Cellular Engineering of Hepatocyte Cell Lines

描述（由申请人提供）：目前，肝衰竭唯一确定的治疗方法是肝移植。克服肝移植技术困难和风险的一种方法是移植分离的肝细胞。然而，目前尚不清楚，如何获得足够数量的人肝细胞，因为可供使用的人肝供应严重有限。鉴于供体肝脏和可用于治疗急性和慢性肝衰竭的人肝细胞的短缺，我们的实验室已经探索了胚胎干细胞（ES）作为供体肝细胞的潜在替代来源。胚胎干细胞具有多能性，可以作为具有正常核型的未分化细胞无限繁殖，可以在体外诱导分化为各种细胞类型，并且在适当条件下能够在体内完全发育为胎儿和成人。在初步研究中，我们已经分化出具有代谢活性和体外合成肝功能的小鼠胚胎干细胞，与原代肝细胞接近，但尚未评估其通过移植纠正肝功能缺陷的全部能力。我们还分化了人类胚胎干细胞，使其表达肝脏特异性基因，分泌白蛋白，并具有与肝功能一致的代谢活性，但与我们在小鼠胚胎干细胞上完成的程度不同。本研究的具体目的是：1)验证es源性肝细胞在肝功能障碍动物移植后能够成熟为功能齐全的肝实质细胞的假设；2)测试成功将小鼠胚胎干细胞分化为肝细胞表型的方案是否对人类胚胎干细胞分化有效，后者将响应可溶性和局部基质相关的肝脏和胚胎信号进行肝脏分化；3)测试体外分化和谱系特异性分离mES-和he -来源的肝细胞是否会降低其畸胎瘤发展的可能性，从而使其更安全地用于临床。ES细胞来源的人肝细胞可用于免疫功能不全小鼠的肝脏重建，为人类肝炎病毒感染和人类药物代谢的研究提供便利，并可用于开发生物人工体外肝支持装置。

DESCRIPTION (provided by applicant): At this time, the only definitive treatment of hepatic failure is transplantation of the liver. One way to overcome the technical difficulties and risks of liver transplantation is to transplant isolated hepatocytes. It is not clear, however, how an adequate number of human hepatocytes might be obtained given the severely limited supply of human donor livers available. Given the shortage of donor livers and, by extension, human hepatocytes available to treat acute and chronic liver failure, our laboratory has explored embryonic stem (ES) cells as a potential replacement source of donor hepatocytes. Embryonic stem cells are pluripotent, can be propagated indefinitely as undifferentiated cells with a normal karyotype, can be induced to differentiate in vitro into various cell types, and are capable, under appropriate conditions, of complete fetal and adult development in vivo. In Preliminary Studies, we have differentiated mouse ES cells with metabolic activity and in vitro synthetic liver function approximating that of primary hepatocytes, but have not assessed their full capacity to correct deficiencies of liver function by transplantation. We have also differentiated human ES cells to express liver-specific genes, secrete albumin, and have metabolic activity consistent with liver function, but not to the same degree as we have accomplished with mouse ES cells. The specific aims of this proposal are: 1) to test the hypothesis that ES-derived hepatocytes can mature into fully functional liver parenchymal cells upon transplantation in animals with liver dysfunction; 2) to test whether protocols successfully used to differentiate mouse ES cells toward a hepatocyte phenotype will be effective in differentiation of human ES cells, which will respond to soluble and local matrix-related hepatic and embryonic signals to undergo hepatic differentiation; 3) to test whether in vitro differentiation and lineage- specific isolation of mES- and hES- derived hepatocytes will reduce their potential for teratoma development, and thus make them safer for clinical use. ES cell-derived human hepatocytes could be used to repopulate the livers of immune incompetent mice, facilitating the study of human hepatitis virus infection and human drug metabolism, and to develop bio-artificial extracorporeal liver support devices.