A NOVEL PHENOTYPE OF LIVER STEM CELLS USING CD133 EXPRESSION

Controversies exist over the phenotype of oval cells (OCs), liver stem cells, and their role in liver regeneration. The goal of this study is to establish the phenotype of OCs. This study compares the proliferation of OCs using two methods of liver damage: 3,5-diethoxycarbonyl-1-4-dihyrdo-collidine (DDC) and 2-acetylaminofluorene with carbon tetrachloride (AAF/CCL4). C57/B6 and immune deficient β2Mnull NOD/SCID mice were fed a DDC 0.1% diet or gavaged AAF with an injection of CCL4 and studied at defined intervals to determine the time course of injury. H&E staining, immunohistochemistry (IHC) co-staining for cytokeratin and BRDU, and IHC for A6, an OC marker all demonstrated proliferation of OCs in DDC damaged livers. These studies indicate that DDC injury is a useful model for future study of OCs. Cytospin of liver non-parenchymal (NP) cells, non-hepatocytes, reveals cells that express A6, c-Met (the Hepatocyte Growth Factor receptor) and αFP (a marker of liver regeneration). Flow cytometry (FACS) analysis determined the percentage of each OC marker as a proportion of CD45- NP cells in DDC damaged livers. (CD45 is a marker of mature hematopoietic cells). CD45- NP (non-hematopoietic) cells expressing CD34 and Thy-1, previously described OC markers, were significantly increased. In addition, CD133+ cells, a marker not previously associated with OCs, were significantly increased in the CD45- NP fraction. CD45+ and CD45- cells expressing these markers were isolated from NP cells for RT-PCR analysis. CD133 + CD45- cells have strong RNA expression of albumin, c-Met, αFP, and HNF4α (a transcription factor of early liver development). The other fractions of CD45- cells, which express Thy-1 or CD34, did not share the same profile of liver gene expression. CD45+ hematopoietic cells do not express liver genes like albumin or HNF4α. Using H&E, IHC and FACS, a comparison of two methods of liver damage demonstrates that DDC is the most efficient method to induce OC proliferation. These studies indicate that murine OCs are within the CD45- fraction of liver NP cells that express the novel marker, CD133.