thus requires lifetime drug therapy. Therefore, the discovery of novel HBV drugs that can eliminate cccDNA is an active area of pharmaceutical drug development. However, such efforts are hampered by the lack of physiologically-relevant model systems that recapitulate critical features of the disease pathogenesis, such as interactions between hepatocytes and relevant liver non-parenchymal cell types (NPCs), and the ability to evaluate patient-specific outcomes. Conducting tests on the chimpanzee, the only animal model for HBV, is prohibitively expensive, severely restricted in the US and Europe, and does not fully mimic human HBV pathogenesis. Thus, human liver culture platforms are the most widely used model for HBV studies. While primary human hepatocytes (PHHs) are the gold standard for HBV studies, they are a scarce resource that does not suffice for high-throughput drug screening and to elucidate the genetic basis of HBV pathogenesis. On the other hand, induced pluripotent stem cell-derived human hepatocyte- like cells (iHeps) are an important patient-specific source from an expandable precursor, thus mitigating many of the limitations with PHHs. We and others have shown that 2D cultures of iHeps can be infected with HBV and used to test drugs. However, absorbed extracellular matrix (ECM) proteins in 2D cultures do not allow adequate elucidation of ECM reorganization/re-modeling during HBV-induced fibrosis progression. Furthermore, existing platforms lack the relevant liver NPCs that interact with HBV-infected hepatocytes in vivo to modulate infection, inflammation, and fibrosis. We have recently pioneered microscale 3D collagen microgels containing iHeps that display adult-like liver functions, including drug metabolism enzyme activities, for several weeks in vitro when co-cultured with primary liver sinusoidal endothelial cells (LSECs), herein referred to as ‘microtissues’. In this proposal, we will test our novel hypothesis that iHep/LSEC 3D microtissues, which display adult-like liver functions, can a) be infected chronically (weeks) with HBV with higher infection levels, spread, and amplification than existing 2D/3D culture platforms (aim 1), and b) when augmented with hepatic stellate cells (HSCs) and Kupffer cells (KCs), display inflammatory and fibrotic signatures correlative of clinical outcomes (aim 2). Our proposal will yield a first-of-its-kind scalable 3D human liver platform containing iHeps and liver NPCs that displays chronic HBV infection while retaining the ability to adequately metabolize drugs for screening. In the future, our HBV platform can be used to study mechanisms underlying hepatocyte-NPC interactions towards discovering novel druggable targets, the inclusion of patient-matched adaptive immune cells to model interaction with resident liver cell types, and gene editing to elucidate genetic determinants of HBV progression.

摘要/项目摘要 乙肝病毒(HBV)慢性感染约2.4亿人，可发展为肝硬化和肝细胞癌 癌症是导致癌症死亡的第三大原因。目前治疗乙肝病毒的药物疗法不能 消除持久的共价闭合环(CCC)DNA，因此需要终生药物治疗。因此， 发现能消除cccDNA的新型乙肝病毒药物是药物研究的一个活跃领域 发展。然而，由于缺乏与生理相关的模型系统，这些努力受到了阻碍 概述疾病发病机制的关键特征，如肝细胞与相关疾病之间的相互作用 肝脏非实质细胞类型(NPC)，以及评估患者特定结果的能力。进行测试 在黑猩猩身上，作为唯一的乙肝病毒动物模型，价格高得令人望而却步，在美国和 欧洲，并不完全模仿人类乙肝的发病机制。因此，人类肝脏培养平台是最多的 广泛应用于乙肝病毒研究的模型。而原代人肝细胞(PHHs)是乙肝病毒的金标准 研究表明，它们是一种稀缺资源，不足以进行高通量药物筛选和阐明 乙肝病毒致病的遗传学基础。另一方面，诱导多能干细胞来源的人肝细胞- 类细胞(IHEP)是来自可扩展前体的重要的患者特有来源，因此减轻了许多 PHH的局限性。我们和其他人已经证明，2D培养的iHeps可以感染乙肝病毒和 用来测试毒品。然而，2D培养中吸收的细胞外基质(ECM)蛋白不允许有足够的 阐明在乙肝病毒诱导的纤维化进展过程中细胞外基质重组/重新建模。此外，现有的 平台缺乏与体内感染乙肝病毒的肝细胞相互作用以调节感染的相关肝脏NPC， 炎症和纤维化。我们最近率先开发了含有iHeps的微型3D胶原微凝胶 在体外数周内表现出成人样的肝功能，包括药物代谢酶活性 与原代肝窦内皮细胞(LSECs)共培养，这里称其为“微组织”。在这 提议，我们将检验我们的新假设，即IHEP/LSec 3D显微组织，它显示成人样的肝脏 功能，a)慢性(数周)感染乙肝病毒，具有更高的感染水平、传播和扩增 比现有的2D/3D培养平台(目标1)和b)增加肝星状细胞(HSCs)和 Kupffer细胞(KCs)表现出与临床结果相关的炎症和纤维化征象(目标2)。我们的 该提案将产生首个可扩展的3D人体肝脏平台，其中包含iHEPS和肝脏NPC， 显示慢性乙肝病毒感染，同时保留充分代谢药物进行筛查的能力。在 未来，我们的乙肝病毒平台可以用来研究肝细胞-鼻咽癌相互作用的机制 发现新的可用药靶点，纳入患者匹配的适应性免疫细胞来模拟相互作用 与常驻肝细胞类型，以及基因编辑，以阐明乙肝病毒进展的遗传决定因素。