Abstract
Purpose :
Usher syndrome (USH) is an irreversible inherited retinopathy. Our study seeks to decipher how the patient-specific mutation USH2A regulated the differentiation and maturation of retinal cells in retinal organoids (ROs) and retinal pigment epithelium (RPE) derived from patient-specific induced pluripotent stem cells (iPSCs) at a late-phase by microfluidic chip.
Methods :
ROs were generated from iPSCs by reprogramming peripheral blood mononuclear cells of two USH2 patients carrying USH2A gene mutation (c.8559-2A>G). The microfluidic chip was designed to co-culture ROs with RPE cells. The morphological, bulk RNA-seq and single-cell RNA-seq (scRNA-seq) of ROs were assayed on D180 and D300.
Results :
The normal 3D ROs (control) exhibited a hierarchical structure similar to the human retina including INL, OPL, and ONL. In contrast, ROs from the USH group did not have distinct OPL and INL layers (Fig. 1A-D). Electron microscopy showed obvious membranous-rich structures at the apical ciliary tip in the control ROs, while all photoreceptor cells in the USH ROs showed apoptotic morphology (Fig. 1E-F). The ROs of USH patients showed less thickness of the outer segment (OS) than those of normal retinal spheroid. Bulk RNA-seq showed that 2787 DEGs were identified in USH ROs versus control at D180. KEGG disease analysis of these DEGs showed that the involved top 15 processes were mainly enriched in the eye, such as retinitis pigmentosa, cone-rod dystrophy, and Usher syndrome (Fig. 1G). The expression of phototransduction-related genes such as SAG and OPN1MW, were lower in USH ROs than those of the control. scRNA-seq analysis showed that about 70% of the cells in both group ROs were photoreceptor cells, among which rod and cone were 40% and 30% at D300, respectively. (Fig. 1H). In the cell-cell contact category, the number of interactions among cone, MG, rod, RPE, RPC, BP, AC, RGC, and HC in control was significantly higher than those in USH ROs (Fig. 1I).
Conclusions :
The ROs in late-phase from USH2A-associated patients display decreased thickness of OS, down-regulated mRNA of phototransduction related, and aberrant cell-cellular contact among retinal cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.