scholarly journals Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T.J Streef ◽  
T Van Herwaarden ◽  
A.M Smits ◽  
M.J Goumans
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Rna Sequencing ◽  
Public Institution ◽  
Funding Source ◽  
Injury Response ◽  
Post Injury ◽  
Paracrine Factors ◽  
Adult Heart ◽  
Single Cell Rna Sequencing

Abstract Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular compositionThe data are further explored to e.g.identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2024 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile, and we are currently investigating these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Dutch Heart Foundation

scholarly journals Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
T J Streef ◽  
T Van Herwaarden ◽  
M J Goumans ◽  
A M Smits
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Rna Sequencing ◽  
Heart Development ◽  
Injury Response ◽  
Post Injury ◽  
Paracrine Factors ◽  
Mesenchymal Transition ◽  
Adult Heart ◽  
Single Cell Rna Sequencing

Abstract Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular composition. The data are further explored to e.g. identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2073 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile and validated these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

DNMT3A clonal hematopoiesis-driver mutations are associated with profound changes in monocyte and T cell signatures in humans with heart failure

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
W Abplanalp ◽  
C Cremer ◽  
S Cremer ◽  
D John ◽  
D John ◽  
...  
Keyword(s):  
Heart Failure ◽  
T Cell ◽  
Chronic Heart Failure ◽  
Single Cell ◽  
Rna Sequencing ◽  
Driver Mutations ◽  
Funding Source ◽  
Clonal Hematopoiesis ◽  
Mutation Carriers ◽  
Single Cell Rna Sequencing

Abstract Background Clonal hematopoiesis (CH) driven by mutations of DNA methyltransferase 3a (DNMT3A) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A CH-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing. Single-cell RNA-sequencing provides a novel opportunity to define subsets of immune cells mediating inflammation in humans. Methods Transcriptomic profiles of peripheral blood mononuclear cells were analysed in N=6 HF patients harboring DNMT3A CH-drived mutations and with HF and N=5 patients with HF and DNMT3A mutations by single-cell RNA-sequencing. Results Monocytes of HF patients carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared to monocytes derived from patients with HF without DNMT3A mutations. Among the specific up-regulated genes were the prototypic inflammatory interleukins (IL) IL1B, IL6, and IL8, the macrophage inflammatory proteins CCL3 and CCL4 as well as restin, which augments monocyte-endothelial adhesion. The classical monocyte subset of DNMT3A mutation carriers showed increased expression of immunoglobulin superfamily members CD80, CD300LB, and SIGLEC12, as well as the cell adhesion molecule CD58, all of which may be involved in monocyte-T cell interactions. DNMT3A mutation carriers were further characterized by increased expression of T cell receptor chains and Th1, Th17, CD8+ and Treg specific signatures. Conclusions This study demonstrates that circulating monocytes and T cells of HF patients harboring CHIP-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic heart failure. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): The German Research Foundation (SFB834, Project B1), and the German Center for Cardiovascular Research (DZHK).

scholarly journals Single Cell RNA-Sequencing Uncovers Paracrine Functions of the Epicardial-Derived Cells in Arrhythmogenic Cardiomyopathy

Circulation ◽  
2021 ◽  
Author(s):  
Ping Yuan ◽  
Sirisha M. Cheedipudi ◽  
Leila Rouhi ◽  
Siyang Fan ◽  
Lukas Simon ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cardiac Myocytes ◽  
Cardiac Dysfunction ◽  
Arrhythmogenic Cardiomyopathy ◽  
Paracrine Factors ◽  
Epicardial Cells ◽  
Single Cell Rna Sequencing ◽  
Genes Encoding

Background: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. Methods: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-Cre ERT2 :Dsp W/F were crossed to Rosa26 mT/mG (R26 mT/mG ) dual reporter mice to tag the epicardial-derived cells (EDCs) with the EGFP reporter protein. Tagged EDCs from adult Wt1-Cre ERT2 :R26 mT/mG and Wt1-Cre ERT2 : R26 mT/mG : Dsp W/F mouse hearts were isolated by FACS and sequenced by single cell RNA-sequencing (scRNA-Seq). Results: WT1 expression was progressively restricted postnatally and was exclusive to the epicardium by postnatal day 21. Expression of Dsp was reduced in the epicardial cells but not in cardiac myocytes in the Wt1-Cre ERT2 :Dsp W/F mice. The Wt1-Cre ERT2 :Dsp W/F mice exhibited premature death, cardiac dysfunction, arrhythmias, myocardial fibro-adipogenesis, and apoptosis. ScRNA-Seq of ~ 18,000 EGFP-tagged EDCs identified genotype-independent clusters of endothelial cells (ECs), fibroblasts, epithelial cells, and a very small cluster of cardiac myocytes, which were confirmed upon co-immunofluorescence staining of the myocardial sections. Differentially expressed genes (DEGs) between the paired clusters in the two genotypes predicted activation of the inflammatory and mitotic pathways, including the TGFβ1 and fibroblast growth factors (FGFs), in the epicardial-derived fibroblast and epithelial clusters but their suppression in the EC cluster. The findings were corroborated by analysis of gene expression in the pooled RNA-Seq data, which identified predominant dysregulation of genes involved in epithelial-mesenchymal transition (EMT), and dysregulation of 146 genes encoding the secreted proteins (secretome), including genes in the TGFβ1 pathway. Activation of the TGFβ1 and its co-localization with fibrosis in the Wt1-Cre ERT2 :R26 mT/mG : Dsp W/F mouse heart was validated by complementary methods. Conclusions: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFβ1 and FGFs, which mediate EMT, and contribute to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of ACM. The findings uncover contributions of the EDCs to the pathogenesis of ACM.

scholarly journals Single cell RNA sequencing identifies transcriptional signature of atherosusceptible endothelium of aorta in diabetes associated atherosclerosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Khan ◽  
S Lee ◽  
A Watson ◽  
S Maxwell ◽  
M Cooper ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Single Cell ◽  
Rna Sequencing ◽  
Gene Expression Signature ◽  
Cell Types ◽  
Funding Source ◽  
Transcriptional Signature ◽  
Single Cell Rna Sequencing

Abstract Background Transcriptomic analyses have provided invaluable information for linking genotypes to phenotypes. However, despite the near identical genotype, each cell types in our body has a unique gene expression signature. Deep sequencing at single cell resolution has provided a unique opportunity to unbiasedly discover cellular heterogeneity, disease associated cell populations and to characterise the cell specific transcriptomic landscape. Cardiovascular (CV) disease, a leading cause of death worldwide, is caused mainly by atherosclerosis, a pathological build-up of plaque within arterial vessel walls. Fluid mechanical forces generated by disturbed blood flow are long known to cause structural and transcriptional changes in the vascular endothelium. Atherosclerosis develops near branches and bends of arteries exposed to disturbed blood flow. Diabetes accelerates atherosclerosis development and indeed, represents an independent risk factor. However, the transcriptional signature of atheroprone endothelium in the diabetic aorta has not been investigated previously for this CV complication. Purpose This study was designed to apply a single cell RNA sequencing (scRNA-seq) approach to identify the transcriptional signature of atherosusceptible endothelium in diabetes associated atherosclerosis. Methods Diabetes was induced with streptozotocin in ApoEs−/− mice and followed for 10 weeks. Cells from digested aortae of control and diabetic mice were FACS-sorted for viable and metabolically active cells. These cells were loaded onto the Chromium Single Cell Controller (10X Genomics) to generate a single cell and gel bead emulsion. ScRNA-seq libraries were prepared with Single Cell 3' Solution V2 kit (10X Genomics) and sequenced with Illumina Nova-seq 6000. We have applied scRNA-seq to identify atheroprone endothelial cells in the aorta. Results and conclusion The atheroprone endothelial cells show distinct transcriptional profile with more than six hundred genes differentially expressed. ScRNA-seq allowed us not only to distinguish the two transcriptionally distinct endothelial subpopulations but also to identify a diabetes associated gene expression signature unique to atheroprone endothelial cells as compared to all other cell types in the aorta. We identified seventeen genes uniquely dysregulated in the diabetic atheroprone endothelial cell (Cut off = FDR s<0.05, Fold change at least 2-fold). This includes Protein C receptor (Procr) which has recently been identified as a marker for blood vascular endothelial stem cells (VESCs). Downregulation of Procr in the atheroprone endothelial cells of diabetic aorta as identified in our scRNA-seq data indicates that diabetes may limit the vascular repair by targeting VESCs thus contributing to accelerated atherosclerosis. These exciting novel findings have uncovered the transcriptomic landscape of atherosusceptible endothelium of aorta at the single cell level as seen in diabetes associated atherosclerosis. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Heart Foundation of Australia; NHMRC National Health and Medical Research Council of Australia

41-OR: Single Cell RNA-Sequencing Reveals the Developmental Heterogeneity of Brown Adipocytes

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 41-OR
Author(s):  
FARNAZ SHAMSI ◽  
MARY PIPER ◽  
LI-LUN HO ◽  
TIAN LIAN HUANG ◽  
YU-HUA TSENG
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Brown Adipocytes ◽  
Single Cell Rna Sequencing
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