June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
In situ hybridization of human trabecular meshwork tissues shows localization of cell-type specific expression of genes identified by scRNA-seq
Author Affiliations & Notes
  • Hua Yang
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Gaurang Patel
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Wen Fury
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Ming Yuan
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Ying Hu
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Jingtai Cao
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Daniel Stamer
    Duke University, Durham, North Carolina, United States
  • Carl Romano
    Ophthalmology, Regeneron pharmaceuticals, Tarrytown, New York, United States
  • Footnotes
    Commercial Relationships   Hua Yang, Regeneron Pharmaceuticals (E); Gaurang Patel, Regeneron Pharmaceuticals (E); Wen Fury, Regeneron Pharmaceuticals (E); Ming Yuan, Regeneron Pharmaceuticals (E); Ying Hu, Regeneron Pharmaceuticals (E); Jingtai Cao, Regeneron Pharmaceuticals (E); Daniel Stamer, None; Carl Romano, Regeneron Pharmaceuticals (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3422. doi:
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      Hua Yang, Gaurang Patel, Wen Fury, Ming Yuan, Ying Hu, Jingtai Cao, Daniel Stamer, Carl Romano; In situ hybridization of human trabecular meshwork tissues shows localization of cell-type specific expression of genes identified by scRNA-seq. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3422.

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

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Abstract

Purpose : Dysfunction of the trabecular meshwork (TM) leads to increased outflow resistance, resulting in elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) patients. We have identified several different cell types in human TM by scRNA-seq. The current study aims to map the molecularly identified cell types anatomically.

Methods : Human donor eyes were processed for paraffin embedding and sectioning for in situ hybridization assay to determine TM target gene expression. Manufacturer’s instruction (Advanced Cell Diagnostics) was followed and RNAscope® 2.5 HD Detection Kit – RED (Cat. No. 322360) was used. Specific human probes were designed for each of the cell clusters identified in TM by scRNA-seq. Negative controls, e.g. DapB and positive probes were also included. Target mRNA can be visualized as red chromogenic dots around tissues and also fluorescence signal detected by a fluorescent microscope. Depending upon numbers of punctate dots present within each cell boundary, a semi-quantitative scoring guideline was used to analyze results.

Results : Human TM scRNA-seq identified 12 distinct cell type clusters. In human TM tissue, RNAscope probes for the Schwann cell-like cluster (AQP7P1 and SCN7A) were localized to ciliary muscle and scleral spur; the myofibroblast cluster (RSPO4) was found in the TM except for the JCT region; fibroblast-like cells (expressing DCN and PDGFRA) were abundant in the TM, including the JCT region; smooth muscle cells (expressing CDH11 and MYH11) reflected ciliary muscle and some TM cells. Macrophage type cell (C1QB, TYROBP) were also abundant in the TM. Most genes were expressed in more than one cluster, for example TAGLN was found in myofibroblast and smooth muscle, and anatomically was found in ciliary muscle and TM. We also found endothelium (FLT1) and lymphatic (LYVE1, FN1, FLT4) cell clusters localized to Schlemm’s canal.

Conclusions : We have identified 12 cell types in hTM with its own specific molecular markers by RNAscope. Our findings illustrate the diversity of cells in the TM and the surrounding regions and lay the groundwork for more mechanistic studies on the physiology and pathology of the human outflow pathway.

This is a 2020 ARVO Annual Meeting abstract.

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