Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line

Valentin M. Sluch, Chung Ha O. Davis, Vinod Ranganathan, Justin M. Kerr, Kellin Krick, Russ Martin, Cynthia A. Berlinicke, Nicholas Marsh-Armstrong, Jeffrey S. Diamond, Hai Quan Mao, Donald J. Zack

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69 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number16595
JournalScientific Reports
Volume5
DOIs
StatePublished - Nov 13 2015
Externally publishedYes

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    Sluch, V. M., Davis, C. H. O., Ranganathan, V., Kerr, J. M., Krick, K., Martin, R., Berlinicke, C. A., Marsh-Armstrong, N., Diamond, J. S., Mao, H. Q., & Zack, D. J. (2015). Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. Scientific Reports, 5, [16595]. https://doi.org/10.1038/srep16595