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 Table of Contents  
Year : 2019  |  Volume : 4  |  Issue : 1  |  Page : 7-12

Application of single-cell sequencing technology in the study of cardiovascular development and diseases

Department of Vascular Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Dongcheng District, Beijing, China

Date of Submission17-Feb-2019
Date of Acceptance22-Mar-2019
Date of Web Publication8-May-2019

Correspondence Address:
Dr. Yuehong Zheng
Department of Vascular Surgery, Peking Union Medical College Hospital, No 1. Shuaifuyuan, Dongcheng District, Beijing
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ts.ts_2_19

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Single-cell sequencing technology has developed rapidly in recent years and has demonstrated its power in many fields, especially in the field of tumor-, immunity-, and stem cell-related researches, where it explains many biological laws that remained uncovered. In terms of cardiovascular disease, in spite of application in studies of atherosclerotic plaque and explaining the cellular heterogeneity in plaque formation, single-cell sequencing technology is relatively limited and is in its infancy. This article reviews the application of single-cell sequencing technology in cardiovascular field in recent years and makes a brief comparison with its application in other fields, hoping to point out the way for future research.

Keywords: Cardiovascular diseases, single-cell sequencing, stem cell

How to cite this article:
Lei C, Chen S, Zheng Y. Application of single-cell sequencing technology in the study of cardiovascular development and diseases. Transl Surg 2019;4:7-12

How to cite this URL:
Lei C, Chen S, Zheng Y. Application of single-cell sequencing technology in the study of cardiovascular development and diseases. Transl Surg [serial online] 2019 [cited 2020 Sep 30];4:7-12. Available from: http://www.translsurg.com/text.asp?2019/4/1/7/257802

  Introduction of Single-Cell Sequencing Top

Next-generation sequencing technology has been proved to be a powerful tool, which revealed the complexity of gene expression in mammalian cells. The most powerful characteristic of next-generation sequencing is the ability to process large amount of sequence reads in parallel,[1] which makes it an efficient instrument for high-throughput transcriptome or epigenomic analysis.

Along with the development of next-generation sequencing, single-cell sequencing was first applied in 2009 to a single mouse blastomere. In addition to the genes previously discovered by microarray technology, an extra 5270 genes were detected.[2] With this technology, researchers have investigated small group of differentiating cells, searched for new biomarkers, and described gene regulation networks. That is, single-cell sequencing provides an efficient way to study the function and genetic information of individual cell in different tissues or organs, and it has already been applied in cancer studies, stem cells or development researches, and many other fields. However, while sample sizes used for bulk tissue sequencing are considerable, a single mammalian cell contains only <10 pg DNA, which is a major challenge for this technology.

Single-cell sequencing consists of several steps. In general, it includes isolating single cell for sequencing, amplifying the low signals, and data processing.[3] To capture a single cell, many effective methods such as flow-assisted cell sorting and micropipetting have been applied.[4]

  A Brief Review of Single-Cell Sequencing Application in Cancer, Immunology, and Stem Cells' Researches Top

Single-cell sequencing identified intratumor heterogeneity

Cancer is now widely recognized as a disease caused by genetic mutations, and human cancers comprise different mutant clones. Single-cell sequencing has shown dramatic effects in cancer studies due to its high resolution. By using this technology, researchers identified rare subpopulations of cells in cancer lesions and found that intratumor heterogeneity is strongly associated with the higher possibility of postsurgical relapse of lung adenocarcinomas.[5] On the other hand, single-cell sequencing has also been applied in primary cancer cells[6] and metastatic tumor cells' ecosystems in the head and neck carcinoma,[7] primary glioblastoma,[8] and breast cancer[9] to study further on intratumor heterogeneity, which is significantly related with prognosis, treatment choice, and other medical processes. Importantly, considering the heterogeneity and subpopulations of tumor cells and their different genetic background, it is essential to guarantee their sensitivity to a specific drug therapy. Otherwise, small surviving subpopulations of cancer cells might influence the efficiency of antitumor therapy.[10] For example, Kim et al. performed single-cell RNA sequencing (RNA-seq) on patient-derived xenograft (PDX) tumor cells from a lung adenocarcinoma patient to reveal the heterogeneity of lung cancer. They identified a subgroup of tumor cells that were associated with anticancer drug resistance through single-cell RNA-seq on surviving PDX cells against treatment in vitro.[11] Therefore, single-cell sequencing provides a powerful method to detect unique subpopulations and gene markers in tumors, which could be the target of anticancer therapy.

Single-cell sequencing applied in immunology

The immune system is composed of various cells, tissues and organs, which protects and monitors the body. Although the process where immune system response can be complex, it is initiated by a limited number of cells, which is appropriated for single-cell sequencing technology. For example, Shalek et al. performed single-cell transcriptomics on mouse bone marrow-derived dendritic cells (DCs) in response of lipopolysaccharide and revealed unobserved bimodality in expression and splicing in these cells.[12] Several uncovered cell subpopulations were defined by single-cell sequencing, which provides possible aspects to understand the complex regulatory framework of immune system.[13]

Single-cell sequencing in stem cells' study and developmental biology

Stem cells are characterized by their capacity of unlimited self-renewal and differentiation into specialized cell types. The number of cells in mammalian preimplantation development is fairly limited; however, global gene expression changes during this process, which displays significantly heterogeneous to generate different cell populations and form the segregation of trophectoderm and inner cell mass. Therefore, single-cell RNA-seq provides unprecedented opportunities to detect gene expression in this process.[14] Moreover, due to its high resolution, we can uncover the regulatory framework at early embryonic state and the differences between human and mouse embryos.[15],[16],[17] Single-cell RNA-seq was also applied on the study of embryonic stem cells,[18] primordial germ cells,[19] and tissue-specific stem cells to identify the novel stem cell types,[20] reveal dynamic gene expression during differentiation, and dissect heterogeneity within a “homogenous” stem cell population.[21]

  Single-Cell Sequencing Application in Cardiovascular Disease Top

Although we have revealed some processes of heart development, it is actually very difficult to understand cardiac tissue formation due to its structural and functional complexity. Single-cell sequencing technology provides an efficient and promising method to uncover the rare subpopulation of cells during the development and pathogenesis, which can be potential therapeutic targets.

Single-cell sequencing classified cell populations during cardiogenesis

Mammalian heart transformation from a linear tube into four chambers is controlled by the complicated gene expression and signaling interactions. Conventional transcriptome analysis is difficult to unmask this relationship, as multiple cell populations involve in the heart development.[22] DeLaughter et al. performed single-cell RNA-seq of >1200 murine cells isolated at embryonic day 9.5 (E9.5) to postnatal day 21 (P21), spanning primordial heart tube to mature heart, to investigate the cellular heterogeneity and established developmental ages for cardiomyocytes (CMs) during cardiogenesis. The research identified transcriptional heterogeneity among CMs; a rarely reported subpopulation (CME+) expressing ECM proteins were disclosed by this technology. Interestingly, this group of CMs kept increasing until P3, after which CME+ cells were hardly detected. Therefore, the researchers speculated that CME+ cells provide a developmental scaffold to form and maintain the architecture of heart during development.[23]

Mutations of cardiac transcriptional factors (TFs) were already proved to be connected with congenital heart diseases (CHDs), including NKX2.5, GATA4, and TBX5.[24],[25] NKX2-5 heterozygous mutations in humans are highly associated with atrial septal defect.[26] However, heterozygous murine models with mutation of NKX2-5 show less profound cardiac malformation and low disease penetrance.[27] Single-cell RNA-seq revealed that haploinsufficiency of NKX2-5 causes cardiac malformation by delaying maturation in both CMs and endothelial cells (ECs).[23] To reveal how genes were regulated in an individual cell during cardiac development, Li et al. performed single-cell RNA-seq on murine cardiac cells at E8.5, E9.5, and E10.5. They determined the spatial origin of single cardiac cells and partitioned an embryonic heart into 17 distinct subpopulations and generated a comprehensive transcriptomic database, which can be used to identify molecular defect of CHD before the morphological abnormality is apparent.[28] GATA4 is one of the earliest TFs expressed in precardiac splanchnic mesoderm at E7.0,[29] and in GATA4 null embryos, 2 promyocardial primordia fail to migrate and fuse ventrally, remaining lateral and finally generating 2 independent heart tubes.[30],[31] GATA4 has always been chosen as marker for genetic screen of CHD.[32] Therefore, single-cell sequencing might accelerate the preimplantation genetic diagnosis and help mutation-carrying parents to deliver normal babies.[33]

Although induced pluripotent stem cells have been discovered for a decade,[34] it remains intractable to generate mature adult CMs from human PSCs. Some studies reported the critical role of microenvironment in PSC-CM maturation; Cho et al. transplanted human PSC-CMs into rat neonatal myocardium and incubated for a month. Single-cell RNA-seq analysis was performed on these cells and normal heart, and 312 differentially expressed genes were identified. Hierarchical clustering analysis revealed a closer relationship between in vivo-matured CMs and adult CMs than in vitro-matured CMs. Gene ontology analysis suggested that those differently expressed genes were related to mitochondrial function and muscle contraction, which indicated the increase in mitochondrial and sarcomeric organization during maturation.[35]

Single-cell sequencing showed innate heterogeneity in normal hearts

It is widely accepted that mammalian heart is a nonregenerative organ, due to a specific number of CMs at birth.[36] However, there is some evidence supporting that CMs continuously renew by preexisting CMs at a low rate,[37],[38] and embryonic heart holds the ability to restore extensive tissue loss by robust CM proliferation.[39] Sereti et al. identified the heterogeneity of these proliferative cells through single-cell RNA-seq. They conducted single-cell RNA-seq on αMHC+ cells at E9.5, E12.5, and P1 and found that genes encoding cell cycle, cell differentiation, and migration in P1 CMs were downregulated, which is distinguishable from E9.5 and E12.5 CMs. On the other hand, P1 CMs mature following the upregulated expression of genes encoding specific structural protein and cellular metabolism in P1 CMs.[40] Due to the heterogeneity of bulk tissue, common methods of determining the transcriptional profiles were prone to mask heterogeneous cells that had a small proportion but whose function was completely different from others. Single-cell sequencing provided a powerful solution to this issue.

Study revealed that dermal fibroblasts could directly transform into induced CMs (iCMs) under the influence of 3 transcription factors (i.e., Gata4, Mef2c, and Tbx5), which had a global gene expression proflile similar to CMs.[41],[42] However, it still remained unknown how the conversion skips the differentiation process. Researchers found the heterogeneity of initial fibroblasts, and the reprogramming population consists of fully, partly, and unconverted cells. Using bulk tissue sequencing may cover the real process of conversion; so, Liu et al. conducted single-cell transcriptomics analysis to reconstruct the reprogramming trajectory, identified novel markers, and uncovered the regulators involved in iCM induction.[43]

On the other hand, cardiac nonmyocyte cells also play an important role in cellular composition and function. These cells included cardiac fibroblasts expressing cardiogenic genes related to heart development or repairment[44] and immune cell populations contributing to heart remodeling.[45] Skelly et al. characterized single-cell transcriptional profiles of the murine nonmyocyte cardiac cellular landscape by single-cell sequencing technology, which provided a more precise strategy to identify cardiac mural cells, fibroblasts, and other cell populations. At least five previously undiscovered subtypes of macrophages were uncovered according to the single-cell sequencing data, which changed our previous understanding of the composition of heart cells. In fact, the composition of these cells is more complicated. The regulation mechanism of this complex composition and its relationship with disease should be highlighted in our next research focus.[46]

Single-cell sequencing identified heterogeneity in failing hearts

CMs in adult mice and humans hold the capacity of renewing by mitosis of preexisting CMs during normal aging,[37],[38] but the rate is too low to provide adequate regeneration to repair during cardiac diseases.[39],[47] Therefore, whether some subpopulations among adult CMs can reenter the cell cycle become the key issue. To answer this question, See et al. undertook single CM nuclear RNA-seq of healthy and failing mouse hearts and discovered dramatic increase in the number of differently expressed genes (1435 genes) in hearts between 8-week posttransverse aortic constriction (TAC) and 1-week post-TAC. When single nuclear RNA-seq is conducted on the CMs of human end-stage failing left ventricle (nonischemic dilated cardiomyopathy), similar core genes were found highly expressed, and heterogeneity was reduced. Thanks to single-cell sequencing technology, some new markers were identified associated with proliferation and differentiation of CMs. For example, DSTN, a dedifferentiation marker, was detected at single nuclear level, while hidden in bulk tissue RNA-seq. Many other markers of “proliferative regulators” and “negative regulators of proliferation” that were masked by bulk tissue were identified due to the high resolution of single-cell sequencing. Among all these markers, Ccnd2 and Ccng1 were the most differently expressed ones. These results proved the hypothesis that stressed nuclei exhibiting the fetal gene response would coexpress dedifferentiation markers, which can only be detected by single nuclear analysis.

Single-cell sequencing identified heterogeneity in vascular cells

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors in eukaryotes,[48] and in the vascular system, GPCRs regulate critical parameters, such as endothelial permeability and vessel tone;[49],[50] GCPR expression was previously studied in bulk cDNA level; however, the heterogeneity of GPCR expression in individual vascular cells is poorly understood. Kaur et al. performed single-cell reverse transcription polymerase chain reaction in vascular smooth muscle cells of skeletal muscle and aortal smooth muscle cells (SMao) of healthy adult mice; different results were observed for both tissues in bulk tissue sequencing and innate heterogeneity. In Smao, 76 GPCRs were detected in individual cell, but only 19 of them were expressed in over 50% cells; 8 of them (Lphn1, Lgr6, F2r, Adra1d, Cd97, Gpr107, Gpr108, and Mrgprf) were expressed in over 90% cells. In skeletal smooth muscle cells (SMsk), GPCR expression differed strongly from Smao. Among the majority GPCRs in SMsk, the largest group was peptide hormone receptors (e.g., receptors for angiotensin II [Agtr1a] and endothelin [Ednra, Ednrb]). Ex vivo culture of primary Smao showed that the expression of majority of GPCRs was strongly increased, while some GPCRs' expression decreased. GPCRs' heterogeneity was also verified in all types of ECs. Single-cell expression analysis allows us to estimate the degree of GPCR heterogeneity within a cell population, which is especially meaningful for pharmacotherapy. Recent GPCR expression data rely on the assumption that cells in the same population are equally expressed; however, single-cell analysis disproved this assumption and provided possibility to target pathologically converted cells selectively based on their specific GPCRs.[51] As receptors for angiotensin II, catecholamines, and histamine, GPCRs are crucial therapeutic targets of many diseases. These data might provide new aspects to treat vascular diseases.

Atherosclerosis is a chronic inflammatory disease characterized by leukocyte-rich plaque in intimal layer of large- and medium-sized arteries.[52],[53] Winkels et al. performed single-cell RNA-seq and mass cytometry in mouse and human aortic atherosclerotic samples, and 11 distinct leukocyte clusters were defined, while healthy aortas showed less diversity. T-cell and myeloid cells (macrophages and monocytes) were dominated species, and single-cell RNA-seq revealed the cell type-specific genetic pathways. For example, genes associated with lipid metabolism and cholesterol metabolism, apoptosis, and inflammasome activation were overexpressed in myeloid cells while genes related to acute myocardial infarction were upregulated in monocytes. These results provided a method to define aortic leukocyte subsets, which may assist in evaluating progression and regression of atherosclerosis or predict plaque vulnerability.[54] Cochain et al. focused on the heterogeneity of macrophages in murine atherosclerosis, and single-cell RNA-seq was performed on myeloid cell populations extracted from aortic CD45+ cells in nondiseased and atherosclerotic mice to establish gene expression profile. Three major macrophage populations in atherosclerotic aortae were discovered, resident-like macrophages, inflammatory macrophages, in which the most significantly enriched genes included various pro-inflammatory chemokines (i.e., Cxcl2, Ccl3, and Ccl4), and previously uncovered TREM2hi macrophages with the most significantly enriched Trem2 gene. A cluster of monocyte-derived DCs/DCs (MoDCs/DCs) was identified as an atherosclerosis-associated cell population (14.9%), and all three groups of macrophages and MoDC/DC existed in advanced atherosclerosis. Previous study proved that total macrophage frequency among CD45+ leukocyte increased with disease progression.[55] Higher resolution technology revealed that this increase is largely due to the expansion of resident-like macrophages and inflammatory macrophages, suggesting a shift of macrophage populations in advanced atherosclerosis. On the other hand, TREM2 expression was universally correlated with plaque stability,[56] and the gene expression signature of these TREM2 hi macrophages was highly similar to osteoclasts and disease-associated microglia, which was associated with calcification in advanced atherosclerotic lesion and amyloid-β plaques in Alzheimer's disease, respectively.[57]

Although atheromatous plaques take root at the intima of arteries, inflammation and pathogenesis involved all three layers of arteries.[58] Progenitor or precursor cells exist in Murine adventitia and express most stem cell antigen 1 (SCA-1) and involved with adventitia cell proliferation in atherosclerosis.[59],[60] Kokkinopoulos et al. employed single-cell sequencing on SCA-1+ cells from adventitia of wild-type and atherosclerosis-prone (ApoE-deficient) mice, and a series of genes controlling cell migration and matrix protein degradation were detected. Further study revealed that in majority of adventitia, SCA-1+ (AdvSCA-1+) cells were local resident progenitors, while a very small proportion of AdvSCA-1+ expressed bone marrow markers. By single-cell gene expression analysis, potential molecules regulating AdvSCA-1+ migration were identified, for instance, microRNA 29b was increasingly expressed due to lipid loading, which can induce sirtuin-1 and matrix metalloproteinase-9 level to promote cell migration. These mechanisms revealed by single-cell sequencing might be potential target of treatment in vascular diseases.[61]

  Limitations and Prospects of Single-Cell Sequencing in Cardiovascular Research Top

Single-cell RNA-seq has shown its power to identify novel cell populations and considerable heterogeneity even in previously known as “homogenous” groups. However, almost all of the researches focus on single-cell RNA-seq, and there are few studies applying single-cell epigenome sequencing to reveal the influence of epigenetic modification during the pathogenic process of cardiovascular diseases. In addition, epigenetic modification has been proved associated with heart failure.[62]

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