Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

Abstract

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (M_r) 55 000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of M_r 52 000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-M_r 55 000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and para-formaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types. By extending the use of this cell typing assay to three established rat liver cell lines of epithelioid morphology (RLEC, MLC, and T51B), we found that all of them reacted with the anti-cytokeratin antibodies, showing that they were indeed of epithelial cell origin. Moreover, these cells were stained with the anti-vimentin antibodies, which yielded differential reactivity patterns equivalent to those of epithelial cells from either the bile ductular structure or the Glisson capsula. Interestingly, the analysis with the antibodies against the exposed surface components revealed a reaction of the anti-ductular oval cell antibody on T51B cells, but not on the other cell lines. This strongly suggests that the T51B epithelial cell type is of bile ductular origin, while the latter cell lines, which exhibit a mesothelial-like phenotype in culture, most likely originate from the Glisson capsula.