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

摘要/项目摘要 B型肝炎病毒（HBV）慢性感染约2.4亿人，并可进展为肝硬化和肝细胞性肝癌。 肝癌（HCC）是癌症死亡的第三大原因。目前治疗HBV的药物疗法不能 消除持久的共价闭合环状（ccc）DNA，这因此需要终生药物治疗。因此，我们认为， 能够消除cccDNA新型HBV药物的发现是药物的活跃领域 发展然而，这种努力受到缺乏生理学相关模型系统的阻碍， 概括疾病发病机制的关键特征，例如肝细胞与相关细胞之间的相互作用， 肝脏非实质细胞类型（NPC），以及评估患者特异性结局的能力。进行测试 在黑猩猩身上，HBV的唯一动物模型，是非常昂贵的，在美国受到严格限制， 欧洲，并且不完全模拟人HBV发病机制。因此，人类肝脏培养平台是最 广泛用于HBV研究的模型。虽然原代人肝细胞（PHH）是HBV的金标准， 研究，它们是一种稀缺资源，不足以进行高通量药物筛选和阐明 HBV发病机制的遗传学基础。另一方面，诱导多能干细胞衍生的人肝细胞- 类细胞（iHep）是来自可膨胀前体的重要的患者特异性来源，因此减轻了许多 PHH的局限性我们和其他人已经表明，iHep的2D培养物可以感染HBV， 用来测试毒品然而，在2D培养物中吸收的细胞外基质（ECM）蛋白不允许足够的细胞外基质（ECM）蛋白的存在。 阐明HBV诱导的纤维化进展期间ECM重组/重塑。此外，现有 平台缺乏与HBV感染的肝细胞在体内相互作用以调节感染的相关肝NPC， 炎症和纤维化。我们最近开创了含有iHep的微尺度3D胶原微凝胶， 显示成人样肝功能，包括药物代谢酶活性，在体外几周， 与原代肝窦内皮细胞（LSEC）（本文中称为“微组织”）共培养。在这 我们将测试我们的新假设，即显示成人样肝脏的iHep/LSEC 3D微组织 功能，可以a）慢性（周）感染HBV，感染水平较高，传播和扩增 与现有的2D/3D培养平台相比（目的1），和B）当用肝星状细胞（HSC）扩增时， 枯否细胞（KCs）显示与临床结果相关的炎症和纤维化特征（目的2）。我们 该提案将产生首个可扩展的3D人类肝脏平台，其中包含iHep和肝脏NPC， 显示慢性HBV感染，同时保留充分代谢药物以进行筛查的能力。在 未来，我们的HBV平台可用于研究肝细胞-NPC相互作用的机制， 发现新的药物靶点，包括患者匹配的适应性免疫细胞来模拟相互作用 与常驻肝细胞类型和基因编辑，以阐明HBV进展的遗传决定因素。

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.