Abstract
Purpose :
Retinal ganglion cells (RGCs) collect and transmit optical information to the brain and are implicated in various ocular pathologies. Although RGCs are classified into various subtypes based on morphology, function, and gene expression signatures, a consensus classification scheme is still lacking. We utilized single-cell RNA sequencing (scRNA-seq) to characterize subtype diversity in RGCs directly converted from human pluripotent stem cells (PSCs).
Methods :
SIX6-p2A-h2b-eGFP/POU4F2-p2A-tdTomato dual reporter PSCs with a Tet-On transgene cassette controlling RGC enriched transcription factors were pre-patterned with a BMP inhibitor and differentiated into neurons. Individual cells at days 7, 14, and 21 were lysed and processed for library preparation using microfluidics-free particle-templated instant partition sequencing (PIP-seq) technology. cDNA was assessed for quality/quantity and libraries were sequenced to obtain >200 million reads/sample. Error-corrected reads were aligned to the human genome with STARsolo and distinct cells were called using PIPseeker. Scanpy was employed for analysis of the feature-cell barcode matrix, mitochondrial read filtering (to remove dead cells), PCA-based dimensionality reduction, and UMAP generation (to cluster cells).
Results :
Cell clusters identified pan-neuronal (TUBB3, MAP2, and SYT1) and RGC-enriched genes (STMN2, GAP43, and SNCG). Conversely, markers for other retinal cell types (glial, bipolar, amacrine, horizontal, and photoreceptor cells) were negligibly expressed thus supporting an RGC identity. Analysis of functional RGC subtype markers revealed both directionally sensitive (DS) and alpha RGCs with a general trend towards the upregulation of DS-like cells. Predictive label transfer analysis classified the cells as 40% midget, 13% parasol, 7% ipRGC, and 40% other RGC subtypes. Moreover, heterogeneous expression was noted for genes ascribed to RGC injury susceptibility genes despite persistent expression of survival-enhancing neurotrophic factor receptors.
Conclusions :
scRNA-seq of the RGC-induced neurons indicated RGC heterogeneity based on gene expression signatures. For each classification scheme, markers were co-expressed across several clusters which suggested that these groupings may not be as binary or discrete in developing RGCs in vitro as is thought to occur in other species warranting further analysis in other developmental models.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.