Fuchs' corneal endothelial cells transduced with the human papilloma virus E6/E7 oncogenes

We sought to develop corneal endothelial cell cultures with extended lifespan from corneas with Fuchs' dystrophy. Descemet's-endothelial cell explants from histology confirmed recipient corneas of two patients with Fuchs' dystrophy were cultured. After a small number of corneal endothelial cells with irregular, endothelial morphology migrated from the explants onto the culture plate, the cells were transduced with a disabled retrovirus (pLXSN16E6/E7) coding for the human papilloma virus type 16 transforming oncoproteins E6 and E7. Expression of E6/B7 mRNA in the cell cultures with extended lifespan was monitored by RT-PCR. In vitro labeled cellular protein patterns of the Fuchs' E6/E7 cell cultures with extended lifespan were compared with those from normal human corneal endothelial E6/E7 cell cultures with extended lifespan using two-dimensional gel electrophoresis. Two endothelial cell cultures with extended lifespan were derived from Fuchs' corneas. The morphology of the Fuchs' cells expressing E6 and E7 was similar to that of normal corneal endothelial cells expressing E6 and E7. Surprisingly, the rate of proliferation of Fuchs'-derived cells was similar to that of normal endothelial cells transduced with E6/E7. Proliferation of each Fuchs' cell culture with extended lifespan continued for over 30 population doublings. There were limited quantitative differences in the two- dimensional gel electrophoretic protein patterns of the Fuchs'-derived and normal endothelial cell cultures with extended lifespan, respectively. Retroviral integration is dependent on cell proliferation. Thus, cells that migrated from the Fuchs' Descemet's explants were undergoing at least limited in vitro proliferation when the retroviral vector coding for E6/E7 integrated. Fuchs' corneal endothelial cells expressing E6 and E7 had similar proliferation, cellular morphology, and two-dimensional gel protein electrophoretic patterns to normal corneal endothelial cells expressing the same oncoproteins.