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S.-Z. Wang, W. Ma, R.-T. Yan; Exploring the Rpe as a Convenient Source of Developing Photoreceptor Cells - Direct Reprogramming in the Eye. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3060.
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A strategy to produce developing photoreceptors bears clinical implications, as it directly impacts cell-replacement studies. This study tests the hypothesis that developing photoreceptor cells can be produced in vivo, de novo from the RPE through direct reprogramming with a specific proneural gene.
To identify molecules that elicit the RPE to give rise to developing photoreceptor cells, we first used dissociated RPE cell cultures to assay 5 growth factors (added to culture medium) and 22 regulatory genes (retrovirally transduced) known or implicated to play important roles in the formation/wellbeing of the eye, the retina, or photoreceptors. The growth factors included BNDF, CNTF, bFGF, GDNF, and NGF. The regulatory genes include homeodomain genes cbx, chx10, crx, msx1, msx2, pax6, rax, raxL, six3, and six9, and proneural bHLH genes ash1, Xash3, ash4, ash6, ath3, ath5, NSCL1, NSCL2, neuroD, ngn1, ngn2, and ngn3. Genes displaying activities were then selected for in vivo studies, in which the developing eye (including the RPE) was infected with RCAS expressing each individual gene, followed by immunostaining for photoreceptor marker visinin, a chick equivalent of mammalian recoverin.
None of the 5 growth factors and none of the 10 homeodomain genes induced the expression of visinin. Among the 12 bHLH genes, 6 showed activities of inducing de novo genesis of photoreceptor-like cells from the otherwise non-neural, RPE cell cultures, with an order of their induction activities of ngn1 > ngn3 > ngn2 > neuroD > ath5 ≈ ash1. Among those 6 "competent" genes, only ngn3 was able to induce the RPE in the eye to expression visinin. In normal chick eye and in the control eye infected with RCAS-GFP, visinin+ cells were absent from the RPE and were confined within the neural retina at the location where the outer nuclear layer (ONL) would form. In eyes infected with RCAS-ngn3, visinin+ cells were present in both the RPE and the prospective ONL. Visinin+ cells in the RPE were unequivocally shown in regions where the RPE and the retina were separated (or detached). We estimated that ~35% of the cells in the RPE were visinin+. Morphologically, some of the visinin+ cells appeared more similar to neurons than to RPE. Some retained black pigmentation typical of RPE cells.
These results show that it is possible to reprogram RPE in the eye to give rise to developing photoreceptor cells, and thus the RPE may be explored as a convenient source of developing photoreceptors for replacement study.
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