There is increasing evidence that progression to cancer is restricted to a small subpopulation of cells within a tumor, the so called Cancer Stem Cells (CSC). The existence of CSC has been demonstrated in many solid tumors, including human hepatocellular cancer (HCC). So far, none of the putative CSC have been shown to possess identical functional or antigenic properties, indicating that the CSC population is heterogeneous and that the ultimate CSC still has to be identified. To characterize the CSC population, we studied the effect of epigenetic modulation on CSC defined by the functional side population (SP) approach. The HCC cell line Huh7 was treated with Zebularine (ZEB), an effective DNA-methyltransferase inhibitor. ZEB-treated and untreated Huh7 cells were then stained with Hoechst-33342-dye and analyzed by flow cytometry (FACS). The clonogenic and tumorigenic capacity of FACS-sorted SP, non-SP and parental cells was investigated in vitro and in vivo after transplantation into NOD/SCID mice, respectively. All isolated cell fractions were examined for differences in the expression of selected genes by qRT-PCR. In addition, antigenic characteristics were evaluated by both IHC and FACS. ZEB treatment significantly reduced the size of the SP fraction. Also, it resulted in an overall decreasein cell proliferation due to a block in G2/M progression. Moreover, ZEB treatment differentially changed the expression profile of selected genes in parental cells and FACS sorted cell fractions. Untreated cells showed a very uniform expression pattern across all examined cell populations. In striking difference, ZEB-treated parental and non-SP cells showed altered expression of selected genes, e.g., downregulation of ABCG2 and EpCAM, while the persisting SP cells remained unaffected. Furthermore, stemness- and CSC-associated genes, such as Nanog, Oct4, CD133 and Bmi1, were highly expressed only in the SP cells. Our results provide evidence for the heterogeneity within the CSC population. Epigenetic modification of HCC cells selects for a more homogeneous group of cells within the SP fraction with more conserved stemness and/or CSC properties. This approach may facilitate the purification and isolation of a unique CSC population. Further investigation will concentrate on genomic characterization of these cells and the validation of the results in different cancer cell lines. Cross-comparison of gene expression of human HCC samples, rat fetal hepatoblasts and mouse HCC revealed that individuals with HCC sharing a gene expression pattern with fetal hepatoblasts have a worse prognosis. The latter suggested that this HCC subtype may originate from hepatic progenitor cells. The objectives of this study were to develop constitutive lentiviral vectors (LV) carrying oncogenic Ras and/or Simian virus 40 large T antigen (SV40 LT) in combination with luciferase/EGFP double reporter gene as a tool to address the tumorigenic competence of different stages of hepatocyte differentiation. Lentiviral vector system was chosen for its high transduction efficiency and low cell toxicity for ex vivo transduction of primary cells. Hepatocytes were isolated from wild type and HNF4a conditional knockout mice to address more directly the role of hepatocyte differentiation in transformation. Fetal (E16), young (2 weeks) and adult (3 months) hepatocytes were infected ex vivo with lentiviral vectors, and the fate of genetically modified cells was followed by engraftment into NOD/SCID mice and a bioluminescence imaging. Lentiviral vectors encoding Ras and SV40 LT transduced both fetal and adult hepatocytes within the first 16-24 h of primary culture and persistently expressed both reporter genes. Intrasplenic injection of adult hepatocytes expressing either Ras or SV40 alone did not result in liver tumor formation. Introduction of Ras together with SV40 LT into both young and adult hepatocytes significantly accelerated the engraftment and hepatic tumor growth. Ongoing work includes analysis of biological phenotype and genotype of the resulting tumors.