The research is focused on defining the cellular and molecular biology of the hepatic and pancreatic stem cell compartments in normal and neoplastic organs. We examined the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and urokinase-type plasminogen activator (uPA) by in vivo infusion on the proliferation of ductal and periductal cells following their activation with 2-acetylaminofluorene (2-AAF). Infusion of EGF, HGF, uPA or any combination thereof for up to 7 days resulted in increased numbers of 3H-thymidine-labeled ductal and periductal cells expanding into the liver acinus. Although the growth factors all increased the number of labeled cells, they preferentially acted on different cell populations. While exposure to 2-AAF alone or combined with infusion of HGF resulted in proliferation of almost equal numbers of ductal and Ito cells, infusion of EGF and any combination thereof resulted in 75-80% of labeled cells having a ductal phenotype. Also, infusion of EGF and HGF resulted in decreased numbers of cells undergoing apoptosis in response to 2-AAF. Our results demonstrate that while 2-AAF acts as a mitogenic stimulus for ductal and periductal cells, growth factors are necessary for survival, motility, and expansion of these cells into the liver acini. We have utilized 3H-thymidine labeling of newly synthesized DNA to examine the earliest effects of 2-AAF on the mitotic activation of cells in the adult rat liver, and in situ hybridization analysis to study the expression of three transcription factors (HNF1-beta, HNF3-gamma, and HNF4), and two of the genes (alpha-fetoprotein [AFP] and albumin) regulated by these factors. A low dose of 2-AAF (and its analogs, 2-AF and N-OH-2-AAF) elicited a mitogenic response in ductal cells and nondescript periductular cells within 24 hours following administration. The compounds also induced the expression of HNF1-beta, HNF3-gamma, AFP, and albumin in ductal structures but had no detectable effect of HNF expression. In contrast, initiation of bile duct proliferation by ligation of the common bile duct had no effect on the expression of these genes in ductal cells. Oval-type cells were isolated by novel methods employing magnetic affinity cell sorting (MACS) followed by analysis and isolation by a multiparametric fluorescence activated cell sorting system (FACS). Using oval cell specific antibody OV-1 and a c-kit antibody, the cell population from both normal and partial hepatectomized (PH) liver from rats were isolated and studied. These were analyzed for proliferative and differentiation markers such as cytokeratins, gamma-glutamyl transferase, AFP, and albumin. These cells and those isolated from PH liver were more clonogenic.
