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

Research output: Contribution to journalArticle

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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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Clustered Regularly Interspaced Short Palindromic Repeats
Retinal Ganglion Cells
Cell Line
Pluripotent Stem Cells
Nanofibers
Optic Nerve Diseases
Colforsin
Blindness
Optic Nerve
Cell- and Tissue-Based Therapy
Glaucoma
Cell Differentiation
Flow Cytometry
Cell Death
Stem Cells
Cell Culture Techniques
Wounds and Injuries
Population
Genes

ASJC Scopus subject areas

  • General

Cite this

Sluch, V. M., Davis, C. H. O., Ranganathan, V., Kerr, J. M., Krick, K., Martin, R., ... 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

Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. / Sluch, Valentin M.; Davis, Chung Ha O.; Ranganathan, Vinod; Kerr, Justin M.; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A.; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S.; Mao, Hai Quan; Zack, Donald J.

In: Scientific Reports, Vol. 5, 16595, 13.11.2015.

Research output: Contribution to journalArticle

Sluch, VM, Davis, CHO, Ranganathan, V, Kerr, JM, Krick, K, Martin, R, Berlinicke, CA, Marsh-Armstrong, N, Diamond, JS, Mao, HQ & Zack, DJ 2015, 'Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line', Scientific Reports, vol. 5, 16595. https://doi.org/10.1038/srep16595
Sluch, Valentin M. ; Davis, Chung Ha O. ; Ranganathan, Vinod ; Kerr, Justin M. ; Krick, Kellin ; Martin, Russ ; Berlinicke, Cynthia A. ; Marsh-Armstrong, Nicholas ; Diamond, Jeffrey S. ; Mao, Hai Quan ; Zack, Donald J. / Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. In: Scientific Reports. 2015 ; Vol. 5.
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