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Dominic Eberle, Thomas Kurth, Sandra Schubert, Denis Corbeil, Marius Ader; Photoreceptor Precursor Cell Transplantation: The Outer Segment In Focus. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2248.
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Retinitis pigmentosa is characterized by a significant progressive loss of photoreceptor cells with no effective treatment available up to date. Current studies are focussing on a cell transplantational approach to replace degenerated photoreceptors. The key feature of every photoreceptor, to have a functional outer segment (OS) with properly aligned disc membrane staples, has not been studied in detail following transplantation so far. In our study we show an ultra-structural analysis of OS integrity on the background of cell transplantational approaches.
As reporter animals we used knock-in human rhodopsin-GFP fusion construct mice (rhoGFP) and double transgenic mice composed of the rhoGFP mouse plus a chicken beta-actin promoter driven DsRed transgene. Transplanted cells were gained from postnatal day 4 reporter mouse retinas. Young photoreceptors were enriched prior to transplantation using CD73 antibody and magnetic associated cell sorting. Finally, donor cells were transplanted into the sub-retinal space of adult wild-type mice. Further analysis was done by immunohistochemical staining of 30µm vibratome sections and ultra-thin cryosections. For electron microscopy analysis a protein A gold staining was applied on labelled cryosections.
Donor photoreceptors were detectable due to their reporter protein expression (DsRed, GFP) in host tissue. The transplanted cells which integrated into the outer nuclear layer (ONL) frequently started to develop an OS with well aligned membrane disc staples. Surprisingly, a lot of cells remaining in the subretinal space were also successful in forming OSs with intact and organized disc membrane staples.
We could show that our method is very suitable for detailed examination of OSs of transplanted photoreceptor cells. Integrated cells develop OSs with native aligned disc staples. Surprisingly, cells which stay in the subretinal space develop OS, too. This leads to the conclusion that OS formation of transplanted photoreceptor precursor cells is independent of an integration of these cells into the ONL.
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