Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Sandeep Kumar; Aitor Andueza; Nicolas Villa-Roel; Juyoung Kim; Dong-Won Kang; Hanjoong Jo

doi:10.3791/63128

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Method Article

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

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DOI:

10.3791/63128

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October 4th, 2021

Sandeep Kumar^*¹ , Aitor Andueza^*¹ , Nicolas Villa-Roel¹ , Juyoung Kim¹ , Dong-Won Kang¹ , Hanjoong Jo¹^,²

¹Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, ²Division of Cardiology, Georgia Institute of Technology and Emory University

^* These authors contributed equally

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Summary

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Abstract

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

Introduction

This laboratory previously demonstrated that induction of d-flow leads to quick and rugged atherosclerosis development in hyperlipidemic mice¹^,². The novel mouse model of d-flow-induced atherosclerosis was possible using partial carotid ligation (PCL) surgery ³. PCL surgery induces low and oscillatory blood flow condition or d-flow in the ligated left carotid artery (LCA). In contrast, the contralateral right carotid artery (RCA) continues to face stable laminar flow (s-flow). Previously, to understand the effect of d-flow on endothelial cell....