Abstract
Purpose :
In dry age-related macular degeneration (AMD), loss of retinal pigment epithelium cells in the macula results in irreversible damage to the overlying photoreceptor cells, leading to central vision loss. Therefore, potential treatments aim at preventing photoreceptor death. To contribute to this objective, it is crucial to establish pathophysiologically relevant disease models for the evaluation of these therapeutic strategies. The purpose of this study is to develop a human induced pluripotent stem cell (hiPSC)-derived retinal organoid (RO) model of photoreceptor cell death that mimics aspects of AMD pathophysiology and that can be applied to therapeutic development. Since smoking is one of the main environmental factors associated with AMD, we have devised a strategy involving the treatment of human ROs with cigarette smoke extract (CSE). We validated this model by assessing its impact on oxidative stress, mitochondrial health, and cell death mechanisms, and we developed assays for the quantification of outcome measures in live ROs.
Methods :
Two hiPSC lines were differentiated into ROs for 180 days to allow for the development of relatively mature photoreceptor cells. ROs were treated with different concentrations of CSE or vehicle control, and evaluated at 0, 6, 24, 48 and 72 hrs. Inflammation, cell death, and photoreceptor markers were analyzed by ELISA, immunofluorescence, confocal microscopy, and quantified using Fiji software. Mitochondrial health and oxidative damage were assessed in live ROs through JC1 and DHE assays and evaluated by 3D automated reporter quantification using a TECAN spark plate reader.
Results :
Our results showed an increase in TUNEL staining as well as caspase-3 and caspase-8 activation in CSE-treated ROs compared to controls. Damage was mostly localized to photoreceptor cells and could be regulated by titration and timing. CSE treatment also increased ROS production and mitochondrial membrane depolarization.
Conclusions :
We report the first human RO model that recapitulates aspects of AMD pathophysiology. The combination of this model with our quantitative outcome measures in live organoids constitutes a powerful paradigm for the evaluation of potential therapeutics for the preservation of photoreceptor health. This valuable tool will complement animal studies, accelerating the development of new treatments for patients suffering from vision loss due to dry AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.