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Sci Rep. 2015 Nov 13;5:16595. doi: 10.1038/srep16595.

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

Author information

1
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Baltimore, MD 21287.
2
Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD, 21205.
3
Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
4
Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
5
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
6
Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
7
Department of Materials Science and Engineering, Whiting School of Engineering, and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218.
8
The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205.
9
Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287.

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.

PMID:
26563826
PMCID:
PMC4643248
DOI:
10.1038/srep16595
[Indexed for MEDLINE]
Free PMC Article

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