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Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD.

ABSTRACT: In examining the prospect of producing functional photoreceptors by reprogramming the differentiation of RPE progeny cells, this study was conducted to investigate whether reprogrammed cells can develop highly specialized ultrastructural and physiological traits that characterize retinal photoreceptors.Cultured chick RPE cells were reprogrammed to differentiate along the photoreceptor pathway by ectopic expression of neuroD. Cellular ultrastructure was examined with electron microscopy. Cellular physiology was studied by monitoring cellular free calcium (Ca(2+)) levels in dark-adapted cells in response to light and in light-bleached cells in response to 9-cis-retinal.Reprogrammed cells were found to localize red opsin protein appropriately to the apex. These cells developed inner segments rich in mitochondria, and while in culture, some formed rudimentary outer segments, analogous to those of developing photoreceptors in the retina. In response to light, reprogrammed cells reduced their Ca(2+) levels, as observed with developing retinal photoreceptors in culture. Further, on exposure to 9-cis-retinal, the light-bleached, reprogrammed cells increased their Ca(2+) levels, reminiscent of visual cycle recovery.These results indicate the potential of reprogrammed cells to develop advanced ultrastructural and physiological traits of photoreceptors and point to reprogramming progeny cells of embryonic RPE as a possible alternative in producing developing photoreceptors.

SUBMITTER: Liang L

PROVIDER: S-EPMC2570253 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD.

Liang Lina L Yan Run-Tao RT Li Xiumei X Chimento Melissa M Wang Shu-Zhen SZ

Investigative ophthalmology & visual science 20080509 9

<h4>Purpose</h4>In examining the prospect of producing functional photoreceptors by reprogramming the differentiation of RPE progeny cells, this study was conducted to investigate whether reprogrammed cells can develop highly specialized ultrastructural and physiological traits that characterize retinal photoreceptors.<h4>Methods</h4>Cultured chick RPE cells were reprogrammed to differentiate along the photoreceptor pathway by ectopic expression of neuroD. Cellular ultrastructure was examined wi ...[more]

PMID: 18469196

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Project description:Iron has been implicated in the pathogenesis of retinal degenerative diseases, including ocular siderosis. However, the mechanisms of iron-induced retinal toxicity are incompletely understood. Previous work shows that intravitreal injection of Fe2+ leads to photoreceptor (PR) oxidative stress, resulting in PR death within 14 days, and cones are more susceptible than rods to iron-induced oxidative damage. In order to further investigate the mechanism of intravitreal iron-induced retinal toxicity and shed light on mechanisms of iron-induced retinopathy in other mouse models, Fe2+, Fe3+, or saline were injected into the vitreous of adult wild-type mice. Pre-treatment with Ferrostatin-1 was used to investigate whether iron-induced retinal toxicity resulted from ferroptosis. Color and autofluorescence in vivo retinal imaging and optical coherence tomography were performed on day 2 and day 7 post-injection. Eyes were collected for quantitative PCR and Western analysis on day 1 and for immunofluorescence on both day 2 and 7. In vivo imaging and immunofluorescence revealed that Fe2+, but not Fe3+, induced PR oxidative damage and autofluorescence on day 2, resulting in PR death and retinal pigment epithelial cell (RPE) autofluorescence on day 7. Quantitative PCR and Western analysis on day 1 indicated that both Fe2+ and Fe3+ induced iron accumulation in the retina. However, only Fe2+ elevated levels of oxidative stress markers and components of ferroptosis in the retina, and killed PRs. Ferrostatin-1 failed to protect the retina from Fe2+-induced oxidative damage. To investigate the mechanism of Fe2+-induced RPE autofluorescence, rd10 mutant mice aged 6 weeks, with almost total loss of PRs, were given intravitreal Fe2+ or Fe3+ injections: neither induced RPE autofluorescence. This result suggests Fe2+-induced RPE autofluorescence in wild-type mice resulted from phagocytosed, oxidized outer segments. Together these data suggest that intraretinal Fe2+ causes PR oxidative stress, leading to PR death and RPE autofluorescence.

Project description:BackgroundThe retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols.MethodsSphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches.FindingsWe provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming.InterpretationThe Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. FUND: National Natural Science Foundation of China and the National Institute of Health USA.

Project description:PurposePhotoreceptor genesis in the retina requires precise regulation of progenitor cell competence, cell cycle exit, and differentiation, although information around the mechanisms that govern these events currently is lacking. In zebrafish, the basic helix-loop-helix (bHLH) transcription factor NeuroD governs photoreceptor genesis, but the signaling pathways through which NeuroD functions are unknown. The purpose of this study was to identify these pathways, and during photoreceptor genesis, Notch signaling was investigated as the putative mediator of NeuroD function.MethodsIn embryos, genetic mosaic analysis was used to determine if NeuroD functions is cell- or non-cell-autonomous. Morpholino-induced NeuroD knockdown, CRISPR/Cas9 mutation, and pharmacologic and transgenic approaches were used, followed by in situ hybridization, immunocytochemistry, and quantitative RT-PCR (qRT-PCR), to identify mechanisms through which NeuroD functions. In adults, following photoreceptor ablation and NeuroD knockdown, similar methods as above were used to identify NeuroD function during photoreceptor regeneration.ResultsIn embryos, NeuroD function is non-cell-autonomous, NeuroD knockdown increases Notch pathway gene expression, Notch inhibition rescues the NeuroD knockdown-induced deficiency in cell cycle exit but not photoreceptor maturation, and Notch activation and CRISPR/Cas9 mutation of neurod recapitulate NeuroD knockdown. In adults, NeuroD knockdown prevents cell cycle exit and photoreceptor regeneration and increases Notch pathway gene expression, and Notch inhibition rescues this phenotype.ConclusionsThese data demonstrate that during embryonic development, NeuroD governs photoreceptor genesis via non-cell-autonomous mechanisms and that, during photoreceptor development and regeneration, Notch signaling is a mechanistic link between NeuroD and cell cycle exit. In contrast, during embryonic development, NeuroD governs photoreceptor maturation via mechanisms that are independent of Notch signaling.

Dataset Information

Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD.

Publications

Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD.

Similar Datasets

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