Efficient generation of laminated and light responsive retinal organoids for use in toxicological assays

Abstract

Purpose : To assess the variability between iPSC lines to differentiate to laminated, light responsive retinal organoids and assess their response to known neurotoxicants.

Methods : Five iPSC cell lines from patients (retinitis pigmentosa and age related macular degeneration) and unaffected subjects were differentiated to retinal organoids using three dimensional culture conditions. After 18 days in culture, the organoids were kept as pooled (6 well) or in single (96 well) conditions. High resolution scanned images were obtained to quantify the emerging retinal neuroepithelium and Retinal pigmented epithelium in each organoid; immunofluorescence and qPCR determined the cell types present, while a multi-electrode array was used for functional assays. Two known neurotoxicants, chloroquine and moxifloxacin, were applied to retinal organoids to assess their response and validate their usefulness for toxicology and pharmacology screening. The proteomic response of the organoids to both neurotoxicants was assessed utilising liquid chromatography coupled to mass spectrometry.

Results : All iPSCs generated retinal organoids (within an efficiency range of 45-89%) containing the key retinal cell types that formed functional synapses and responded to light and chemcial stimuli. We succesfully transferred this differentiation protocol to 96 well plates, whilst maintaining lamination, ultrastructural features and light responses, allowing for the scalability and automation required for toxicology and pharmacology studies. Toxicology screening for chloroquine indicated an acute response in the ganglion cell like layer and increases in proteins such as spermine synthase and Alpha-2-HS-glycoprotein. Moxifloxacin affected the photoreceptors, ganglion cells and increased secretion of proteins like Retinol binding protein 1 and Sec1 family domain-containing protein 1.

Conclusions : Our data show that light responsive retinal organoids can be generated from iPSC lines in a format which allows for easy scalability and automation, resulting in better drug discovery and delivery rates bridging the gap between compound screening and clinical trials.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.