Abstract
AbstractEndothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.Understanding of endothelial functions would be accelerated by methods for the specific isolation of these cells from archived human specimens. Here, the authors use the endothelial transcription factor Erg to isolate nuclei from mouse and human tissues, paving the way for high-throughput characterization of the function of endothelium in homeostasis and disease.
Highlights
Endothelial cells line the vasculature of the brain, where they contribute to the function of a wide range of fundamentally important tissues including maintenance of the blood brain barrier, immune cell surveillance, blood flow, and the maintenance of neural stem cells
To determine whether endothelial transcripts were enriched, and to see whether other cell populations might be enriched, we looked for the enrichment of RNA markers of specific cell types based on a meta-analysis of single-cell data from mouse brain samples[17]
We examined the top cell-specific transcripts enriched in major brain cell types, as we had done in the mouse brain, by focusing on only splice junction reads (Fig. 3B–D and SI Table 3)
Summary
Endothelial cells line the vasculature of the brain, where they contribute to the function of a wide range of fundamentally important tissues including maintenance of the blood brain barrier, immune cell surveillance, blood flow, and the maintenance of neural stem cells. Despite their critical brain functions, endothelial cells are a relatively small proportion of all cells in the brain, with ~2–4% in total area coverage[1], and ~0.2–7% as a total proportion of all brain cells based on single-cell sorting experiments[2,3,4,5,6]. Together with the limited availability of acutely isolated human brain samples, these complications have severely limited the analysis of endothelial cell transcripts in human brain pathology
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