Abstract
Purpose :
To assess landscapes of exosomal miRNAs in retinal organoids (ROs) of different qualities to detect exosomal miRNAs related to better and worse differentiation process of ROs.
Methods :
The ROs were differentiated from hiPSCs according to a retinal differentiation protocol. Exosomes were extracted from the supernatant of the retinal-organoids culture medium using an exosome isolation kit. Nanoparticle tracking analysis (NTA) was performed to determine the concentration and size of organoid exosomes. Exosomes derived from ROs were morphologically analyzed by transmission electron microscopy. The western blot was used to determine the exosome protein markers. The total and exosomal RNAs were extracted, RNA quality was assessed and RNA quantification was performed.
Results :
The number of expressed miRNA species were significantly higher in exosomes produced by better-quality ROs than those of worse-quality ROs (406.8 ± 15.4 vs. 278.0 ± 13.7, p<0.05). To identify specific exosomal miRNAs produced by better-quality ROs, a differential expression gene (DEG) analysis was conducted. In the exact test, 10 miRNAs were found to be upregulated, while 40 miRNAs were downregulated in the exosomes derived from better-quality ROs in comparison to those from inferior ROs (FDR q<0.05). Next, a non-parametric Mann-Whitney's U test was conducted to identify markers for assessing the quality of ROs. The results showed that 63 genes were upregulated, and 21 miRNAs were downregulated in the exosomes derived from better-quality ROs compared to those from worse-quality ROs. (p<0.05). To validate the potential of miRNA expression levels as markers for better-quality ROs, quantitative real-time PCR was conducted on the target miRNAs using additional exosome samples isolated from conditioned media of independent better-quality and worse-quality ROs. The results indicated that the level of hsa-miR-654-3p in better-quality ROs was 3.47 ± 0.92 times higher than that in worse-quality ROs (p=0.019). Hsa-miR-3605-3p displayed a trend towards an increase of 2.10 ± 0.48 times in better-quality ROs compared to worse-quality ROs (p=0.077).
Conclusions :
Properly differentiated ROs showed higher diversity of exosomal miRNAs, and has-miR-654-ep and has-miR-451a were considered potential biomarkers for RO differentiation grading.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.