Background & Aims: Although expandable hepatic progenitors provide renewable cell sources for treatment of hepatic disorders, long-term cultivation of hepatic progenitors may affect proliferation and differentiation abilities, and even initiate the formation of malignant cancer stem cells. This study aims to determine characteristics of primary cultured hepatic oval cells after prolonged cultivation in vitro. Methods: Hepatic oval cells isolated from rats fed with a choline-deficient, ethionine-supplemented diet were continuously propagated every 5-7 days, to 100 passages over two years. Hepatocytic differentiation was induced by sodium butyrate and characterized using western blot, periodic acid Schiff assays, albumin secretion and urea production. Proliferation capacity was, evaluated using growth-curve and cell-cycle analysis; anchorage-independent growth and tumorigenicity were determined using soft agar and xenograft assay. Results: After 2 years of serial passages, hepatic oval cells with typical epithelial morphology continuously expressed OV-6, BD-1, BD-2, and Dlk as markers for hepatic progenitors, cytokeratin 19 as a cholangiocyte marker, and alpha-fetoprotein and albumin as hepatocyte markers. Furthermore, sodium butyrate could induce these cells to become glycogen-storage cells with the functions of albumin secretion and ureagenesis from ammonia clearance, indicating hepatocytic differentiation. Although proliferation slightly accelerated after the 50th passage, hepatic oval cells stayed diploid cells with features of chromosomal stability, which did not acquire anchorage-independent growth capacity and caused no tumor in immunodeficient mice, suggesting no spontaneous malignant transformation. Conclusions: Hepatic oval cells retain the progenitor cell features without spontaneous malignant transformation after prolonged cultivation, and thus may serve as an expandable cell source for future exploitation of stem cell technology. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.