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Hany Abdelgawad, Michael J Young; Enrichment of mouse cone photoreceptors derived from pluripotent cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4579.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative disorders such as macular degeneration are characterized by irreversible changes in the photoreceptor (PR) and retinal pigment epithelium (RPE) layers. While PR precursors and progenitors give rise to rods and cones after transplantation (e.g. in rodent and porcine recipients), the alignment and number of integrated cells do not yet permit the full restoration of vision. Although published work shows it is relatively easy to generate large numbers of photoreceptor precursors from pluripotent human and mouse cells in 3D culture, the transition to differentiated cones has remained elusive.
In this study, we used mouse induced pluripotent stem cells (mIPSC) line labeled with s-opsin green fluorescent protein (GFP). Optic cups were generated in-vitro using a three-dimensional approach using Matrigel®. A NOTCH pathway inhibitor (DAPT) was added to the culture from day 12 to day 15 and s-opsin GFP+ cells were isolated by FACS at day 21 and day 28 of culture. Subsequently, the isolated s-opsin GFP+ cell populations were characterized by RT-PCR and Flow-Cytometry for expression of cone photoreceptor markers.
S-opsin GFP+ isolated cells expressed a number of s-opsin cone photoreceptor specific markers including OPN1SW, ARR3, GNGT2, CNGB3, PRPH2, SALL3 and DLG4.
This report provides a novel strategy for the generation of large numbers of mouse s-opsin cones that can be used for transplantation paradigms, as well as the development of high throughput screening (HTS) assays for cone development and survival. This will enable the development of tissue engineering and drug therapies to treat retinal degeneration diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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