Ethanol effects on primate embryonic stem cells

描述(由申请人提供)：原位肝移植(OLT)治疗肝病具有相当高的发病率和死亡率。此外，由于器官短缺，每年有数千人在没有移植的情况下死亡。因此，更安全、更方便的替代疗法将使许多需要肝移植的人受益。一种可能解决这一问题的方法是开发一种能够表达肝脏特异性基因的增殖性细胞系，这种基因可以用于细胞移植或生物人工肝。从猴子胚胎干细胞(ESC)开发这样的株系将提供一个对药理学和毒理学研究有价值的细胞系，并建立一种可以用于人类细胞的方法。具体目标：1)建立和鉴定恒河猴来源的干细胞；2)确定定向分化为肝细胞系的条件，并评估乙醇对分化过程的影响；3)阐明乙醇对干细胞来源的细胞在肝脏重新填充能力的影响；4)描述细胞在体内肝损伤模型中的治疗作用；以及5)评估基因转染方案在体外和体内系统治疗乙醇诱导的肝损伤中的有效性。方法：初步实验将包括开发和 胚胎干细胞的特征及其向肝细胞系的分化。将开发单细胞克隆，试图建立具有高水平肝脏特异性功能的统一系。乙醇对ESC来源的细胞的影响将通过在体外系统中监测肝脏特异性功能、生长曲线和致癌潜力来确定。乙醇对细胞植入能力、增殖能力和体内功能的影响也将被评估。将使用脂质体或病毒载体将细胞外超氧化物歧化酶或过氧化氢酶运送到ESC来源的细胞，以试图抑制乙醇诱导的损伤。与健康相关：如果研究成功，它将为发展可用于毒理学和药理学研究的无限来源的分化灵长类肝细胞提供基础，并为建立类似的人类细胞系提供基础，然后可用于人体肝细胞移植研究。

DESCRIPTION (provided by applicant): Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from monkey embryonic stem cells (ESC) would provide a cell line valuable for pharmacology and toxicology studies, as well as establishing an approach that could be employed in human cells. Specific Aims: 1) to develop and characterize ESC derived from rhesus monkeys; 2) to determine conditions for directing the cells into a hepatocyte lineage, and to assess the effects of ethanol administration on the differentiation process; 3) to elucidate the effects of ethanol on the ESC-derived cells' ability to repopulate the liver; 4) to delineate the therapeutic utility of the cells in an in vivo model of liver injury; and 5) to assess the effectiveness of gene transfection protocols in treating liver injury induced by ethanol in in vitro and in vivo systems. Methods: Initial experiments will include the development and characterization of ESC and their differentiation towards a hepatocyte lineage. Single cell clones will be developed in an attempt to establish uniform lines with high levels of liver-specific function. The effects of ethanol will be determined on the ESC-derived cells by monitoring liver-specific function, growth curves, and oncogenic potential in in vitro systems. The effects of ethanol on the ability of the cells to engraft, proliferate, and function in vivo will also be assessed. Liposomes or viral vectors will be employed to deliver extracellular superoxide dismutase or catalase to the ESC-derived cells in an attempt to inhibit ethanolinduced injury. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated primate hepatocytes that can be used for toxicology and pharmacology studies, and provide the basis for the establishment of similar lines from human cells which could then be employed in liver cell transplantation studies in man.