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J. Greenwood, A.A. Vugler, E. Miljan, S. Hassan, M. Patel, S. Hines, J. Sinden, S.E. Moss; Transplantation of Human Foetal Retinal Cells Into the Developing and Degenerating Rat Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5760.
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© ARVO (1962-2015); The Authors (2016-present)
To characterise human foetal retinal progenitor cells in vitro and to asses post–graft survival following transplantation into the developing or degenerating retina.
Primary retinal cells were harvested from 10 and 12 week gestation human foetal eyes. Cells were expanded as a monolayer on fibronectin coated dishes in defined neural medium supplemented with bFGF, EGF and 5% FCS. These conditions supported successful expansion of cells to passage 10 at which time they were characterised using immunocytochemistry for various retinal cell markers. Passage 10 cells were also transplanted into the vitreous space of postnatal day 3 rats (n=12) and into both the vitreous and subretinal space of 8 week old RCS dystrophic rats (n=8). Host animals were immune–suppressed prior to and following grafting. Anatomical assessment of graft survival up to 26 days post–graft was carried out using immunohistochemistry and confocal microscopy.
At passage 10 the majority of cells were nestin positive with evidence of continued cell division. Approximately 10% of cells were found to be positive for the photoreceptor markers recoverin, s–opsin and ML–opsin whereas approximately 30% expressed the ganglion cell markers neurofilament 160, 200, ßIII tubulin and melanopsin. A few cells (<2%) were positive for the RPE marker keratin 8 which, on occasions, co–expressed melanopsin. All cells were negative for rhodopsin and GFAP. When grafted into neonatal and RCS dystrophic animals the cells survived up to 26 days post–graft. In neonates, grafted cells were observed in the vitreous and attached to the lens. In RCS dystrophic rats, no human cells were observed in the subretinal space irrespective of the site of grafting. Surviving cells at 26 days post–graft were seen clumped together on the inner limiting membrane. Preservation of the RCS host retinal photoreceptors was substantial in the vicinity of the graft. Grafted human cells were positive for nestin, but largely negative for Ki67 and retinal specific markers. Grafted cells were intimately associated with host ED1 positive macrophages/microglia.
Human foetal retinal cells can be cultured and expanded for extended periods with a proportion exhibiting a phenotypic profile consistent with ganglion cells and cone photoreceptors. Cells surviving transplantation failed to integrate into the host retina and remained largely nestin positive indicating a lack of differentiation.
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