Stem cell-derived retinal ganglion cell differentiation and its transplantation

Abstract

Purpose : In this study, we aimed to establish a rapid protocol for retinal ganglion cell (RGC) differentiation from human induced pluripotent stem cell (hiPSC)/embryonic stem cell (hESC) and for cell transplantation.

Methods : To shorten the time for stem cell differentiation to neural progenitor cell, we utilized lentivirus to introduce neurogenin 2(Ngn2) in hiPSC/hECS for facilitating induced-RGC (iRGC) differentiation. We also co-transfected cells with Sox4, a gene that is required for and strongly promotes RGC fate. RGC markers Brn3a and RBPMS were detected in iRGCs by immunofluorescence (IF) staining and QPCR. IRGCs were co-cultured with explanted retina or injected intravitreally in the adult rat eye, and co-stained with RBPMS and human nuclei markers. Calcium imaging stimulated by GABA agonist muscimol was used to confirm GABA_Areceptor expression and calcium reversal potential as a marker of electrophysiologic maturity in iRGCs. RGC neuroprotection after iRGC transplant was tested after optic nerve crush (ONC). All experiments were conducted at least three times independently. Data were analyzed by ANOVA post-hoc t-test with Tukey correction, with Pvalue of <0.05 considered statistically significant. All research was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Results : We found that overexpression of Ngn2 promotes neural cells differentiation to iRGCs from both hiPSC and hESC. Starting from day 6, we were able to detect neural marker Tuj-1 and RGC marker Brn3a in iRGCs by IF and QPCR. We further observed that iRGCs survive on mouse explanted retina and extend their axons 7 days after transplant. In vivo, we observed iRGC survival and growth in the ganglion cell layer in rat retina 7 days after transplant. IRGCs showed a muscimol-induced calcium imaging pattern similar to immature primary mouse RGCs. In addition, iRGC transplant protected against RGC loss 7 days after ONC. GFP-labeled iRGCs showed elongated neurites toward the optic nerve head in vivo.
Conclusion:

Conclusions : This rapid protocol shortens the time for human iRGC differentiation from stem cells, which used to take 2 weeks to months. Invivodata also support that iRGC transplant could be a therapeutic strategy to protect RGCs from degeneration in RGC-related diseases such as glaucoma.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.