This proposal represents a continuation of our efforts to investigate fundamental mechanisms regulating gene expression in normal and regenerating liver. During the past 5-10 years, the emphasis of our research has changed in part and our goals now are 1) to identify and/or clone genes involved in early events in proliferation/differentiation of liver nonparenchymal epithelial cells; 2) to determine the specific gene expression patterns in liver cell populations enriched for activated hepatocyte progenitor cells; 3) to culture or immortalize and culture nonparenchymal epithelial cells in the hepatocyte lineage; 4) to determine the growth properties, gene expression patterns and the differentiation program of immortalized hepatocyte progenitor cell lines; 5) to transplant putative progenitor cells or progenitor cell lines into a congenic strain of rats to obtain proliferation and differentiation of transplanted cells. These studies will use an inbred strain of rats, Fischer 344, in which we have a normal (wt) strain and a mutant strain deficient in a liver membrane surface enzyme, dipeptidyl peptidase IV (DPPIV-) which will be utilized to follow the success of progenitor cell transplantation into various host sites and proliferation of transplanted progenitor cells, if this occurs. Molecular, histochemical and immunohistochemical markers will be employed to identify cells of various lineages, particularly nonparenchymal epithelial cells which have the ability to differentiate into hepatocytes. These studies will take advantage of our recent observation that injection of the non-carcinogenic agent, D-galactosamine, into rats in a single large dose causes activation, proliferation and differentiation of progenitor cells into hepatocytes during the course of liver regeneration following injury with this agent. Our hypothesis is that activation of hepatocyte progenitor cells in vivo by D-galactosamine administration will provide us with large numbers of progenitor cells which can be isolated, cultured, genetically engineered and effectively transplanted into suitable recipient hosts. We also anticipate that these cells will continue to proliferate in recipient animals or that we will be able to identify conditions in recipient animals in which proliferation can be induced. These events will be monitored by molecular and histochemical methods which have been fully established in our laboratory. Our final hope is that these studies will point the way toward developing appropriate liver cell transplantation and/or somatic gene therapy methods for eventual use in man.
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