Modeling alcohol toxicity in human hepatocytes

项目摘要 在美国，酒精导致了近一半的肝脏相关死亡，其中大部分是肝硬化 和肝细胞癌。尽管大量的人滥用酒精，只有一小部分人 发展成肝病。更好地了解导致肝细胞损伤的机制 酒精是有道理的。 小鼠模型是进一步了解酒精对肝细胞作用的一种工具。 然而，部分原因是物种差异和人类遗传倾向无法建模， 在小鼠中，乙醇在小鼠中的研究具有有限的转化用途。另一种研究 酒精对人肝细胞的影响是肝嵌合小鼠。这些模型基于 将原代人肝细胞移植到具有肝损伤的免疫缺陷小鼠中， 人细胞增殖并重新填充小鼠肝实质。这些都有很多限制 模型，其中之一是只有非常健康的肝细胞移植。肝细胞来自 发炎的肝脏或肝硬化通常不能移植，这限制了我们创造疾病特异性的能力。 嵌合模型这个问题的可能解决方案将是遗传操纵原代肝细胞 或者用多能干细胞制造嵌合小鼠。然而，操纵原代人类细胞， 然而，这仍然具有挑战性，多能干细胞在很大程度上未能重建肝脏嵌合模型。 我们最近已经创建了有效地移植肝脏嵌合模型的方案， 多能干细胞或在培养物中遗传改变的原代细胞。 嵌合动物，其中大部分肝脏已被人源化。此外，我们还可以分离出大量的 从这些小鼠中获得大量人肝细胞，以产生原代人肝细胞培养物， 稳定数月。这些进展，结合新的类器官系统，为我们提供了独特的工具， 研究肝细胞对酒精的反应 我们在这里建议联合收割机这些进展来测试酒精对人类肝细胞的影响。 我们将使用原代人肝细胞和多能干细胞衍生的肝细胞来建立 肝细胞中的酒精毒性系统，并测试一种使人类易患 酒精性肝炎此外，新的类器官技术将使我们能够测试酒精对 肝细胞再生 在这些研究完成后，我们将建立起一个系统， 可以对人肝细胞的长期酒精毒性进行建模。这些系统将进一步推动 创建酒精性肝病的患者特异性嵌合模型，并可能促进干细胞的使用 用于患者的治疗用途的细胞疗法。

Project Summary Alcohol contributes to nearly half the liver related deaths in the United States, mostly from cirrhosis and hepatocellular cancer. Even though large numbers of people misuse alcohol, only a small fraction develops liver disease. A better understanding of the mechanisms that contribute to hepatocyte damage from alcohol is clearly warranted. Mouse models are one tool to further our understanding of the role of alcohol on hepatocytes. However, in part due to species differences and because human genetic predispositions cannot be modeled in mice, ethanol studies in mice have been of limited translational use. An alternative approach to study the effects of alcohol on human hepatocytes are liver chimeric mice. These models are based on the transplantation of primary human hepatocytes into immunodeficient mice with liver injury, after which the human cells proliferate and repopulate the mouse liver parenchyma. There are many limitations with these models, one of them being that only very healthy hepatocytes engraft. Hepatocytes from patients with inflamed livers or cirrhosis generally fail to engraft, which has limited our ability to create disease-specific chimeric models. Possible solutions to this problem would be to genetically manipulate primary hepatocytes or to create chimeric mice with pluripotent stem cells. However, manipulating primary human cells has remained challenging and pluripotent stem cells have largely failed to reconstitute liver chimeric models. We have recently created protocols to efficiently engraft a liver chimeric model with induced pluripotent stem cells or with primary cells that were genetically altered in culture These protocol leads to chimeric animals in which the majority of the liver has been humanized. In addition we can isolate large numbers of human hepatocytes from these mice to create primary human hepatocyte cultures that are stable for months. These advances, combined with a new organoid system, provides us with unique tools to study various hepatocyte responses to alcohol. We here propose to combine these advances to test the effects of alcohol on human hepatocytes. We will use both primary human hepatocytes and pluripotent stem cell-derived hepatocytes to establish systems of alcohol toxicity in hepatocytes, and test a genetic variant that predisposes humans to develop alcoholic hepatitis. In addition the new organoid technology will allow us to test the effects of alcohol on hepatocyte regeneration. At the completion of these studies we will have established systems with which the effects of long- term alcohol toxicity on human hepatocytes can be modeled. These systems will further advance the creation of patient-specific chimeric models for alcoholic liver disease, and may advance the use of stem cell therapies for therapeutic use in patients.