Immortalization of human fetal sinusoidal liver cells by polyoma virus large T antigen

Fetal sinusoidal liver cells were isolated from human liver explant cultures and transfected with pCMVLT, a plasmid containing the immediate early promotor of cytomegalovirus (CMV) and the large tumor antigen (LT) coding part of the polyoma virus (py) genome. Whereas nontransfected cells stopped proliferating after 4 weeks, the transfected sinusoidal cells were stimulated to divide more quickly without changes in their morphology. Up to now, cells have been permanently cultured for more than 18 months and passaged over 130 times, corresponding to around 400 generations. This allows them to be regarded as "immortalized" cells. The presence of LT protein in the cells has been documented by means of immunoprecipitation and immunofluorescence. Expression of the v. Willebrandt factor VIII was the main criterion for classifying the cell population as endothelial cells. The presence of cytokeratins 7, 8, and 18 in these cells underlines their close ontogenic and functional relationship to mesothelial cells. Sinusoidal endothelial cells (SECs) synthesize vimentin and the typical extracellular matrix components collagen IV and fibronectin, but are negative for laminin and entactin. We used immortalized SEC's in co-culture experiments with fresh fetal human hepatocytes and adult mouse hepatocytes. They promoted survival of both types of hepatocytes over a period of 8-10 weeks. Control human fetal liver explant culture cells survived for only 3-4 weeks, whereas control adult mouse liver cells retained vitality for 8-10 days only.