Stem Cell-Based In vivo Models of Human Genetic Liver Diseases

描述（由申请人提供）：在过去的二十年中，许多人类遗传疾病的遗传基础已经被阐明，并且从任何患有遗传疾病的个体中产生iPS细胞的能力使得研究遗传变化对体外细胞功能的影响成为可能。然而，由于再生医学的主要目标是修复和替代人体组织，表征影响器官发育和功能的遗传因素至关重要。此外，在许多情况下，动物模型，包括基因工程小鼠，不能准确反映人体器官生理学。由于这些障碍，建立一个研究人体器官发育和功能的实验平台，利用从患有遗传疾病的个体产生的iPS细胞，将代表着具有深远影响的变革性进步。我们最近开发了一种新的人类肝脏再生小鼠模型，以及将人类干细胞祖细胞分化为肝细胞的新方法，该方法可以有效地在该模型中重建人类肝脏。这些高度创新的方法将结合起来，创建第一个基于人类干细胞的体内模型，用于治疗导致肝脏病理的两种人类遗传疾病：Alagille综合征（ALGS）和Alpers综合征。如果成功，该平台可用于创建许多其他人类遗传疾病的体内模型。我们还将改进最近开发的基因工程方法，以有效地在人类干细胞中引入（或恢复）致病突变，并将表征改变的等位基因（相对于等基因细胞）对体内人类肝脏疾病相关表型的影响。这些疾病的病理生物学不能使用细胞或小鼠基因敲除模型进行适当的分析。例如，ALGS模型将用于回答关于关键信号通路（Notch）在人体实体器官（肝脏）发育过程中所起作用的基本问题，并描述由患者中看似相似的遗传改变引起的高度可变疾病表现的机制。除此之外，这些基于干细胞的模型还有其他变革性的应用。由于许多药物意外地在人类中引起严重的线粒体毒性，我们目前缺乏预测性筛选方法来识别它们，我们将确定Alpers综合征模型（或使用具有致病突变的iPS细胞的体外版本）是否可以用于预测人类药物诱导的线粒体毒性。

DESCRIPTION (provided by applicant): Over the last two decades, the genetic basis for many human genetic diseases has been elucidated, and the ability to generate iPS cells from any individual with a genetic disease has made it possible to investigate the impact that genetic changes have on cell function in vitro. However, since the primary objectives of regenerative medicine are to repair and to replace human tissues, it is of critical importance to characterize genetic factors affecting organ development and function. Furthermore, in many situations, animal models, including genetically engineered mice, do not accurately reflect human organ physiology. Because of these roadblocks, the creation of an experimental platform for studying human organ development and function, which utilizes iPS cells generated from individuals with genetic disease, would represent a transformative advance with far reaching impact. We have recently developed a novel murine model for human liver regeneration, and novel methodology for differentiating human stem-progenitor cells into hepatic cells that can efficiently reconstitut human liver in this model. These highly innovative methods will be coupled to create the first human stem cell-based in vivo models for two human genetic diseases that cause liver pathology: Alagille syndrome (ALGS) and Alpers syndrome. If successful, this platform could be used to create in vivo models for many other human genetic diseases. We will also improve upon recently developed genetic engineering methodology to efficiently introduce (or revert) disease-causing mutations in human stem cells, and will characterize the effect of the altered allele (relative to isogenic cells) on human liver disease-related phenotypes in vivo. The pathobiology for each of these diseases cannot be properly analyzed using cellular or murine genetic knockout models. For example, the ALGS model will be used to answer fundamental questions about the role that a key signaling pathway (Notch) plays in the development of a solid human organ (liver), and to characterize the mechanisms underlying the highly variable disease manifestations that arise from seemingly similar genetic alterations in patients. Beyond this, these stem cell-based models have other transformative applications. Since many drugs have unexpectedly caused severe mitochondrial toxicity in humans, and we currently lack predictive screening methods to identify them, we will determine if the Alpers syndrome model (or an in vitro version using the iPS cells with the causative mutations) can be used to predict drug-induced mitochondrial toxicity in humans.