Building a functional biliary system from hepatocytes

项目总结/摘要 该应用针对的是胆管缺乏患者未满足的医疗需求，这些患者经常发生严重的 目前只能通过肝移植治愈的胆汁淤积性肝损伤。我们的应用程序建立在我们的发现之上 肝细胞可以形成肝内胆管（IHBDs），其功能是逆转小鼠的胆汁淤积 IHBD严重缺乏的模型。肝细胞向胆管细胞的转化已有报道，但我们的小鼠 该模型建立了肝细胞可以从头开始建立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IHBD缺乏的严重性及其独特的遗传组成，影响了NOTCH和 TGF β信号传导，胆管发育的主要调节因子。我们建议确定机制 在我们的小鼠模型中负责肝细胞自发IHBD恢复，长期目标是 启发组织工程方法并开发胆管相关疾病的治疗方法 匮乏。在目标1中，我们将定义NOTCH和TGF β驱动肝细胞转化之间的相互作用- 至胆管细胞并组装成IHBD。为此，我们将使用体内小鼠模型来执行 调节TGF β信号传导的药理学和遗传学方法用于检查“步骤”（转化 和形态发生）。此外，我们将调查 TGF β效应子的缺失影响转录顺式调节因子与转录调控因子之间的关联的机制 复合物诱导肝细胞向胆管细胞转分化。目标2，其中我们将定义 驱动肝细胞向胆管细胞转化并组装成IHBD的转录网络也将 为这些努力提供信息。为此，我们将研究在体内转化肝细胞时，哪些转录因子是 活性，并验证其在微模式共培养系统中稳定的原代肝细胞中的功效， 胆管球形态发生测定。利用从这些实验中获得的洞察力，我们将表达 使用非整合的、无毒的腺相关病毒在体内对胆管分化和管形成的效应 用于将基因递送至肝细胞的病毒载体。我们的研究将产生新的见解， 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.