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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Differentiation / genetics*
  • Cell Line
  • Cells, Cultured
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism*
  • Gene Expression
  • Genetic Engineering / methods*
  • Humans
  • Immunohistochemistry
  • Membrane Potentials / genetics
  • Mice
  • Microscopy, Fluorescence
  • Retinal Ganglion Cells / cytology
  • Retinal Ganglion Cells / metabolism*
  • Retinal Ganglion Cells / physiology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Thy-1 Antigens / metabolism
  • Time Factors
  • Transcription Factor Brn-3B / genetics
  • Transcription Factor Brn-3B / metabolism

Substances

  • Thy-1 Antigens
  • Transcription Factor Brn-3B