Survival and integration of organoid derived iPSC-RGCs into NHP tissue in an organotypic retinal explant culture system

Abstract

Purpose : Retinal ganglion cells (RGCs) transmit visual information from the eye to the brain. Optic neuropathies can lead to vision degradation through the loss of RGCs. There are currently no treatments to replace lost RGCs. Using non-human primate (NHP) retinal explant culture, we aim to overcome barriers to donor human iPSC (induced pluripotent stem cell)-derived RGC survival and integration.

Methods : NHP eyes were collected 5-30 minutes after euthanasia, transported in CO₂ buffered Ames media, retinas islated and dissected into 4mm pieces, attached to cell culture inserts, and incubated at 37^oC (95/5% O₂/CO₂). Organoid derived iPSC-RGCs edited to express GFP are thawed and seeded on explants (approx. 7500 cells/explant), and supplied growth media every 2-4 days. Individual GFP+ cells were targeted for whole cell recordings at different time points post-seeding to assess the electrical characteristics of donor cells. Tissue was fixed and stained to assess cell survival, growth, and integration into NHP tissue.

Results : Donor RGCs survived 90 days post-seeding (DPS). Of the initial population of ~8K viable cells seeded per explant, 983.12 ± 201 GFP+ cells were detected via GFP immunoreactivity at time points between 24-45 DPS (N=8). GFP signal covered 2.39 ± 0.64% of the explant surface, and these surviving cells represent 13.12±2.7% of the initial viable seeded population. Only a small fraction of the GFP signal was found in the GCL or inner retina. GFP+ donor cells were targeted for whole cell recordings at a range of ages post-seeding. Between 3-21 DPS, GFP+ cells had high input resistance (IR) and lacked active membrane conductances. Between 24 and 31 DPS, there was a significant drop in IR, and given 25-150pA current injections, GFP+ cells began to reach action potential (AP) threshold. IR plateaued around D31 and cells began to fire bursts of APs as explant and donor cell age increased. (D3-21, IR=3.96±0.49 GΩ, N=21; D24-31 IR=1.66±.27 GΩ, N=26; D37-84 IR=.848±.15 GΩ N=21)

Conclusions : The presence of structurally and functionally maturing iPSC-RGCs in explants is encouraging for future studies involving transplantation into in vivo models. The long-term survivability of explants and donor cells will serve as a useful model to evaluate other manipulations, physical, enzymatic, or gene editing, to promote further donor cell integration into appropriate retinal locations.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.