There is a distinct need to identify, isolate and characterize liver stem cells because of implications for: a) converting these dormant stem cells in the adult to commit to regenerate in vivo to replace lost cells and restore function; b)transplanting them into needy hosts; c)using these stem cells for gene therapy; and d)understanding the mechanisms of stem cell aging and the role of this aging in neoplastic conversion. Our research proposal, to isolate liver stem cells from pancreas, is based on a novel copper depletion model system, developed and optimized in our laboratory, which induces hepatocyte differentiation in the pancreas. This pancreatic hepatocyte model system offers several advantages for the search and isolation of liver stem cells over the hepatocyte oval cell system, the most important being that there are no pre-existing hepatocytes within the pancreas to confound the observation of lineage analysis. Two major hypotheses form the basis of studies proposed in this competing renewal application: 1) copper-deficiency induces oxidative damage in adult pancreatic acinar cells by reducing the levels of expression of antioxidant enzymes, and the resultant oxidant damage leads to apoptotic death of acinar cells; and 2) stem cells exist in the adult pancreas, but remain dormant in the normal adult organ, and such cells can be activated to proliferate due to activation of a """"""""stem cell stimulatory signal"""""""" and/or the removal or neutralization of a """"""""stem cell suppressor signal"""""""" provided by the global loss of acinar cells.
The specific aims i nclude: 1) dissection of molecular events leading to the neutralization of stem cell suppressor function in pancreas during copper depletion; 2) isolation of putative pancreatic liver stem cells from the pancreas of F-344 rats and SV40-T antigen expressing transgenic Sprague-Dawley rats maintained for up to 8 weeks on a copper deficient diet; 3) characterization of growth and cytodifferentiation potential of putative pancreatic stem cells in vivo and in vitro, where possible, by tagging these cells with indicator genes, such as Beta-galactosidase or green fluorescent protein; 4)identification of genes whose expression is up-regulated or down- regulated in the pancreas, during copper depletion-induced pancreatic acinar cell loss and putative stem cell proliferation, using the gene expression screen; and 5) explore the malignant potential of putative stem cells and cells derived from them in an attempt to confirm the cytodifferentiation lineage.
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