Building a functional biliary system from hepatocytes

项目摘要/摘要 这一应用针对的是胆管缺乏患者未得到满足的医疗需求，这些患者经常发展为严重的 胆汁淤积性肝损伤，目前只能通过肝移植治愈。我们的应用程序建立在我们发现的基础上 肝细胞可以形成肝内胆管(IHBD)，从而逆转小鼠的胆汁淤积 IHBD严重缺乏的模型。已有肝细胞转化为胆管细胞的报道，但我们的小鼠 模型建立了肝细胞可以从头开始建立一个治疗有效的胆道系统。我们 假设我们的小鼠模型已经揭示了肝细胞向胆管细胞转化的全部潜力 由于其严重的IHBD缺乏和其独特的基因构成，影响开槽和 转化生长因子β信号，胆管发育的主要调节因子。我们建议确定这些机制 负责在我们的小鼠模型中从肝细胞自发恢复IHBD，长期目标是 启发组织工程学方法，开发胆管相关疾病的治疗方法 贫乏。在目标1中，我们将定义Notch和TGFbeta之间的相互作用，以推动肝细胞的转化- 转化为胆管细胞并组装成IHBD。为此，我们将使用活体小鼠模型来执行 药理学和遗传学方法调节转化生长因子β信号以检查“步骤”(转换 和形态发生)的肝细胞来源的从头IHBD形成。此外，我们还将调查 转化生长因子β效应因子缺失影响转录顺式调控关联的机制 诱导肝细胞向胆管细胞转分化的复合体。目标2，我们将在其中定义 驱动肝细胞向胆管细胞转化并组装成IHBD的转录网络也将 告知这些努力。为此，我们将研究在体内转化肝细胞中哪些转录因子 在微图案化共培养系统中稳定的原代肝细胞中激活并验证其有效性 胆管球体形态发生实验。利用从这些实验中获得的洞察力，我们将表达 利用非整合、无毒腺相关的体内胆汁分化和胆管形成的效应因子 用于向肝细胞运送基因的病毒载体。我们的研究将产生对分子的新见解 调节肝细胞特性、胆管发育和再生。

Project Summary/Abstract This application targets the unmet medical needs of patients with bile duct paucity who often develop severe cholestatic liver injury that currently is only curable by liver transplantation. Our application builds on our finding that hepatocytes can form intrahepatic bile ducts (IHBDs) that function to reverse cholestasis in a mouse model of severe IHBD paucity. Conversion of hepatocytes-to-cholangiocytes has been reported, but our mouse model establishes that hepatocytes can build a therapeutically effective biliary system from scratch. We hypothesize that our mouse model has revealed the full potential of hepatocyte-to-cholangiocyte conversion because of the severity of its IHBD paucity and its unique genetic makeup, affecting both NOTCH and TGFbeta signaling, the main regulators of bile duct development. We propose to identify the mechanisms responsible for spontaneous IHBD restoration from hepatocytes in our mouse model with the long-term goal of enlightening tissue engineering approaches and to develop a therapy for diseases associated with bile duct paucity. In Aim 1 we will define the interplay between NOTCH and TGFbeta driving conversion of hepatocytes- to-cholangiocytes and assembly into IHBDs. For this we will use in vivo mouse models to perform pharmacologic and genetic approaches modulating TGFbeta signaling for examining the “steps” (conversion and morphogenesis) of hepatocyte-derived de novo IHBD formation. In addition, we will investigate the mechanism by which loss of a TGFbeta effector influences the association of transcriptional cis-regulatory complexes to induce hepatocyte-to-cholangiocyte transdifferentiation. Aim 2, in which we will define the transcriptional network driving conversion of hepatocytes-to-cholangiocytes and assembly into IHBDs will also inform these efforts. For this we will investigate in converting hepatocytes in vivo which transcription factors are active and validate their efficacy in primary hepatocytes stabilized in a micropatterned co-culture system and a cholangiosphere morphogenesis assay. Using the insight gained from these experiments, we will express effectors of biliary differentiation and tube formation in vivo using non-integrating, nontoxic adeno-associated viral vectors for gene delivery to hepatocytes. Our research will generate new insight into the molecular regulation of hepatic cell identity, biliary development and regeneration.