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Kevin Gregory-Evans, Anat Yanai, Christopher Laver, Cheryl Y Gregory-Evans; Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1840.
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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 a promising and widely applicable approach in the treatment of blinding retinal diseases. It has however been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is extremely low. The factors that inhibit PPC engraftment remain largely unknown in part because so many adverse factors could be at play during in vivo experiments. We therefore propose the hypothesis that an ex vivo model system will help to identify specific factors inhibiting PPC survival and integration into dystrophic retina.
Neural retina harvested from either rat or human eyes was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Histology, TUNEL staining and immunocytochemistry were used to assess functional integration. Images were obtained by confocal scanning microscopy.
Explants co-cultured with hESC-derived RPE maintained normal gross morphology and viability for up to two weeks, whereas the explants cultured on ARPE19 and RPE-J failed by seven days. The proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when co-cultured with hESC-derived RPE (20% and 10% respectively), than when co-cultured with ARPE19 (only 6% and 2% respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs co-cultured with hESC-derived RPE increased to approximately 30% and 15% respectively.
These data demonstrate the utility of an ex vivo model system to study PPC survival in diseased retina, highlight the need for viable, functional RPE in PPC survival and suggest a role for synaptogenic factors such as TSP-1 in the functional integration of PPCs in future in vivo experiments in models of retinal degeneration.
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