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Yossi Mandel, Yoav Chemla, Astar Shamul, Amos Markus, Nairouz Farah, Ron S. Goldstein; Differentiation of Human Embryonic Stem cells into Photoreceptor Precursor – In-Vitro and In-Vivo Study. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1144.
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© ARVO (1962-2015); The Authors (2016-present)
There are several methods for inducing the differentiation of human embryonic stem cells (hESCs) into photoreceptors precursors. Herein, we studied photoreceptors generation in two-dimensional (2D) or a three-dimensional (3D) cell culture model. In addition, we studied the retention of labeled photoreceptor cells in the subretinal space of rats by fluorescence imaging of the retina.
hESCs (H9, US National Stem Cell Bank) differentiation into photoreceptor precursors was investigated in two cell culture models: a 2D model where photoreceptor differentiation was induced in monolayer cultures over 17 days using media including Dkk1, Noggin and IGF1, or a 3D model, where differentiation was induced in suspension cultures over 21 days with media containing endo-IWR 1, SAG, CHIR 99021. Differentiation was studied by immunostaining (CRX, PAX6) and mRNA expression (CRX,VSX2,PAX6). Cells that were subjected to the differentiation protocol were labeled by infection with GFP-expressing adenovirus and then injected into the subretinal space of Long-Evans rats (100,000 cells/8 µl). The retention of the cells was studied over a month by repeated fluorescence imaging of the retina by a Micron-IV Phoenix rodent imaging system.
hESC were differentiated into photoreceptor precursors in the 2D and 3D both models, as was revealed by staining or expression of CRX. Higher efficiency was found in the 3D model as compared to the 2D with up to 80 percent of cells stained for CRX. Transplanted cells diffused evenly in the subretinal space without producing cells clumps. Retention of cells was low with less than 10% of the transplanted cells being be detected by fluorescence imaging at 1 month post transplantation.
Both 2D and 3D protocols were efficient in photoreceptor generation with some advantage of the 3D over the 2D method. Retention of injected cells differentiated from hESC in the subretinal space of rats is poor. Further studies should be conducted in order to improve the transplantation protocol.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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