Abstract
Purpose :
Glaucoma typically progresses slowly, selectively targeting retinal ganglion cell (RGC) axons, and ultimately leads to visual deficits. At this point, traditional therapies may slow further progression, but disease-related damage cannot be reversed. Human embryonic stem cell (hesc) derived RGCs is one potential therapy to restore visual function. However, we are just beginning to understand the characteristics of hesc-RGCs. Here, we sought to examine the morphological and physiological development of cultured hesc-RGCs.
Methods :
Hesc-derived RGCs were obtained from Dr. Donald Zack’s laboratory at Johns Hopkins University. RGCs were plated on laminin-coated coverslips at a density of 50,000 cells/cm2. Cells were cultured in iNS media and fed twice per week. We investigated the morphology and electrophysiological properties of cells after one week (1wk) and four weeks (4wk) in culture. RGCs were identified by td-tomato-tagged Brn3b expression. Td-tomato-positive cells were targeted for recording using borosilicate pipettes filled with (in mM): 100 KMeSO4 5 KCl, 5 NaCl, 10 HEPES, 4 EGTA, 10 Na-Phosphocreatine, 4 Mg-ATP, 0.4 Na-GTP, 1 Lucifer Yellow. We assessed spontaneous spike activity in cells at resting membrane potential (RMP) or clamped at -60mV. Next, we evaluated voltage-gated Na+ and K+ channel activity by clamping cells at -80mV and depolarizing cells with voltage steps from -80 to 30mV in 10mV steps for 50ms. After electrophysiology, cells were fixed for 24hrs in 4% PFA, RGCs were confocally imaged, and we measured soma size.
Results :
Following 1wk in culture, 29% (5/17 cells) of targeted cells exhibited spontaneous spiking activity. After 4wk in culture, 67% (10/15 cells) showed spontaneous activity. Spike rate, after 4wk in culture, significantly increased from 0.16 to 0.5 spikes/s (p=0.03). There was no significant change in RMP between 1wk and 4wk (1Wk=-43±2.2mV, 4Wk=-46.5±2.4mV). The increased spiking observed following 4wk in culture was correlated with significantly larger voltage-gated Na+ and K+ currents compared to 1wk cells (p<0.001). Finally, RGC somas were significantly larger after 4wk in culture (n=46, 172±12.55µm2) compared to 1wk (n=58, 65±2.5µm2) (p>0.001).
Conclusions :
RGCs cultured for 4wk display more mature electrical and morphological properties, which may facilitate integration and function within damaged tissue.
This is a 2020 ARVO Annual Meeting abstract.