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Venkata R M Chavali, Jie He, Sonika Rathi, Jason Mills, Claire H Mitchell, Joan M O'Brien; Induced pluripotent stem cell derived retinal ganglion cells as in vitro model to study molecular pathobiology in Glaucoma.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3796.
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© ARVO (1962-2015); The Authors (2016-present)
To establish and characterize human induced pluripotent stem cells derived retinal ganglion cells as models to investigate mechanism of ganglion cell loss.
Human induced pluripotent stem cells (iPSCs) were differentiated into retinal ganglion cells (RGCs) using a modified chemically defined protocol developed in our lab. This protocol differentiates iPSCs into RGCs without the need for manual selection, organoid generation or CRISPR modulation of the iPSC lines before differentiation. Mature ganglion cells formed after differentiation were evaluated for their RGC related protein expression using immunohistochemistry, physiological function using patch clamp methodology and real-time PCR profiling of gene expression. The RGC health and viability were assessed by documenting morphology and neurite dynamics using Incucyte neuronal analysis software. RGC cultures were further enriched by either treatment with mitotic inhibitors or by magnetic cell sorting (MACS) sorting with CD90.1 antibody. Oxidative stress, qPCR arrays, cell stretch assays and calcium imaging experiments were performed to assess the biology and function of the cells harvested.
We generated over 45-50% iPSC-RGCs from iPSCs in 35 days. Mature retinal ganglion cells exhibited large soma with elongated axonal processes. Treatment of iPSC-RGC cultures for seven days with mitotic inhibitor, cytosine arabinoside (AraC) significantly improved (over 95%) the homogeneity of iPSC-RGC cultures by eliminating mitotically active undifferentiated cells. Alternately, MACS sorting equally enriched the purity of RGC cultures and removed any undifferentiated cells. Differential response of N-methyl D-Aspartate receptor to Ca2+ efflux in iPSC-RGCs demonstrates RGC subtypes generated by our protocol. The real-time PCR arrays demonstrated the presence of RGC-associated gene expression and generation of several RGC subtypes in these cells. Hydrogen peroxide treatment (350uM) in iPSC-RGCs resulted in significantly reduced expression of SOD1, NRF2, IL6, suggesting their susceptibility to oxidative stress.
We have established protocols and methodologies to generate highly reproducible and homogenous RGC cultures from human induced pluripotent stem cells. These mature iPSC-RGCs will serve as ideal in vitro model systems to study the RGC pathobiology.
This is a 2020 ARVO Annual Meeting abstract.
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