Ethanol effects on primate embryonic stem cells

描述（由申请人提供）：肝脏疾病的原位肝移植（OLT）治疗具有相当高的发病率和死亡率。此外，由于器官短缺，每年有成千上万的人死于器官移植。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。一种可能解决这个问题的方法是开发一种表达肝脏特异性基因的增殖细胞系，这种细胞系可以用于细胞移植或生物人工肝。从猴子胚胎干细胞（ESC）中培育出这样的细胞系，将为药理学和毒理学研究提供有价值的细胞系，并建立一种可用于人类细胞的方法。具体目的：1)开发和鉴定恒河猴造血干细胞；2)确定引导细胞分化为肝细胞谱系的条件，并评估乙醇给药对分化过程的影响；3)阐明乙醇对esc来源细胞在肝脏再生能力的影响；4)描述细胞在体内肝损伤模型中的治疗效用；5)评估基因转染方案在体外和体内治疗乙醇肝损伤的有效性。方法：初步实验将包括发展和

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.