Abstract
Purpose :
Although scRNA-Seq enables identification of distinct and changing cell populations during retinal development, the spatial resolution of those cell populations is missing. Spatial transcriptomics (ST) enables the visualisation and quantitative analysis of the transcriptome in spatial location, providing a novel tool for detailed studies of human retinal development. In this study ST is used for the first time on pluripotent stem cell derived retinal organoids to examine the spatial resolution of retinal cell-types during development.
Methods :
Retinal organoids were cultured until day 210 of differentiation and collected at day 10, 20, 35, 45, 60, 90, 150 and 210 for ST and scRNA-Seq. experiments. ST experiments were performed using the 10xVisium Spatial Gene Expression Kit (10x Genomics). For this purpose, retinal organoids were fresh frozen and stored at -80°C. Cryosections of each sample were collected on individual 10xVisium Gene Expression slides and processed according to manufacturer's protocol. Whole transcriptome libraries were sequenced, analysed, and visualized using Space Ranger and Spaniel R package. For scRNA-Seq. retinal organoids were dissociated followed by cell capture and library generation using the Chromium Single Cell 3′ Library & Gel Bead Kit (10x Genomics). Uniform manifold approximation and projection plots were used to visualise the clusters.
Results :
Early retinal organoids (day10) were dominated by a cell cluster expressing eye-field marker genes whereas later stages (day20/35) showed several progenitor cell clusters including retinal progenitor cells. Additionally, ocular surface epithelium and lens/cornea cells were found at these stages. Retinal ganglion cells appeared at day 35 and were detected throughout differentiation. Mid stages of retinal development revealed photoreceptor precursor expression at day 90, which matured to rods and cones in late developmental stages (day150/210), where also a bipolar cell cluster was evident. The comparison of ST and scRNA-Seq. data indicated comparable cluster distribution in development.
Conclusions :
ST provides a powerful tool to investigate the spatial resolution changes within a tissue during development, demonstrating here for the first time on human retinal organoids. Thus, ST offers significant insights in normal retinal developmental and/or disease pathological mechanisms.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.