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Silvia Aparicio Domingo, MIGUEL FLORES-BELLVER, M Natalia Vergara, Minda M. McNally, Joseph A Brzezinski, Valeria Canto Soler; Characterization of hiPSC-derived 3D mini retinas in long term culture. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4558.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative diseases cause the dysfunction and death of retinal photoreceptor cells leading to vision loss, and eventually, blindness. We recently established an efficient strategy for inducing human iPS cells to differentiate into fully laminated 3D mini retinas containing light-sensitive photoreceptors. The purpose of this study is to characterize the viability and differentiation capacity of our 3D mini retinas upon long-term culture in vitro, as a further step towards evaluating their potential for modeling retinal degenerative diseases as well as for their use as cellular products for retinal transplantation.
Differentiation of hiPSC into 3D mini retinas was conducted as previously described (Zhong et al., 2014). hiPSC-derived mini retinas were collected from week 15 to 70 (~4 to 17 months) of differentiation. Cellular composition and three-dimensional organization were evaluated by immunofluorescence detection of molecules associated with differentiation and function of various retinal cell types and three-dimensional confocal imaging. Functional differentiation and maturation was further evaluated at the ultrastructural level by transmission electron microscopy.
hiPSC-derived mini retinas showed highly organized laminae throughout the culture period. Over time however, cells in the inner nuclear layers (i.e. ganglion and amacrine cells) were gradually lost due to apoptotic cell death. The remaining bipolar cell layer and ONL maintained their cellular organization and showed evidence of further maturation, featuring highly differentiated rods and cones of all subtypes, as well as the presence of all major bipolar cell subtypes, including rod, cone ON and cone OFF bipolar cells. Furthermore, we observed evidence of synaptic formation between photoreceptors and cells located in the bipolar cell layer.
Our results demonstrate the feasibility of culturing hiPSC-derived 3D mini retinas for significantly long periods of time. These long-term mini retinas consist of a well-organized ONL and a bipolar cell layer, including their corresponding major cellular subtypes. Our system provides a model to study the mechanisms of human photoreceptor differentiation over a period equivalent to at least 6-8 months after birth. Furthermore, it facilitates evaluating strategies for cell-based therapies involving transplantation of hiPSC-derived photoreceptor sheets.
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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