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L. Liang, H. Zhang, S.–Z. Wang; Exploring RPE as a Source of Photoreceptors: Assessing Cellular Differentiation in vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5541.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate neuroD–induced RPE transdifferentiation into photoreceptor cells using the subretinal space as a "natural" incubator.
RPE cells were dissociated, cultured, and guided to transdifferentiate by infecting them with retrovirus RCAS–neuroD, or RCAS–GFP as a control. The cells were then harvested and microinjected into the subretinal space of day 6–7 chick embryos. In addition to those from Spafas (Charles River Laboratories, CT), embryos of line 72 were also recipients. Line 72 chickens lack cellular receptors for RCAS(A) virus, and thus viral spreading into host cells cannot occur. This made it possible to unambiguously identify the injected cells by the presence of viral protein p27. Experimental eyes were fixed and analyzed for the development and differentiation of injected cells with molecular markers and morphological criteria typical of photoreceptors.
We found that injected cells from the control RPE culture (infected with RCAS–GFP) integrated into the host RPE tissue when present in a small number. At places where a large number of injected cells were present, multi–layers of RPE–like tissues were formed. The RPE–like tissues comprised of injected and host cells. None of the cells from the control culture expressed visinin or other photoreceptor–specific genes. Compared with the control, fewer cells from the RPE cultures infected with RCAS–neuroD partook in the formation of multi–layered, RPE–like structures. Most of the RCAS–neuroD infected cells remained un–pigmented. A large number of them were Visinin+, and the Visinin+ cells aligned along the newly formed RPE–like structure. Injected cells also expressed red opsin, rhodopsin, RXRγ, or synaptic protein PSD–95. A small number of cells displayed outer segment–like structures decorated by antibodies to red opsin.
Our results indicate that those transdifferentiating cells were intrinsically capable of developing a highly ordered structure. Thus, RPE could potentially serve as a source of photoreceptors cells when guided by an appropriate gene. Our data also showed that cultured cells could organize into a structure resembling the native RPE tissue.
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