Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.