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Yongqing liu, Xiaoqin Lu, Wei Wang, Liang Zhou, Li Huang, Henry J. Kaplan, Douglas C. Dean; Long-Term Culture and Efficient Photoreceptor Differentiation of Swine Embryonic Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1318.
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Retina degeneration is a major cause of blindness, and the mammalian retina shows only a limited capacity for regeneration following injury or disease. Therefore, studies are underway assessing the ability of transplanted stem cells to restore retinal function. Stem cell therapies have utilized embryonic stem cells and induced pluripotent stem cells which are pluripotent and must be driven toward a lineage prior to transplantation, and adult stem cells which are progenitors already committed to an organ-specific lineage. Encouraging results are being obtained with adult stem cells, but such adult stem cells have not been isolated from the mammalian eye. However, photoreceptor progenitors are evident in the embryonic eye. But, attempts to maintain and expand these progenitors in culture under conditions where they can readily differentiate into photoreceptors has been largely unsuccessful, leading to a focus on embryonic stem cells and induced pluripotent cells for retinal transplant experiments. The purpose of this study is to optimize conditions for maintaining and expanding swine embryonic retinal progenitor cells capable of efficiently differentiating into photoreceptors.
Primary retinal cultures were generated from swine at different embryonic ages. Culture media and substrate were varied and effects on cell proliferation and ability to differentiate into photoreceptor lineages were assessed by immunostaining with lineage markers.
Based on immunostaining of developing retina, we found Pax6+ retinal progenitors were evident between embryonic days 50 and 85. Primary retinal cultures were generated during this developmental range. We found that it was essential that the cells be maintained with diluted matrigel in neural medium to prevent differentiation. Differentiation into photoreceptor lineages was initiated by forcing the cells to form neurospheres in suspension culture followed by a monolayer culture on matirgel in differentiation medium. The cells could be transplanted into a swine model of rod photoreceptor degeneration, where they integrated into the outer nuclear layer and expressed rhodopsin.
We have devised culture conditions for maintaining embryonic swine retinal progenitor cells, and a protocol for efficient differentiation of these cells into rod photoreceptors. These cells have the potential to integrate into the retina in a swine model of rod photoreceptor degeneration.
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