Abstract
Purpose :
Retinal ganglion cells (RGCs) are the first neurons specified in human retinal organoids, yet they are predominantly lost over long-term culture, compromising retinal stratification and functional maturation in vitro. In the developing vertebrate retina, excess RGCs are eliminated after two waves of programmed cell death. Here, we investigate how inhibition of apoptosis promotes the survival and maintenance of RGCs in human retinal organoids.
Methods :
To study the role of apoptosis in RGC loss, we differentiated retinal organoids from BAX and BAK double knockout (BAX/BAK dKO) human pluripotent stem cells in parallel with the isogenic control through day 200. Retinal organoids were collected, frozen, and sectioned into 10-um slices every 20 days for further analysis. RGC density and cell death were quantified by immunofluorescent staining with RGC markers (SNCG, POU4F1) and cell death markers (cleaved Caspase 3 and propidium iodide). Birthdating experiments were conducted by EdU pulse treatment at several timepoints of organoid differentiation. N = 3 organoids were analyzed per timepoint and at least n = 3 sections per organoid were assessed.
Results :
In this study, we identified three waves of retinal cell death in organoids, including an early wave of apoptosis between days 40-60, a late wave of apoptosis at day 100, and a wave of necrosis between days 100-130 of differentiation. RGC density in organoids peaks between days 60-80, and declines after day 100. The highest proportion of apoptotic RGCs was observed during the late wave of apoptosis, but not the early wave. BAX/BAK dKO nearly abolished apoptosis without reducing necrosis. More EdU-labeled RGCs survived after the peak of RGC generation in BAX/BAK dKO organoids compared to controls. We also observed a transient increase in necrosis in the EdU-labeled RGC population of BAX/BAK dKO organoids. In long-term culture, BAX/BAK dKO organoids displayed a decline in overall RGC density after day 100, but maintained a subpopulation of RGCs at day 200.
Conclusions :
In conclusion, we have shown that apoptosis and necrosis contribute to RGC loss in human retinal organoids. Inhibition of BAX/BAK-mediated apoptosis promotes the survival and maintenance of a subset of RGCs in long-term organoid culture. Apoptosis inhibition also leads to increased necrosis of RGCs, suggesting a compensatory mechanism for RGC death in retinal organoids.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.