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Anat Yanai, Christopher Laver, AMA Bashar, Andrew Metcalfe, Kevin Gregory-Evans; Development of an ex-vivo retinal explant model to assess transplantation efficiency of photoreceptor progenitor cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5818.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is one of the most promising and widely applicable approaches to the treatment of retinal degenerations. However, the factors that promote PPC engraftment are largely unknown. Here we focused on developing a novel ex-vivo system to facilitate investigation and optimization of PPC transplantation.
Harvested neural retinas from adult rats were dissected into equal-sized quadrants and placed directly on i) a hydrophilic Millicell filter (with 0.4 micron pores) of tissue culture inserts, or ii) on monolayers of different retinal pigment epithelium (RPE) cell lines (RPE-J, ARPE19 and hESC derived RPE) growing on the same filter. These explants, with or without RPE, were cultured for up to three weeks in serum-free medium. Tissue microstructure was assessed and outer nuclear layer (ONL) thickness was measured at different time points. Immunocytochemical analysis was performed with markers for different retinal cell types, such as photoreceptors (e.g. CRX and S-Opsin), to validate explants’ viability. To mimic PPC transplantation to the subretinal space, hESC-derived PPCs were inserted between the rat neural explant and underlying RPE/filter combination and their growth and integration was assessed.
Explants co-cultured with RPE maintain normal gross morphology for up to three weeks and continued to express proteins characteristic of rod and cone photoreceptors. ONL thickness and cell viability measurements demonstrate negligible changes over time. We also demonstrate the feasibility of “transplanting” PPCs into this system and assessing their integration efficiency in the presence of selected factors (e.g., isolated components from the interphotoreceptor matrix).
A model system that includes retinal explants, co-cultured with RPE, can be used to screen and define factors that influence integration efficiency of transplanted PPCs. Identifying factors that enhance integration of PPCs into retinal explants in an ex-vivo system can then be examined in in-vivo models of retinal degeneration.
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