Reprogramming Progeny Cells of Embryonic RPE to Produce Photoreceptors: Development of Advanced Photoreceptor Traits under the Induction of neuroD

Photoreceptors are specialized sensory neurons in the retina. Their degeneration is a common cause of blindness from light damage, genetic changes, and aging.1,2 Because they are terminally differentiated and do not reenter the cell cycle for regeneration, photoreceptors that are lost cannot be replenished. The importance of photoreceptors to vision has spurred a spectrum of investigations ranging from photoreceptor rescue by trophic factors,3 supporting cells,4,5 or stem cells6 to photoreceptor replacement through retinal regeneration7–9 and transplantation.10,11 The recent demonstration of successful photoreceptor transplantation in blind mice12 offers hope for vision rescue or restoration through cell replacement. It also heightens the importance of finding a source of photoreceptors, especially developing photoreceptors.13 One approach is to induce photoreceptor genesis through programming or reprogramming the differentiation of cells that can be propagated in large amounts. In recent years, brain, retinal, bone marrow, and embryonic stem cells have been explored in attempts to produce photoreceptors.14–20 Although these stem cells continue to be at center stage, there appears to be a need for alternative, perhaps provocative, sources of developing photoreceptors for cell-replacement studies.21 Unlike retinal neurons, the retinal pigment epithelial (RPE) cells from many species, including human, can reenter the cell cycle. More important, their progenies are able to differentiate into cell types other than RPE,22 raising a question of whether the RPE could be a source of retinal stem cells.23 Making use of RPE cells’ plasticity, we explored the possibility of using a regulatory gene to coax cultured RPE cells into differentiating along the photoreceptor pathway. NeuroD, which encodes a transcriptional factor belonging to the basic helix-loop-helix family, was tested, because of its instrumental role in photoreceptor differentiation and survival.24–27 Ectopic expression of neuroD in RPE cell cultures derived from day-6 chick embryos (E6) leads to de novo appearance of cells expressing photoreceptor-specific genes, including visinin, IRBP, α-PDE, opsins, and RaxL,24,25,28 a homeodomain gene essential for initiating photoreceptor differentiation.29 When grafted into the subretinal space of embryonic chick eyes, those photoreceptor-like cells migrate into the photoreceptor layer of the retina and emanate axonal arborization into the outer plexiform layer.30 Critical to the prospective functionality of such reprogrammed cells is their ability to develop structural features and functional properties characteristic of highly specialized photoreceptors. We report that reprogrammed cells developed ultrastructural features typical of developing photoreceptors. They responded to light by decreasing their cellular free-calcium (Ca2+) levels. On exposure of light-bleached cells to 9-cis-retinal, their Ca2+ levels were elevated, a reaction reminiscent of the Ca2+ increase on visual cycle recovery. These results provide support to the prospect of guiding RPE progeny cells to differentiate into photoreceptors.