Abstract
Purpose :
Glaucoma is characterized by retinal ganglion cell (RGC) death and visual field loss. Preventing neurodegenerative failure in glaucoma through stem cell technologies and preclinical human models presents an important step forward for translational research. Human induced pluripotent stem cell (iPSC) technology allows RGCs to be generated in-vitro from reprogrammed corneal fibroblasts. We investigate the survival of these hiPSC derived RGCs after transplanting them in our ocular translaminar autonomous system (TAS) perfusion model under normal and high intraocular pressure (IOP). This will allow us to validate a potential therapeutic to restore functional capacity in the degenerated human adult retina at late-stage disease when most RGCs are lost.
Methods :
Human iPSCs were generated by reprogramming human donor corneal fibroblasts using Yamanaka factors. These iPSCs were then differentiated into retinal organoids from which RGCs were obtained. The RGCs were transduced with AAV2-GFP and transplanted into control donor human eyes. They were pressurized for approximately 5-7 days (N=4), with left eye maintained at normal and right eye at elevated IOP. Viability was assessed by pro-survival pathways via qRT-PCR, immunohistochemistry staining, and electroretinography for retinal function.
Results :
We successfully transplanted human RGCs into human donor eyes and visualized them after GFP transduction. We maintained a pressure differential between the two eyes for approximately 5-7 days using the TAS model (left=9.5 ±1.45, right=23.1 ±1.5, N=4, p<0.05). After RGC transplantation, increased expression of pro-survival RGC marker (RBPMS, p<0.05) was observed. Additionally, there was a trend towards decreased expression of apoptosis, inflammation, gliotic markers (BAX, CASP3, TLR4, and GFAP) along with decreased trends in fibrosis (FN and COLIV) under normal IOP conditions compared to high IOP (N=4). Further, post-transplantation, decreased amplitudes in both retinal a-wave and b-wave (N=4) were observed under high IOP in contrast to normal IOP.
Conclusions :
Survival of RGCs was more conducive under normal IOP conditions with significantly increased degeneration observed at high IOP. Thus, alluding to the fact that elevated IOP could potentially create a micro-environment that would significantly inhibit successful transplantation of de novo RGCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.