Functional development of cone photoreceptors in human stem cell derived retinal organoids

Abstract

Purpose : Human stem cell derived retinal organoids (ROs) hold promise for therapy and understanding retinal diseases as well as for studying development of human retina in vitro. In fact, recent gene expression studies show that the developmental timeline in ROs closely matches that of human retina in vivo. During differentiation, ROs can be categorized into three distinct developmental stages. However, not much is known about the functional development of neurons in ROs including photoreceptors. Here, we assess in detail the light sensitivity and electrical properties of cones at distinct time points of RO development. In addition, we determine if lack of a defined retinal pigment epithelium (RPE) limits the availability of chromophore for cone function at different stages of RO development.

Methods : We used electrophysiology to record light responses from >500 cones across 8 distinct developmental time points from ROs (170-340 days in vitro). We treat ROs of different ages with 9 cis-retinal and compare cone light sensitivity with those in untreated ROs.

Results : At d170 (stage 2), we did not observe any light-evoked responses from cones in ROs. This is consistent with previous findings that the photoreceptors are not fully developed at stage 2 and may not have the required phototransduction machinery to elicit light responses. Cones in stage 3 ROs (>d200) exhibit light responses with peak sensitivity between d250-d260. Beyond d300, the fraction of light sensitive cones significantly decreases. Despite the differences in light sensitivity, membrane properties of cones remain comparable across different time points of RO development. Cone light responses also demonstrate adaptation at higher luminance like typical vertebrate cones. Addition of 9-cis retinal significantly improved cone light sensitivity across all time points of RO development probed in this study.

Conclusions : By assaying cone function at various stages of RO development, we identified d250-260 as the timepoint of peak cone sensitivity. The timeline of cone functional maturation suggests that cone phototransduction reaches its maximal sensitivity at later stages of fetal development continuing up to early stages of postnatal development, similar to human retina development in vivo. Our results further show that chromophore availability, most likely from the RPE, plays a significant role in cone light sensitivity in ROs during development.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.