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Kirstin Brooke VanderWall, Clarisse M Fligor, Jason S Meyer; Single cell RNA-sequencing elucidates the diversity of human pluripotent stem cell-derived retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4765.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) are the projection neurons of the visual system typically characterized by the transmission of signals from the retina to the brain. However, individual cells within this group may differ in their functional, morphological, and molecular features. Previous studies have explored RGC heterogeneity in a variety of animal models, although these studies remain limited in humans. Human pluripotent stem cell (hPSC)-derived RGCs can serve as a foundational model for elucidating the diverse nature of human RGCs as these cells can be readily differentiated and isolated for high-throughput analyses such as single cell RNA-sequencing. Thus, efforts of the current study examined the diversity and cellular heterogeneity of RGCs derived from hPSCs.
hPSCs were differentiated toward an RGC fate using an established, step-wise protocol yielding retinal organoids containing all of the major cell types of the retina. RGCs were purified and examined using immunocytochemistry and western blot analyses specific to known molecular markers of RGC subtypes in mouse and macaque models. Additionally, the heterogeneity of RGCs was also examined using 10x Chromium single cell RNA sequencing to elucidate molecular differences among the RGC population, with these transcriptional features confirmed by immunocytochemistry and western blot analyses.
hPSC-derived RGCs displayed robust expression of canonical RGC associated proteins including BRN3 and RBPMS. More so, hPSC-derived RGCs expressed a variety of markers previously associated with RGC subtypes in animal models. Single cell RNA-sequencing revealed 16 distinct clusters within the purified RGC population, with the expression of many markers previously identified within non-human primate models. These subpopulations were further examined for novel molecular markers as well as correlations of these subpopulations to their potential functional properties.
Results of the current study reveal the diversity among human RGCs, with results demonstrating close transcriptional similarities to recent non-human primate RGC subtype data. Furthermore, these results can be used as a foundational step toward the discovery and detailed investigation of human RGC subtypes, including their physiological properties as well as how these subtypes may be differentially affected in blinding disorders such as glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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