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Sarah Decembrini, Ute Koch, Freddy Radtke, Alexandre P Moulin, Yvan Arsenijevic; Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1115.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative diseases resulting in the loss of photoreceptors are a major cause of blindness. Recently, different groups validated the possibility to reactivate dormant retinal circuits of degenerating retinas using retinal prosthesis, gene and cell replacement therapy. Photoreceptor replacement therapy may be feasible since transplanted photoreceptors, collected directly from the developing or the adult retina, have been shown to restore some visual function in mice affected by retinal degeneration. Because the developing retina is not a suitable source of renewable photoreceptors, we focused on embryonic stem cells (ESC) for their capacity to generate retinal progenitors and photoreceptor cells in vitro.
In this study, we derived a new transgenic ESC line in which the reporter gene, the Crx-GFP transgene, is expressed in both post-mitotic immature and mature photoreceptors, and assessed the extent to which this protocol recapitulates photoreceptor development in vitro.
Various oxygen concentrations were tested at different development stages to improve photoreceptor production. As observed during the retinogenesis, the optimized 3D-retina induction protocol allows the production of GFP-positive photoreceptors between 12 and 14 days of culture which reach the peak of birth between day 18 and 20 of culture. Similarly the intensity of the GFP signal and their alignment increase over time. We observed that hyperoxic condition improved photoreceptor survival only when present since photoreceptor differentiation onset. Up to ten layers of photoreceptors can be formed in each in vitro-generated retinas. In addition we proved that transplantation of ESC-derived photoreceptors is feasible. No appearance of tumour formation was detected after transplantation of sorted photoreceptor cells. Many Crx-GFP-positive cells show the presence of outer-segments, ribbon synapses, and light signal transduction pathway proteins.
These experiments show the feasibility to reliably generate a large quantity of integration-competent photoreceptors from ESCs. A further characterization of the transplanted photoreceptors to reveal their capacity to mediate light stimuli is underway.
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