Abstract
Purpose :
Advancements in the use of human pluripotent stem cells (hPSCs) have created new techniques to derive complex tissues in vitro and three-dimensional organoid derivation recreates an environment for the development of retinal tissue for real-time analyses. Understanding the molecular mechanisms that occur during retinogenesis, specifically as uncommitted eyefield progenitors progress to retinal progenitors, will aid in creating faithful retinal and photoreceptor cells. MicroRNAs (miRNAs) are non-coding, short RNAs that negatively regulate gene expression by targeting mRNA for degradation or inhibition of translation. Retinal development studies have identified roles for miRNAs in mammals, but the study of miRNAs in human-specific retinogenesis is underappreciated.
Methods :
A SIX6-GFP and VSX2-tdTomato dual reporter was introduced into hPSCs via the CRISPR-Cas system and differentiated into three-dimensional retinal organoids. Organoids were collected at specific time-points during development based on GFP or tdTomato fluorescent expression, and small RNA-seq and bioinformatic analyses were performed.
Results :
Retinal organoids expressed GFP+ and tdTomato+ signals, indicating SIX6 and VSX2 expression, respectively, during development. Transcriptomic analysis identified miRNA expression changes that occur during the transition of early SIX6-GFP+ eye field progenitors to VSX2-tdTomato+ retinal progenitors. Bioinformatic analysis identified putative miRNAs targeting SIX6, VSX2, and other retinal genes, suggesting miRNA involvement in retinal development. Pathway analysis of miRNA targets identified possible involvement in axon guidance and growth factor signaling.
Conclusions :
Further analyses of the roles of miRNAs will lead to better understanding of gene regulation during retinal formation. Elucidating the full breadth of gene expression and regulation in retinal development will contribute to knowledge of the basic mechanisms of both human development and disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.