Liver cell transplantation could potentially be used to treat liver failure and liver-based metabolic diseases. Unfortunately, the number of human livers available for hepatocyte isolation and transplantation is limited by competition for use in whole organ transplantation. A potential alternative to the transplantation of primary hepatocytes would be to use a clonal cell line, which would provide the advantages of availability, uniformity and sterility. Such cells could be grown in unlimited number and at far less cost compared to isolated primary hepatocytes. We hypothesize that human hepatocytes can be conditionally immortalized and engineered to be safe for transplantation in the treatment of liver diseases. Rat hepatocyte cell lines have been generated that are capable of reversible immortalization by site-specific excision of an introduced SV40 T antigen using the loxP/Cre system. Such reversibly immortalized hepatocytes can correct liver function in animal models of acute and chronic liver failure and liver-based metabolic diseases. This proposal will assess whether reversibly immortalized human hepatocytes can be generated that can successfully integrate into the liver parenchyma of Rag2-/- mice following transplantation through the portal circulation. Studies will also examine whether reversibly immortalized human hepatocytes can function to correct the physiologic abnormalities associated with chronic liver decompensation in cirrhotic rats. In order to avoid the need for viral gene transfer technologies, we will also determine whether loxP/Cre generated reversibly immortalized human hepatocytes can be induced to undergo recombination using a TAT-Cre fusion protein. Finally, this proposal will evaluate the tumorigenic potential of loxP/Cre generated human hepatocytes. Evidence of alterations in tumor suppressor mechanisms will be assessed and transformation assays will be used to directly measure the tumorigenic potential of pre- and post-excision immortalized human hepatocytes.