Drs. Sell and Leffert propose to isolate and characterize liver stem cells. This application is based on a model of liver injury in which allyl alcohol is used to injure the periportal area of the liver. This treatment results in proliferation of a population of null periportal cells which the investigators suggest differentiate into a-fetoprotein (AFP)-producing transition hepatocytes and which replace the necrotic periportal tissue by differentiating into mature hepatocytes. The heart of this application is the investigators proposal to isolate and culture these putative stem cells by harvesting rat livers 3-4 days after allyl alcohol administration, at which time these periportal null cells are most abundant. They will also isolate proliferating hepatocytes, which are predominant 1-2 days after the toxic treatment. The two proliferating cell populations will be compared for growth and differentiation characteristics in vitro. Further, they will be transduced using a retroviral vector system with a series of proto-oncogenes and oncogenes, with emphasis on those genes that are often overexpressed in hepatocarcinogenesis and development (fos and jun), as well as those that are associated functionally with liver growth transitions (myc and H-ras). They will also transduce v-src, which has been implicated in the phenotypic commitment process and in gap junction communication. In the final aim, the investigators propose to study the fate and function of candidate stem cells after transplantation into syngeneic rats, to compare their fates and functions to the fate and function of transplanted proliferating hepatocytes. In this fashion these investigators expect to resolve the longstanding and ongoing debate regarding the existence and functionality of liver stem cells and to establish the basis for gene therapy with such cells.
