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Related Experiment Video

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Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Mark I Watson1, Peter Barabas1, Mary McGahon1

  • 1Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland.

Molecular Vision
|December 31, 2020
PubMed
Summary

This study optimized single-cell RNA sequencing for bovine retinal endothelial cells, revealing insights into the genes forming the blood-retina barrier and identifying cell homogeneity related to proliferation.

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Area of Science:

  • Ophthalmology
  • Molecular Biology
  • Genomics

Background:

  • The blood-retina barrier (BRB) is crucial for retinal health.
  • Understanding the molecular components of the BRB is essential for treating retinal diseases.

Purpose of the Study:

  • To characterize retinal endothelial barrier properties.
  • To analyze individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs).

Main Methods:

  • Single-cell RNA sequencing (scRNA-seq) workflow optimization.
  • Utilized Fluidigm C1, Nextera XT, and Illumina NextSeq for library preparation and sequencing.
  • Data analysis with R packages (Scater, SC3, Seurat), ASAP, Outrigger, and Cytoscape.

Main Results:

  • RMECs form a homogeneous population in culture, primarily differing in proliferation status.
  • Identified capillary origin for most cells and developed an in silico BRB model.
  • Discovered a subgroup of barrier genes correlated with PECAM-1 and observed extensive alternative splicing in barrier genes.

Conclusions:

  • Optimized scRNA-seq workflow for primary RMECs.
  • Provided fundamental insights into genes involved in retinal microvascular barrier formation.