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Promotion of Growth and Differentiation of Rat Ductular Oval Cells in Primary Culture¹

Laval University Cancer Research Center, Hôtel-Dieu Hospital, and Department of Medicine, Laval University, Québec, G1R 2J6 Canada

Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to -fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45: 673–681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of M_r 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS₇) and normal hepatocytes (HES₆) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES₆ antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10⁷ cells, of which 95% expressed CK39, CK52, and BDS₇, 84% -glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [³H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 µM bilirubin, 10 ng/ml cholera toxin, 1 µM dexamethasone, 2.5 µg/ml insulin, 50 mM ß-mercaptoethanol, and 5 µg/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor. Cell morphology varied depending on the combinations of factors used. Most of the oval cells still expressed the three cytokeratins, BDS₇, -glutamyl transpeptidase, and some vimentin after 5 days in culture. Culture in medium MX in the absence of sodium butyrate resulted in the emergence of a small subpopulation of BDS₇ negative/-glutamyl transpeptidase positive fusiform cells, which by day 5 constituted a distinct epithelial cell population that expressed CK39 and vimentin and which could be subcultured. The addition of dimethyl sulfoxide or sodium butyrate at day 1 in the presence of dexamethasone differentially modified the phenotype of the ductular oval cells cultured in growth factor-supplemented medium. Of major interest was the finding that the presence of sodium butyrate and dexamethasone led to a massive production of albumin over a 3-day period and a progressive induction of tyrosine aminotransferase activity. Fourteen days after the injection of freshly isolated oval cells into the fat pads of syngeneic rats, the cells were arranged as clusters similar to bile ductular structures. These findings indicate that oval cells isolated from 3-methyl-4-dimethylaminoazobenzene-treated rat liver which exhibit the phenotypic characteristics of bile ductular epithelial cells after in vivo transfer, produce albumin, cease AFP production, and express tyrosine aminotransferase activity in primary mass culture, properties of facultative hepatocytic precursors.

¹ Supported by the National Cancer Institute of Canada. Presented in abstract form at the 77th annual meeting of the American Association for Cancer Research, May 7–10, 1986, Los Angeles, CA.

² Recipient of a studentship from the Medical Research Council of Canada and Fonds de la Recherche en Santé du Québec.

³ To whom requests for reprints should be addressed, at Laval University Cancer Research Center, Hôtel-Dieu de Québec Hospital, 11 Côte du Palais, Québec, G1R 2J6, Canada.

Received 12/29/86. Revised 6/29/87. Revised 10/15/87. Accepted 10/19/87.

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