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Yvonne Ou, Karen L Chu, Erik M Ullian; Generation and characterization of retinal ganglion cells from glaucoma patient iPSCs. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1361.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma, the leading cause of irreversible blindness worldwide, is a degenerative optic neuropathy characterized by gradual loss of peripheral vision and eventual blindness. The underlying cause of visual dysfunction is the progressive loss of retinal ganglion cells (RGCs) and their axons. Recently, retinal ganglion cell-like cells have been derived from human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). However, the proportion of cells in these cultures that are iPSC-derived RGCs (iPSC-RGCs) is typically low. Here we describe a modified protocol based on existing methods to increase the proportion of iPSC-RGCs derived from glaucoma patients in order to establish a human glaucoma cell culture model.
Skin biopsies were obtained from glaucoma patients seen at the UCSF Department of Ophthalmology’s Glaucoma Service. Multiple hiPSC lines were established using nonintegrating episomal reprogramming vectors consisting of OCT 3/4, c-Myc, KLF4, and SOX2. The glaucoma patient-derived hiPSCs were differentiated into RGCs in the presence of various BMP and Wnt inhibitors, as well as IGF-1 and Sonic hedgehog (Shh). The retinal progenitor cells and differentiated neurons produced were then assayed using RT-PCR and immunofluorescence microscopy.
Glaucoma patient-derived hiPSC lines were established and then differentiated into retinal progenitors as assayed by expression of eye-field transcription factors. The addition of DAPT, a Notch signaling pathway inhibitor, increased the proportion of cells expressing neuronal and RGC-specific markers. The inclusion of Shh increased the proportion of Brn3a-expressing cells in a concentration dependent manner (25.4% at 100 ng/ml vs. 11.4% at 10 ng/ml; p<0.05). These glaucoma patient-derived iPSC-RGCs were also capable of forming anatomic synapses by synaptic marker expression.
We have modified existing protocols to enhance the differentiation of RGCs from glaucoma patient-derived hiPSCs, providing an abundant source of cells for the establishment of an in vitro cell culture model of human glaucoma.
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