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A zerbib, S Porte, G Puel, A David, M Neuner-Jehle, S Paisant, F Martin, A Degeorges, J Greenwood, J Crouzet; Cell Therapy: New Genetically "Immortalized" Human RPE Cell Lines . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4629.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Neurotech is developing cell-based therapies for the treatment of ophthalmic disorders with a special interest in retinal diseases. One approach consists in using immortalised human retinal pigment epithelium (RPE) cell lines for replacement therapies in humans. This approach could be used in AMD or in genetic diseases associated with RPE cell impairment. Methods: Primary RPE cells were isolated from a human donor eye and immortalised cell lines were obtained by stepwise transfection with two genes: first, the SV40 large T antigen gene, then the gene encoding the catalytic component of human telomerase (hTERT). Proliferative and phenotypic characteristics were analysed at different steps of the immortalisation process. Results: Expression of the SV40 large T antigen gene was necessary to bypass senescence, and the obtained cell line retained typical properties of primary RPE culturesa. However, for bypassing cell crisis and maintaining cell proliferation and morphology, transfection of the hTERT gene was necessary. Four populations were independently derived, they retain the large T expression and display telomerase activity. All of them exhibit a cobblestone-like morphology, express cytokeratin 8 and 18, and display a junctional distribution of ZO-1 as detected by immonufluorescence. Cell clones were isolated from these populations: they are contact inhibited, serum dependent and could proliferate 50 population doublings beyond the cell crisis undergone by the large T positive / HTERT negative parental cell line. Conclusion: Transfection with two immortalising genes (SV40 large T Antigen and hTERT) was necessary to immortalise this primary culture and maintain typical RPE properties. aKanuga et al. IOVS, in press.
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