Abstract
Purpose :
To characterize and uncover potential regulators of human retinal ganglion cell (RGC) differentiation.
Methods :
H9 human ESCs with a POU4F2-P2A-tdTomato-P2A-Thy1.2 reporter were differentiated into RGCs according to a small molecules-based method established in our lab. On day 28, 35, 42, and 49 of RGC differentiation, cells were dissociated and purified by MACS with mouse CD90.2 beads. Purified RGCs from each of these time points were prepared for single-cell RNA sequencing using the Drop-seq protocol and sequenced on a Novaseq 6000. Reads were aligned to reference GRCh38/hg38. Data processing and analysis were done with the Seurat package. By label transfer analysis, cell identity probabilities and predicted labels were assigned to each cell using a human fetal retinal single-cell RNA-seq dataset as a reference.
Results :
After quality control, a total of 3,883 cells were included in the downstream analysis. Purified cells were clustered into 5 clusters (clusters 0 – 4). Clusters 0 and 1, which made up the majority of the population, were enriched in RGC markers. Cluster 2 and Cluster 4 expressed retinal progenitor cells (RPCs) markers. Label transfer analysis showed concordant cell type annotation with that based on cell marker expressions. Most cells in clusters 2 and 4 were predicted to be retinal precursor cells (RPCs), while most cells in clusters 0 and 1 were labeled as RGCs.
Clustering of transcription factor (TF) expression revealed multiple TFs that were highly enriched in RGC clusters yet have not been previously shown to be related to RGC differentiation (e.g. PBX3, ST18, and ZBTB38). Co-expression analysis showed that PBX3, ST18, and ZBTB38 were also co-expressed with RGC markers POU4F2 and ISL1 in some RGCs. Given the read depth and sparse nature of single-cell RNA-seq data, it is reasonable to expect a higher co-expression level using other methods, such as IHC, suggesting that they may participate in RGC differentiation.
Conclusions :
PBX3, ST18, and ZBTB38 are expressed in hESC-derived RGCs and are co-expressed with RGC markers in human RGCs, suggesting that these TFs potentially may play a role in human RGCs. Gene expression gain- and loss-of-function experiments are being planned to more directly assess the potential roles of these genes in human RGC differentiation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.