Accelerated Retina Organoid Differentiation and Maturation through a Brief Exposure to Electrical Field

Abstract

Purpose : Previous studies demonstrated that electrical stimulation (EF) promotes the differentiation of human embryonic stem cells (hESCs) into a diverse range of neuronal cell types compared to growth factor-induced differentiation, which leads to more limited neuronal cell types. This study aims to investigate the potential of biphasic electric stimulation to expedite photoreceptor lineage commitment and maturation and augment neuronal development within retinal organoids(RO).

Methods : Retina organoids(hESC-RO, Day 90) were subjected to electrical stimulation using a custom-designed EF setup. EF parameters including frequency, amplitude, and duration were optimized through preliminary immunohistochemistry and qPCR experiments. Organoids were analyzed for neuronal development, and photoreceptor maturation using whole RNA sequencing. Experimental groups used were Control (n=50), Biphasic group 1 (n=50), Biphasic group 2 (n=50), and direct current(DC, n=12).

Results : EF significantly accelerated the differentiation and maturation of ROs. Immunohistochemical analysis and qPCR demonstrated abundant expression of photoreceptor markers such as general photoreceptor markers (Recoverin, CRX), rod-specific markers(Rhodopsin), and cone-specific markers (SAG, red-green opsin, blue opsin, cone arrestin) and increased expression of RGC markers Muller cell markers. A higher number of photoreceptors were there in biphasic group 1 than in biphasic group 2, DC, and control(P≤0.01). Gene expression profiling(whole RNA seq) confirmed the upregulation of key genes associated with retinal development, neuronal activity, retinal homeostasis, retinal layer formation, neuronal function, and visual perception after EF.

Conclusions : Our findings demonstrate that EF exerts a profound impact on the differentiation and maturation of ROs derived from hPSCs. In previous studies using retinal organoids, various signaling factors/small molecules were used at various time points and concentrations in augmenting RO maturation with varying success. Electrical stimulation showed a better way for consistently higher expression of photoreceptor markers enabling efficient harvesting of the desired cell types with higher yield. This approach holds promise for applications in disease modeling, in vitro retinal model systems, drug screening, epigenomics, and regenerative medicine for retinal disorders.

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