Preparation of single cell suspensions enriched in mouse brain vascular cells for single-cell RNA sequencing

Abstract Aims The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. Methods and results Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. Conclusion Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

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Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

10.1101/2020.06.04.135277 ◽

2020 ◽

Author(s):

Sunny Z. Wu ◽

Daniel L. Roden ◽

Ghamdan Al-Eryani ◽

Nenad Bartonicek ◽

Kate Harvey ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

High Throughput ◽

Cell Suspensions ◽

Tumour Heterogeneity ◽

Fresh Tissue ◽

Preservation Methods ◽

Human Cancers ◽

Cryopreserved Cell ◽

Single Cell Rna Sequencing

AbstractBackgroundHigh throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms.ResultsWe show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of ∼120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing.ConclusionsOur study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

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Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing

STAR Protocols ◽

10.1016/j.xpro.2020.100144 ◽

2020 ◽

Vol 1 (3) ◽

pp. 100144

Author(s):

Hugo Lee ◽

Feyza Engin

Keyword(s):

Single Cell ◽

Pancreatic Islets ◽

Rna Sequencing ◽

Cell Suspensions ◽

Single Cell Rna Sequencing ◽

Mouse Pancreatic Islets

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Single-cell RNA sequencing identifies senescent cerebromicrovascular endothelial cells in the aged mouse brain

GeroScience ◽

10.1007/s11357-020-00177-1 ◽

2020 ◽

Vol 42 (2) ◽

pp. 429-444 ◽

Cited By ~ 3

Author(s):

Tamas Kiss ◽

Ádám Nyúl-Tóth ◽

Priya Balasubramanian ◽

Stefano Tarantini ◽

Chetan Ahire ◽

...

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Mouse Brain ◽

Aged Mouse ◽

Single Cell Rna Sequencing

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Single-cell analysis of salt-induced hypertensive mouse aortae reveals cellular heterogeneity and state changes

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00704-w ◽

2021 ◽

Author(s):

Ka Zhang ◽

Hao Kan ◽

Aiqin Mao ◽

Li Geng ◽

Xin Ma

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Salt Intake ◽

High Salt ◽

Gene Set Enrichment Analysis ◽

Cellular Heterogeneity ◽

Vascular Cells ◽

Mesenchymal Transition ◽

Single Cell Rna Sequencing

AbstractElevated blood pressure caused by excessive salt intake is common and associated with cardiovascular diseases in most countries. However, the composition and responses of vascular cells in the progression of hypertension have not been systematically described. We performed single-cell RNA sequencing on the aortic arch from C57BL/6J mice fed a chow/high-salt diet. We identified 19 distinct cell populations representing 12 lineages, including smooth muscle cells (SMCs), fibroblasts, endothelial cells (ECs), B cells, and T cells. During the progression of hypertension, the proportion of three SMC subpopulations, two EC subpopulations, and T cells increased. In two EC clusters, the expression of reactive oxygen species-related enzymes, collagen and contractility genes was upregulated. Gene set enrichment analysis showed that three SMC subsets underwent endothelial-to-mesenchymal transition. We also constructed intercellular networks and found more frequent cell communication among aortic cells in hypertension and that some signaling pathways were activated during hypertension. Finally, joint public genome-wide association study data and our single-cell RNA-sequencing data showed the expression of hypertension susceptibility genes in ECs, SMCs, and fibroblasts and revealed 21 genes involved in the initiation and development of high-salt-induced hypertension. In conclusion, our data illustrate the transcriptional landscape of vascular cells in the aorta associated with hypertension and reveal dramatic changes in cell composition and intercellular communication during the progression of hypertension.

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