Abstract
Purpose :
During development, the proper histoarchitecture of the retina is achieved through a delicate balance between proliferation, differentiation, and cell death. Retinal ganglion cells (RGCs) in particular increase in numbers during development except for two periods of sharp decline that appear conserved in vertebrates. The mechanisms responsible for these phenomena are incompletely understood. Notably, we observed that the first wave of RGC death is replicated within human stem-cell derived retinal organoids. Thus, we took advantage of this model to investigate the mechanisms of RGC death in the developing human retina.
Methods :
Retinal organoids were differentiated from human induced pluripotent stem cells (hiPSCs) and collected weekly between weeks 6 and 10 of differentiation. The timeline of the first wave of RGC death was characterized by immunofluorescent staining of RGCs and further confirmed in organoids generated from a fluorescent reporter line. Potential microglia involvement was evaluated at the transcriptional and translational levels. TUNEL and activated caspase 3 immunostaining were performed to assess for apoptotic cell death, and Western blot was used to identify the intracellular pathways involved.
Results :
Retinal ganglion cell numbers consistently decreased at week 8 of differentiation, as observed by different quantitative methods. Immunofluorescent staining for IBA1 and RT-PCR for microglia markers verified the absence of microglia in this model, while a spike in cell death coinciding with the trough in RGC numbers was observed by TUNEL staining and cleaved caspase 3 immunofluorescence. Interestingly, we observed a decrease in the BAX/BCL2 ratio and a marked increase in cleaved caspase 8, suggestive of the involvement of the extrinsic apoptotic pathway.
Conclusions :
Human retinal organoids recapitulate the first wave of developmental ganglion cell death in vitro. The absence of microglia in this model and the evidence of caspase-dependent apoptosis revealed an additional contributor to regulating RGC numbers in human retinal development. The activation of caspase 3 and caspase 8 suggest the recruitment of the extrinsic apoptotic pathway. This knowledge can lead to new insights regarding congenital retinal abnormalities.
This is a 2021 ARVO Annual Meeting abstract.