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Marcela Garita, Francisco Díaz-Corrales, Slaven Erceg, Shomi Bhattacharya; TRANSPLANTED mESC-DERIVED RETINAL PROGENITORS DIFFERENTIATE TO MATURE PHOTORECEPTORS IN VIVO, MIGRATE AND INTEGRATE IN THE MICE RETINA. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3988.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal dystrophies characterized by the progressive degeneration of photoreceptors are the leading causes of incurable blindness. Due to the limited capacity of the mammalian retina to regenerate using embryonic stem cells (ESC) as an unlimited source to replenish the lost cells has represented a main objective for the scientific community. Despite great advances in the field of differentiation of ESC towards photoreceptors in the recent years, few drawbacks remain unresolved. Such as, efficiency, purity of the population and the constant worry that once differentiated, cells could not be able to integrate into the host retina
We optimized a new protocol to differentiate mouse ESC (mESC), involving the control of the oxygen tension to mimic the retinal niche conditions, as well as the manipulation of key signaling pathways involved during normal retinal development. The retinal progenitor cells generated were subretinally transplanted in order to evaluate their migration and integration capacities by immunohistochemistry analysis
Hypoxia increases the efficiency of differentiation towards photoreceptors, but as well it improves the modeling of retinogenesis in vitro, by decreasing the time necessary to acquire each specific phenotype. Transcription factors associated to each stage of retinal differentiation such as eye field (Rax, Six3), optic cup (Pax6, Mitf, Chx10), photoreceptor precursors (Nrl, Crx) and even mature photoreceptors (Rhodopsin, Recoverin and Opsin-S) were upregulated earlier and their levels of expression were significantly higher than those reached under normoxic conditions as determined by qPCR. Moreover, when photoreceptor precursors derived from mESC cells following our protocol under hypoxia were transplanted into the subretinal space of wild type mice they differentiated mainly towards Recoverin/Rhodopsin positive cells, migrate and integrate into the host retina and in some cases acquired the morphology of mature rods with formation of structures suggesting outer segments
Our results demonstrate that hypoxia increases the yield of retinal phenotypes from mESC, including mature phenotypes. Moreover, in vivo experiments demonstrated that hypoxia promotes the survival of the grafted cells, allowing the retinal progenitors to differentiate towards photoreceptors, migrate and integrate in the mice retina
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