biostudies-literatureSluch VMNEI NIH HHSPHS HHS16595https://www.ebi.ac.uk/biostudies/studies/S-EPMC4643248biostudies-literatureUnknown5Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.Scientific reportsDifferentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.PMC4643248R01EY0237545P30EY001765R01 EY0237545T32EY007143T32 EY0071431R01EY02268001P30 EY001765T32-90040730Mao HQMarsh-Armstrong NDavis CHKrick KDiamond JSSluch VMBerlinicke CAZack DJRanganathan VKerr JMMartin RfalseDifferentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.2015-01-01T00:00:00Z2015 Nov2025-04-04T13:25:45.773Z2019-03-27T02:01:52ZS-EPMC46432482656382610.1038/srep16595