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Jee Min, Joe Phillips, Patrick Barney, Anna Petelinsek, Abbey Cash, Tasnia Tabassum, David M Gamm; Promoting photoreceptor genesis and reducing proliferation in human induced pluripotent stem cell derived-optic vesicles. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3601.
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© ARVO (1962-2015); The Authors (2016-present)
Human induced pluripotent stem cell-based (hiPSC) photoreceptor replacement strategies will require enriched photoreceptor production, as well as the removal of proliferative retinal progenitors prior to transplantation. The purpose of this study is to quantify the effects of known pro-photoreceptor and anti-proliferation reagents in our culture system.
Using our established protocols, hiPSC cultures were differentiated to a retinal lineage and optic vesicle (OV)-like structures were isolated. Anti-proliferative reagents tested included cytarabine, DAPT and aphidicolin. For these experiments, hiPSC-OV cultures were dissociated at day 70-100 of differentiation, plated on a 96 well plate, and treated for 1 week. Optimal dosage, proliferation reduction, and photoreceptor production/preservation was determined for each drug. To test potential pro-photoreceptor reagents, free floating OVs were treated for 30 days with taurine, retinoic acid, or DAPT. Following treatment, OVs were sectioned and immunolabeled for confocal microscopy analysis, or dissociated and plated for quantitative analysis. For both proliferation and photoreceptor generation experiments, quantitative immunocytochemistry analysis was performed on the Operetta High Content Screening System (Perkin Elmer).
Short term cytarabine, DAPT, or aphidicolin treatment each reduced the number of proliferative retinal progenitors when compared to controls, with aphidicolin providing the greatest reduction in proliferation. Meanwhile, 30 day treatment with retinoic acid or DAPT both resulted in increased photoreceptor precursor production, while taurine treatment increased the production of rods specifically.
We have demonstrated the reduction of proliferation and the enhancement of photoreceptor production in our culture system. Current experiments are underway to uncover the optimal timing of supplementation with pro-photoreceptor and anti-proliferation molecules, as well as an investigation of their combinatorial effects.
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