This work is based on the hypothesis that the fetal liver contains a subset of hepatic stem/progenitor cells that have not reached terminal differentiation and arrest in the adult quiescent liver as dormant/slow cycling, unidentifiable stem cells or partially differentiated progenitor cells. Under certain conditions, when the liver parenchyma is extensively damaged, liver stem/progenitor cells can be activated by yet unknown mechanisms to proliferate and differentiate into mature hepatocytes and biliary epithelial cells and repopulate the liver or, alternatively, undergo malignant transformation. Because of the biological and pathobiological significance and applicability of liver stem/progenitor cells, our long-term objectives are: (i) identification and isolation of these cells from fetal and adult liver, (ii) maintenance/propagation of liver stem/progenitor cells in culture, and (iii) elucidation of molecular mechanisms leading to their activation, proliferation and differentiation and the genes involved in these processes. To achieve these goals, modern methods of gene analysis, suppression-subtractive hybridization (SSH) and cDNA microarray technology, are being utilized. Five fetal liver epithelial cell specific clones have been identified with the SSH technology and will be analyzed further. Markers for identification and isolation of liver stem and progenitor cells will be identified and isolated using cDNA microarrays. Gene expression profiles of differentiating fetal liver epithelial cells in vivo will be studied and the clones obtained sorted into functional groups and clusters. Those implicated in epithelial cell growth, proliferation and differentiation will be subjected to functional analysis. The cell specificity of the identified markers and their applicability for isolation of the target cells will be tested employing in situ technique of analysis of gene expression in the individual cells. The biological function of different growth factors and the ECM protein tenascin C (for which induced expression in ED13 fetal liver was observed), on maintenance, proliferation and differentiation of fetal liver epithelial cells in culture will be studied. Upon completion of the experiments on the gene expression profile of liver stem cells, a molecular model of activation, proliferation and differentiation of these cells will be drawn. The liver stem and progenitor cell specific markers identified will enable the isolation of these cells and with the knowledge of the specific growth factors/cytokines and extracellular matrix proteins necessary for their growth and differentiation, their propagation in vitro.
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