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.
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