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Valeria Marigo, Claudia Aruta, Gian Carlo Demontis, Antonella Comitato, Francesca Giordano, Emeline F. Nandrot; Molecular And Functional Properties Of Retinal Pigment Epithelium And Photoreceptors Derived From Retinal Neurospheres. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3180.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cells have been proposed as a prospective source for retinal transplantation. Nevertheless, turning retinal progenitors into transplantable photoreceptors is a complex process. The application of retinal stem cells (RSC) for regenerative medicine will require an enriched and homogeneous population of cells able to become functional RPE or rods in the retina. To this purpose we have developed protocols to in vitro differentiate RSC derived from adult ciliary epithelium into either RPE or rod-like photoreceptors and tested their molecular and physiological properties.
RSC, derived from the ciliary body of adult mice, were differentiated in the presence of selective differentiating factors. Gene expression was analyzed by Real-time PCR; protein localization by immunofluorescence; morphology by EM; functionality of RPE by phagocytosis assay; rod functionality by patch-clamp.
During the first weeks of in vitro differentiation, cells expressed progenitor markers. Treatment with serum and specific hormones allowed RPE differentiation with active melanogenesis and phagocytosis capacity. Rod photoreceptor differentiation was achieved by treatment with a defined medium in the absence of serum. Co-expression of rod proteins, such as Rho and Gnat or Rho and Pde6b, was detectable in 60% of the cells after 20 days of differentiation. 30 days in vitro allowed 80% of cells to co-express rod markers. Electrophysiological analysis demonstrates cGMP-gated currents after 3 weeks in vitro, suggesting that expression of cGMP-gated channels starts later than rhodopsin. Most importantly, cells, after 40 days of differentiation, generated an electrical response to light stimuli consistent with closure of cGMP-gated channels.
Specific differentiation protocols allowed differential differentiation of RSC into either RPE or rods. RPE in vitro forms a monolayer with active melanogenesis that will allow studies on diseases affecting this process, such as albinism. Expression, subcellular localization and electrophysiology suggested that we in vitro generate high percentage of cells acquiring rod-like properties. Long differentiation times are required to achieve high degree of maturation.
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