Abstract
Purpose :
The progressive death of cones ultimately causes loss of vision in patients with retinal degenerative diseases, and there is currently no treatment available. Differentiation of human-induced pluripotent stem cells (hiPSCs) into a cone-rich cell population represents a potential therapy for cone/retinal degeneration. COCO is a multifunctional antagonist of the WNT, TGF-β, and BMP pathways and has been shown to promote cone differentiation from hiPSCs. NRL and NR2E3 are key transcription factors for photoreceptor differentiation. Thyroid hormone is known to orchestrate cone development. This work investigated the potential of differentiating hiPSCs into a cone-rich cell population by targeting these different regulatory pathways.
Methods :
Wild-type iPS(IMR90)-4 cells and mutant iPS(IMR90)-4 cells with deletion of NRL or NR2E3 were used. NRL or NR2E3 knockout cell lines were generated using Synthego Gene Knockout Kit V2 and confirmed by Sanger sequencing. Embryonic bodies generated from the wild-type and mutant cells were cultured in a retinal induction medium containing COCO (30 ng/ml) for 4 weeks. Neuronal retinal vesicles formed were then transferred into bioreactors (Synthecon) and cultured in the COCO differentiation medium containing triiodothyronine (T3, 5 nM) for another 2-3 months, followed by evaluation of the expression of photoreceptor genes and differentiation of cones using qRT-PCR and immunofluorescence labeling.
Results :
Treatment with COCO enhanced differentiation of IMR90-4 cells into cones and increased expression of the photoreceptor precursor genes (PAX6, CRX, and RXR) and the cone-specific genes (OPN1SW, ARR3, and GNAT2). Deletion of NRL or NR2E3 further enhanced the gene expression and cone differentiation. COCO plus thyroid hormone induced differentiation of IMR90-4 cells into M-cones and increased expression of OPN1MW, compared with cells treated with COCO only.
Conclusions :
COCO induces differentiation of IMR90-4 cells into retinal/photoreceptor precursors and cone photoreceptors. Deletion of NRL or NR2E3 promotes cone differentiation. Thyroid hormone promotes differentiation of M-cones from IMR90-4 cells. These findings highlight the potential of differentiation of hiPSCs into a cone-rich cell population by targeting multiple regulatory pathways.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.