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Donald J Zack, Valentin Sluch, Vinod Ranganathan, Cynthia Berlinicke; Differentiation of Human Stem Cells to Retinal Ganglion-like Cells using a CRISPR/Cas9 Engineered Reporter Line. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2671.
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© ARVO (1962-2015); The Authors (2016-present)
A number of diseases lead to retinal ganglion cell (RGC) death and vision loss, the most common of which is glaucoma. Once the optic nerve is damaged, few avenues of intervention exist since the mammalian optic nerve does not regenerate. One ambitious goal is to use cell replacement therapy to restore vision. Human pluripotent stem cell (hPSC) differentiation could, in theory, provide the necessary RGCs for this strategy. In addition, human stem cell-derived RGCs could make possible an approach for medically relevant drug screening that would have advantages over the current use of rodent RGCs. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we describe a protocol for differentiation of hPSCs to RGC-like cells that express a variety of RGC-enriched markers and exhibit spontaneous transient calcium activity typical of neurons.
H7 human embryonic stem cells were genetically engineered using the CRISPR/Cas9 nuclease system to contain a membrane Cherry fluorescent reporter downstream of BRN3B, an RGC-enriched transcription factor. This augmented cell line was differentiated in N2B27 neuronal medium in the presence of 2% matrigel. Following differentiation, we were able to use fluorescent activated cell sorting (FACS) to isolate the RGC-like cells. We analyzed the cells using qPCR, immunostaining, and calcium imaging.
By day 30 of differentiation, BRN3B reporter expression was evident and the cells displayed long neurite projections. The cultures were enriched for BRN3B, BRN3A, SNCG, NEFH, NRN1, and RBPMS expression by qPCR. Additionally, we were able to detect low levels of melanopsin expression. Calcium imaging of day 35 cultures showed spontaneous calcium activity, typical of neuronal electrical activity. Immunostaining confirmed the presence of cells double-labeled for MAP2 and BRN3A, neuronal and RGC-enriched markers, respectively.
We were able to differentiate hPSCs to RGC-like cells. Additionally, through the use of CRISPR technology, we generated a stem cell line containing a human RGC reporter. These cells can be FACS-sorted to obtain isolated RGCs. We believe that our protocol lays the groundwork for further experiments on stem cell-derived human RGCs which can be used for drug screening, developmental and biological studies, as well as cell replacement experiments.
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