Novel insights into retinal microvascular endothelial cells revealed through single cell RNA-seq analysis.

Purpose: To better characterise the population structure of primary bovine retinal microvascular endothelial cells (RMECs) based upon their individual transcriptomes. Methods: Individual RMECs were captured on the Fluidigm C1 system (Fluidigm), cDNA libraries were prepared using a Nextera XT kit and sequencing performed on a NextSeq (Illumina). Results: Application of a single cell RNA-seq analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main subgroup being proliferating cells. Expression of markers from along the arteriovenous tree suggests that most cells originate from capillaries. An in silico model of the blood retinal barrier was created, including junctional proteins not previously reported within the retinal vasculature. Numerous alternative splicing events involving exons within microvascular barrier genes were observed and in many cases individual cells expressed exclusively one isoform. Conclusions: We have optimised a workflow for single-cell transcriptomics in primary RMECs. Our results have provided fundamental insights into the genes involved in retinal microvascular barrier formation. Overall design: Single cell RNA-seq analysis of retinal microvascular endothelial cells from two separate Fluidigm C1 runs, 49 cells from run 1 and 51 cells from run 2.