A considerable body of indirect evidence has been generated that supports the existence of hepatic stem-like cells capable of differentiation along hepatocytic and biliary lineages and progression during carcinogenesis to hepatocellular carcinoma. Direct proof of transplantation of stem-like cells isolated according to their expression of stage specific cell surface phenotypes, the approach used for hematopoietic and neural stem cells, will require antibodies against lineage related cell surface antigens. We have identified a panel of 7 monoclonal antibodies against surface antigens that identify 5 distinct stages in liver development.
In Aim 1, we will continue ongoing efforts to clone cDNAs for these antigens using the following three strategies: PCR or RT-PCR using a cDNA library or total RNA from prostate or bile duct epithelial cell lines as template and degenerate primers synthesized according to primary amino acid sequence obtained from mass spectrometry or N-terminal sequence analysis of tryptic peptides; screening plaque lifts from a lambda phage cDNA library with cDNA probes prepared by subtractive PCR or differential display PCR; expression cloning using eukaryotic cDNA expression libraries prepared from poly A RNA isolated from bile duct epithelial or prostate epithelial cell lines.
In Aim 2, we will assess the role of MAB defined antigens in bile duct morphogenesis by examining the effects of MAb, PAbs or ScFvs (single chain antibodies) on the formation of duct-like structures in vitro. We will determine the effects of sense and antisense expression vectors for cDNAs cloned in Aim 1 on ductal morphogenesis in cultured and transplanted bile duct epithelial cells(BDEC). The methods proposed for inducing ductal morphogenesis in vitro have been used successfully by several investigators and functional inhibition with antibodies is a widely used, straight forward strategy.
In Aim 3, we will explore the role of OV6+ periportal hepatocytes(PPH) in liver carcinogenesis. We will determine whether these OV6+ PPH are progenitors of OV6+, GGT+ foci generated by the Solt/Farber carcinogenesis protocol, by tracing the fate of these cells in situ during the early stages of the Solt/farber regimen and after transplantation into DENA/AAF or retrorsine treated rats. All of the proposed studies under Aim 3, with the exception of the immunoisolation protocol with MAB OV6, are based on established protocols. These studies will provide new insights into the role of hepatic progenitor cells in liver development, renewal and carcinogenesis.
