June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Single-cell RNA-seq of Human Embryonic Stem Cell-derived Retinal Ganglion Cells Unravels New Potential Regulators of Retinal Ganglion Cell Differentiation
Author Affiliations & Notes
  • Weifeng Li
    Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Cynthia A Berlinicke
    Department of Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Jie Cheng
    Department of Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Xiaoli Chang
    Department of Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Felipe Takaesu
    Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States
  • Donald J Zack
    Department of Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
    The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Weifeng Li None; Cynthia Berlinicke None; Jie Cheng None; Xiaoli Chang None; Felipe Takaesu None; Donald Zack None
  • Footnotes
    Support  National Eye Institute (NIH), Research to Prevent Blindness, Edward N. & Della L. Thome Memorial Foundation, Guerrieri Family Foundation
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3204. doi:
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    • Get Citation

      Weifeng Li, Cynthia A Berlinicke, Jie Cheng, Xiaoli Chang, Felipe Takaesu, Donald J Zack; Single-cell RNA-seq of Human Embryonic Stem Cell-derived Retinal Ganglion Cells Unravels New Potential Regulators of Retinal Ganglion Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3204.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

 

 

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