Transcriptomic analysis of the harvested endothelial cells in a swine model of mechanical thrombectomy

Abstract

PurposeIn mechanical thrombectomy (MT) for ischemic stroke, endothelial cells (ECs) from intracranial blood vessels adhere to the stent retriever device and can be harvested. However, understanding the molecular biology and the role of the endothelium in different pathological conditions remains insufficient. The purpose of the study was to characterize and analyze the molecular aspect of harvested ECs using cell culture and transcriptomic techniques in an MT swine model relevant to clinical ischemic stroke.MethodsIn swine, preformed thrombi were injected into the external carotid and subclavian arteries to occlude their branches. MT was performed according to clinical routine. The stent retriever device and thrombus were treated with cell dissociation buffer. The resulting cell suspension was analyzed by immunohistochemistry and was cultured. Cultured cells were analyzed using single-cell RNA sequencing (scRNA-seq) after fluorescence-activated cell sorting (FACS).ResultsA total number of 37 samples were obtained containing CD31-positive cells. Cell culture was successful in 90% of samples, and the cells expressed multiple typical EC protein markers. Eighty-nine percent of the sorted cells yielded high-quality transcriptomes, and single-cell transcriptomes from cultured cells showed that they expressed typical endothelial gene patterns. Gene expression analysis of ECs from an occluded artery did not show distinctive clustering into subtypes.ConclusionECs harvested during MT can be cultured and analyzed using single-cell transcriptomic techniques. This analysis can be implemented in clinical practice to study the EC gene expression of comorbidities, such as hypertension, diabetes mellitus, and metabolic syndrome, in patients suffering from acute ischemic stroke.

Isolation and identification of leukocyte populations in intracranial blood collected during mechanical thrombectomy

“…These cells were present only in the intracranial blood, and presumably live based on viability dye. While live endothelial cells can be harvested and cultured from the stent retriever in a swine model of thrombectomy, 45 we collected the intracranial blood prior to stent placement. Circulating endothelial cells are associated with small vessel vasculitis 46 and may have been an underlying, albeit undiagnosed, condition in this patient that increased the propensity toward or severity of hemorrhagic transformation.…”

An Introduction to the Analysis of Single-Cell RNA-Sequencing Data

“…Even though FACS has been shown to have a minimal effect on gene expression, 67 , 68 sorting prolongs the time that cells are not in optimal culture conditions and kept in a single-cell suspension, which could stress the cells and possibly alter mRNA and mitochondrial mRNA expression. 68 , 69 , 70 , 71 Also, passing of cells in small chambers, or through microfluidics or a cell sorter can cause shear stress and impact some cell types more than others in terms of causing cell stress or death, especially because the cells are vulnerable in a single-cell suspension. 72 , 73 , 74 Therefore, delicate cell types might be under-represented in droplet-based single-cell sequencing experiments, especially if the cells were sorted prior to single-cell isolation.…”

Endovascular Device for Endothelial Cell Sampling

“…[2,3] Previous attempts to sample endothelial cells through endovascular sampling strategies, e.g., with coils, stents, stent retrievers, or guidewires, suffer from poor selectivity and a low or highly variable cell yield. [8][9][10][11][12][13][14][15][16] When harvesting ECs from stent retrievers used for mechanical thrombectomy, there was a low EC yield or high contamination of thrombotic material. [15,16] In these studies, fluorescence-activated single-cell sorting (FACS) was used to sort the sample, and in one study, ECs were cultured to allow cytological analysis and single-cell RNA sequencing.…”

“…[8][9][10][11][12][13][14][15][16] When harvesting ECs from stent retrievers used for mechanical thrombectomy, there was a low EC yield or high contamination of thrombotic material. [15,16] In these studies, fluorescence-activated single-cell sorting (FACS) was used to sort the sample, and in one study, ECs were cultured to allow cytological analysis and single-cell RNA sequencing. In another study, a guidewire was navigated in the antecubital veins and yielded a significant number of ECs.…”