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Abstract #65929 Published in IGR 17-3
Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line
Sluch VM; Davis CH; Ranganathan V; Kerr JM; Krick K; Martin R; Berlinicke CA; Marsh-Armstrong N; Diamond JS; Mao HQ; Zack DJScientific reports 2015; 5: 16595
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.
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Baltimore, MD 21287.
Full articleClassification:
2.17 Stem cells (Part of: 2 Anatomical structures in glaucoma)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.6 Cellular biology (Part of: 3 Laboratory methods)
11.8 Neuroprotection (Part of: 11 Medical treatment)
