Abstract
Purpose :
The common pathology of retinal ganglion cell (RGC) degeneration in different types of glaucoma, associated with different risk factors, suggests an intrinsic vulnerability of these cells to disease. We have recently shown that RGCs derived from SIX6 risk allele glaucoma patient-specific induced pluripotent stem cells (iPSCs) develop abnormally and display morphological and functional deficits, which might make them susceptible to disease if maintained into adulthood. Here, we used single cell transcriptome analysis to determine where in the RGC developmental trajectory the effect of the risk allele was manifested and whether or not it influenced the RGC sub type diversity.
Methods :
RGCs generated from age- and sex-matched control and SIX6 risk allele POAG-specific iPSCs at 15DIV were subjected to single cell RNAseq (scRNAseq) using the 10X Genomics Chromium System. The clustering methods included K means and unsupervised graph-based method. t-stochastic neighbor embedding (t-SNE) was carried out to visualize the data in 2D space. Immunocytochemical analysis was carried out to corroborate subtype diversity.
Results :
t-SNE plots identified 9 and 13 distinct clusters for control and Six risk allele RGCs, respectively, each characterized by unique set of differentially expressed genes (DEG). Gene ontology analysis of DEGs in controls identified developmentally related clusters that led to a trajectory, beginning with clusters with pan neural stem cell identity to mature RGCs with intervening progenitor/precursor clusters in different stages of transition. The developmental trajectory of SIX6 risk allele RGCs were similar to controls but the majority of cells belonged to progenitor/precursor clusters and RGC cluster was immature in terms of RGC specific gene expression and subtype diversity.
Conclusions :
Single cell transcriptome analysis of SIX6 risk allele RGCs revealed that the risk allele negatively affects the developmental trajectory of RGCs, specifically, the transition of the RGC precursors toward maturity, compared to controls. This developmental barrier is reflected in subtype diversity, which may influence RGC and optic nerve susceptibility/resistance to degenerative changes in glaucoma.
This is a 2020 ARVO Annual Meeting abstract.