Mapping the structure and function of whole-mount retinal organoids

Abstract

Purpose : Cell therapy is a therapeutic avenue for retinal degenerative diseases. Photoreceptor transplantation faces challenges in obtaining high-quality and mature photoreceptors that can effectively integrate and function, facilitating regeneration. For designing therapies we will produce retinal organoids (RO) from ihPSC. To enhance our understanding of RO maturation in a 3D and dynamic context, we aim to characterize the structure and function of retinal neurons. This will help identifying the optimal timing for harvesting photoreceptors for subsequent implantation. In this work, we focus on the characterization of RO derived from ihPSC from healthy and vision-impaired individuals, specifically Retinitis Pigmentosa type 25

Methods : We tracked calcium activity in 3D with light-sheet fluorescence microscopy (LSFM). This optical configuration allows fast tracking of dynamics (10 Z-planes at 5 Hz) occurring on a volumetric scale (600x830x100µm) with minor photo-toxic effects. For visualizing changes in the calcium influxes, we used viral vectors encoding for GCaMP6s. Changes in fluorescence intensity were quantified and analysed in a 3D fashion to determine the pattern and frequency of calcium waves in the neuronal network. Subsequently to the life dynamic recordings, and to characterize the structure of whole-mount mature ROs (up to day 250), we optimized an optical clearing method (FluoClear BABB) in combination with improved antibody permeabilization

Results : We imaged in high-resolution the calcium activity of RO derived from healthy and vision-impaired donors and quantified differences in the functional communities of neurons by using transfer entropy. Using our clearing protocol, we identified the morphology and population of the three neuron-path that provide the direct route for visual information transmission: cone and rod photoreceptors, bipolar and ganglion cells

Conclusions : RO are promising in vitro models for studying retinal diseases. Our findings shed more light on the temporal, spatial and functional organization of retinal cells within the organoid. Our LSFM can be applied to study fast events occurring in mm range samples with cellular resolution

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Comparison of 3x clearing methods and identification of retinal cells

 

Functional connectivity matrix study from calcium imaging data