Abstract
Purpose :
Retinal organoids (ROs) derived from human induced pluripotent cells (hiPSC) are a valuable model to study development and diseases of the human retina in vitro. While many studies focus on the structure of ROs, far fewer have addressed retinal function. When dark-adapted ex vivo retinas from animals are incubated in FM dyes in the dark, photoreceptor synapses uptake the dye as synaptic vesicles are recycled, labeling the photoreceptor terminals in the OPL. We have studied synaptic vesicle recycling and release in the outer plexiform layer (OPL) of developing ROs using FM dyes.
Methods :
ROs (D180-220 of differentiation) were incubated in 30 mM FM14-3X (a fixable analog) at 37 oC in Brain Phys media or 0 Ca2+ media. Excess FM14-3 was quenched in 1 mM advasep-7 in 0 Ca2+ Ringer’s solution for 30 minutes. Live ROs were mounted on filter paper and sliced at 100 mm on a tissue chopper. Slices were imaged by confocal microscopy and high K+ BP was added to depolarize cells to release vesicles. The slices were imaged for 20 minutes in High K+ (50 mM K+) and fluorescence intensity in the OPL was measured across time to calculate decay constants. Some organoids were fixed in 4% PFA, cryosectioned and counterstained with DAPI. Fluorescence intensity was measured in the OPL and compared to organoids incubated in FM14-3 in 0 Ca2+ media.
Results :
FM14-3 labeling was observed in the OPL of ROs after incubation in FM143 for 30 minutes, 1 hour and 2 hours. FM14-3 labeling showed exponential decay over time in the presence of 50 mM K+ with an average time constant of ~20 minutes. Background decay was linear and with a significantly different slope than the OPL. In fixed ROs, fluorescence intensity was greater in the OPL of ROs incubated in Ca2+ containing media.
Conclusions :
Synaptic vesicle recycling and release occurs in ROs derived from hiPSC. The time constant of vesicle release is ~20 minutes, slower than calculated for animal retinae from multiple species which is ~5 minutes. Using fixable analogs, fluorescence intensity is measurable in the OPL of cryosections. These findings suggest that photoreceptors in ROs derived from hiPSC have functional synapses in the OPL that can be studied in both live and fixed ROs. This functional assay may be useful in comparing ROs derived from patients with retinal diseases with those derived from controls as well as in drug validation studies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.