Abstract
Purpose :
Human retinal ganglion cells (RGCs) can be generated in human stem cell-derived retinal organoids. During long-term organoid culture, RGCs are predominantly lost, limiting studies of RGC maturation and functionality in vitro. In the vertebrate retina, RGCs are first generated in an overabundance, and excess RGCs are eliminated by two waves of programmed cell death during development. In organoids, limited oxygen and nutrient diffusion in the innermost regions leads to the formation of necrotic cores, a potential cause for RGC loss. Here, we sought to investigate what causes RGC loss during organoid differentiation, focusing on roles for apoptosis and necrosis.
Methods :
We differentiated experimental and control human pluripotent stem cells into retinal organoids through day 200 and examined RGCs and cell death every 20 days. To study RGC developmental dynamics, we grew organoids that express an RGC-specific fluorescent reporter and characterized RGC morphologies and distribution with live imaging. To study the role of apoptosis, we differentiated BAX/BAK double knockout (BAX/BAK dKO) organoids and quantified cell death with cleaved Caspase 3 and propidium iodide staining.
Results :
We characterized four phases of RGC development in organoids: RGC generation, lamination, migration, and degeneration. RGCs were first generated in the inner neuroblastic layer and then joined the putative ganglion cell layer (GCL). During the migration phase, some RGCs remained in the GCL while others migrated to other retinal layers and non-retinal regions (e.g., necrotic cores). Though most RGCs underwent degeneration, we observed a small number of RGCs in day 200 organoids. We identified three waves of cell death in organoids, including an early wave of apoptosis during RGC generation/lamination, a late wave of apoptosis during RGC degeneration, and a wave of necrosis in the core. Double knockout of BAX and BAK nearly abolished apoptosis but did not significantly increase RGC density in organoids. Interestingly, a higher proportion of RGCs remained in the GCL in BAX/BAK dKO organoids than in wildtype, suggesting apoptosis plays a role in RGC localization.
Conclusions :
Retinal organoids recapitulate the temporal generation and developmental cell death of RGCs observed in the retina despite the formation of necrotic cores. Preventing apoptosis by BAX/BAK dKO does not alter RGC density in organoids, supporting the role for necrosis in RGC loss.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.