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Tave van Zyl, Alexi McAdams, Wenjun Yan, Karthik Shekhar, Yirong Peng, Mallory Laboulaye, Aviv Regev, Joshua Sanes; Molecular Classification of Cell Types in the Human Trabecular Meshwork and Surrounding Structures using Single-Cell RNAseq. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1787.
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© ARVO (1962-2015); The Authors (2016-present)
Trabecular meshwork (TM), a complex tissue responsible for IOP homeostasis, is composed of cells whose molecular identities are incompletely characterized. We sought to generate a comprehensive cell atlas of the human TM and surrounding structures in the iridocorneal angle.
We used high-throughput single-cell RNA sequencing (scRNA-seq) to characterize tissue samples obtained via blunt dissection from the iridocorneal angle of 5 post-mortem eyes from 4 genetically unrelated individuals (ages 44 to 78) without personal history or clinical evidence of eye disease or prior ocular surgery. Tissue processing and RNA isolation were performed within ~10 hours of death. We used immunohistochemistry and RNA in situ hybridization on separate donor tissues to validate specific markers for each cell type identified through computational analysis.
Unsupervised clustering of >10,000 high quality cell profiles revealed 15 transcriptionally distinct cell types which were classified based on canonical markers as being endothelial (5 clusters), smooth muscle (2), immune (4), pigment epithelial (1), or neuronal/neuroendocrine/glial (3). Among endothelial cell clusters, Schlemm canal endothelium was identified as a single cluster with distinct markers including PECAM1/CD31, FLT1/VEGFR1 and TIE1. Four additional, transcriptionally distinct clusters of endothelial cells were identified, all of which shared the markers FBLN1, EDN3, PDPN, and LOXL1. Using this cell atlas, we mapped expression of glaucoma susceptibility genes to specific cell types. A single endothelial cluster uniquely expressed ANGPTL7, CHI3L1 and COCH; another cluster with smooth muscle characteristics uniquely expressed NDUFA4L2, GUCY1A3, NOTCH3 and COX4I2. Immune cell clusters included LYVE1+/CD163+ macrophages, CD27+ B cells, NKT cells, and mast cells.
Trabecular meshwork cells, previously described by their morphological and spatial characteristics and bulk RNA seq, can be assigned precise molecular profiles based on differential gene expression determined by scRNA-seq.Unique markers of distinct cell types within the TM identified through scRNA-seq can be used for more targeted investigation into TM dysfunction.Identification of cell-type specific expression of established glaucoma susceptibility genes within the TM may reveal more precise targets for therapeutic development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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