Retinal Ganglion Cells can be More Efficiently Generated From Adult Human Retinal Stem Cells After Atoh7 or Brn3b Gene Transduction

Abstract

Abstract: : Purpose: It has been shown that the ciliary margin of the mammalian eye (including the human eye) contains retinal stem cells (RSCs), that show the two cardinal principles of stem cells: self–renewal and the multipotentiality to make all of the cell types of the retina including retinal ganglion cells. Visual impairment in the human glaucomatous eye is associated with retinal ganglion cell loss and optic nerve atrophy, and thus our focus here is to drive human RSC progeny to take on retinal ganglion cell phenotypes. Methods: To enhance the production of retinal ganglion cells that differentiate from human RSC, we transfected two critical genes for retinal ganglion cell development, using lentiviral vectors expressing the mouse Atoh7 or mouse Brn3b gene. Atoh7 is crucial for retinal ganglion cell determination and Brn3b for retinal ganglion cell differentiation. To test the role of each factor in retinal ganglion cell specification, transfected clonal human RSC spheres were differentiated in culture. Results: Differentiated retinal ganglion cells from human RSC progeny expressed the retinal ganglion cell marker, neurofilament–M, and their morphology appears to be similar to endogenous retinal ganglion cells. The number of differentiated retinal ganglion cells in Atoh7– (11%) or Brn3b–expressing (6%) human RSC progeny was significantly higher than in those found in the control GFP–expresing clonal progeny (0.5%). Conclusions: These results suggest that human RSCs with appropriate gene transduction may be valuable in treating humans with retinal ganglion cell loss.

Keywords: retinal development • gene transfer/gene therapy • ganglion cells