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Sarah Ohlemacher, Akshayalakshmi Sridhar, Yucheng Xiao, Anthony Baucum, Theodore Cummins, Jason S Meyer; Analysis of Retinal Ganglion Cell Development and Maturation From Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1369.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) serve as the primary connection from the eye to the brain, allowing for normal visual processing. Loss of RGCs leads to devastating blinding disorders termed optic neuropathies. Human pluripotent stem cells (hPSCs) allow for the development of a variety of translational applications for optic neuropathies, including cell replacement, drug screening, and disease modeling. To date, however, little attention has been paid to the degree of maturation of these hPSC-derived RGCs, limiting their ability to serve as effective tools for the study and/or treatment of optic neuropathies.
A stepwise differentiation protocol was utilized to direct the differentiation of hPSCs toward a retinal lineage, as previously described. Within the first 40 days of differentiation, RGCs could be definitively identified by gene expression and morphological characteristics. RGCs were grown in culture for up to 12 weeks, at which point the degree of maturation of these RGCs was monitored by immunocytochemistry, electrophysiology, and western blot analyses.
Initially, hPSC-derived RGCs were identified based on the expression of BRN3. Following the dissociation and plating of these cells, morphological and phenotypic features associated with RGCs arose over time. Throughout the course of 12 weeks, the degree of dendritic and axonal extensions were analyzed, with corresponding increases in complexity over time. Additionally, the survival and proliferation of RGCs was monitored by the expression of activated caspase-3 and Ki-67, respectively. Furthermore, the ability of these RGCs to adopt features of developing synapses was analyzed based on the expression of synaptic proteins by Western blot. Ongoing experiments aim to test the ability to enhance the timing and efficacy of RGC maturation from hPSCs.
The results presented above demonstrate the ability to reliably and efficiently derive RGCs from hPSCs. Prolonged differentiation of these RGCs resulted in increased complexity of RGC morphologies, as well as a temporal increase in the expression of synaptic markers. Overall, the results of this study indicate the ability of hPSC-derived RGCs to acquire advancing features of maturation, with important implications for disease modeling, drug screening, and cellular replacement therapies.
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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