There are no A1 and A2 astrocytes in brain

2021 ◽  
Author(s):  
Shuang-qi Gao
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Sorting ◽  
Reactive Astrocytes ◽  
Marker Genes ◽  
Set Up ◽  
The Brain

Abstract Objectives The subsets of astrocytes in the brain have not been fully elucidated. Using bulk RNA sequencing, reactive astrocytes were divided into A1 versus A2. However, using single-cell RNAseq (ScRNAseq), astrocytes were divided into over two subsets. Our aim was to set up the correspondence between the fluorescent-activated cell sorting (FACS)-bulk RNAseq and ScRNAseq data. Results We found that most of reactive astrocytes (RAs) marker genes were expressed in endothelial cells but not in astrocytes, suggesting those marker genes are not suitable for astrocytic activation. The absence of A1 and A2 astrocytes in the brain.

scholarly journals Molecular characteristics and spatial distribution of adult human corneal cell subtypes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ann J. Ligocki ◽  
Wen Fury ◽  
Christian Gutierrez ◽  
Christina Adler ◽  
Tao Yang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cross Sections ◽  
Cell Types ◽  
Marker Genes ◽  
Molecular Characteristics ◽  
Transcriptional Level ◽  
Human Cornea ◽  
Adult Human ◽  
And Migration

AbstractBulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.

scholarly journals Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas S. Tombor ◽  
David John ◽  
Simone F. Glaser ◽  
Guillermo Luxán ◽  
Elvira Forte ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Endothelial Cell Migration ◽  
Metabolic Adaptation ◽  
Endothelial Cell Proliferation ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing

AbstractEndothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

scholarly journals Modeling cellular crosstalk and organotypic vasculature development with human iPSC-derived endothelial cells and cardiomyocytes

2020 ◽  
Author(s):  
Emmi Helle ◽  
Minna Ampuja ◽  
Alexandra Dainis ◽  
Laura Antola ◽  
Elina Temmes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Tissue Growth ◽  
Cell Phenotype ◽  
Cell Populations ◽  
Pump System ◽  
Single Cell Rna Sequencing

AbstractRationaleCell-cell interactions are crucial for the development and function of the organs. Endothelial cells act as essential regulators of tissue growth and regeneration. In the heart, endothelial cells engage in delicate bidirectional communication with cardiomyocytes. The mechanisms and mediators of this crosstalk are still poorly known. Furthermore, endothelial cells in vivo are exposed to blood flow and their phenotype is greatly affected by shear stress.ObjectiveWe aimed to elucidate how cardiomyocytes regulate the development of organotypic phenotype in endothelial cells. In addition, the effects of flow-induced shear stress on endothelial cell phenotype were studied.Methods and resultsHuman induced pluripotent stem cell (hiPSC) -derived cardiomyocytes and endothelial cells were grown either as a monoculture or as a coculture. hiPS-endothelial cells were exposed to flow using the Ibidi-pump system. Single-cell RNA sequencing was performed to define cell populations and to uncover the effects on their transcriptomic phenotypes. The hiPS-cardiomyocyte differentiation resulted in two distinct populations; atrial and ventricular. Coculture had a more pronounced effect on hiPS-endothelial cells compared to hiPS-cardiomyocytes. Coculture increased hiPS-endothelial cell expression of transcripts related to vascular development and maturation, cardiac development, and the expression of cardiac endothelial cell -specific genes. Exposure to flow significantly reprogrammed the hiPS-endothelial cell transcriptome, and surprisingly, promoted the appearance of both venous and arterial clusters.ConclusionsSingle-cell RNA sequencing revealed distinct atrial and ventricular cell populations in hiPS-cardiomyocytes, and arterial and venous-like cell populations in flow exposed hiPS-endothelial cells. hiPS-endothelial cells acquired cardiac endothelial cell identity in coculture. Our study demonstrated that hiPS-cardiomoycytes and hiPS-endothelial cells readily adapt to coculture and flow in a consistent and relevant manner, indicating that the methods used represent improved physiological cell culturing conditions that potentially are more relevant in disease modelling. In addition, novel cardiomyocyte-endothelial cell crosstalk mediators were revealed.

scholarly journals Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

2019 ◽  
Author(s):  
Suraj Kannan ◽  
Matthew Miyamoto ◽  
Brian Lin ◽  
Renjun Zhu ◽  
Sean Murphy ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Sorting ◽  
Large Particle ◽  
Fluorescence Activated Cell Sorting ◽  
High Quality ◽  
Adult Cardiomyocytes ◽  
Single Cell Rna Sequencing ◽  
Mitochondrial Transcripts ◽  
Manual Methods

ABSTRACTRationaleSingle cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs).ObjectiveWe investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs.Methods and ResultsWe found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties.ConclusionsOur study enables high quality dissection of adult CM biology at single-cell resolution.

scholarly journals Integrated Single Cell Atlas of Endothelial Cells of the Human Lung

Circulation ◽  
2021 ◽  
Author(s):  
Jonas C. Schupp ◽  
Taylor S. Adams ◽  
Carlos Cosme Jr. ◽  
Micha Sam Brickman Raredon ◽  
Yifan Yuan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Single Cell ◽  
Differential Expression ◽  
Human Lung ◽  
Differential Expression Analysis ◽  
Cell Types ◽  
Marker Genes ◽  
Lung Endothelium ◽  
Lung Endothelial Cells

Background: The cellular diversity of the lung endothelium has not been systematically characterized in humans. Here, we provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single cell RNA sequencing (scRNAseq) data from six datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in-vitro was performed. The signaling network between different lung cell types was studied. For cross species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs, we found previously indistinguishable subpopulations: among venous EC, we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that the endothelial diversity is maintained in pulmonary hypertension. Our manuscript is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusions: Our integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

scholarly journals Evaluation of single-cell classifiers for single-cell RNA sequencing data sets

2019 ◽  
Vol 21 (5) ◽  
pp. 1581-1595 ◽  
Author(s):  
Xinlei Zhao ◽  
Shuang Wu ◽  
Nan Fang ◽  
Xiao Sun ◽  
Jue Fan
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Reference Data ◽  
Predictive Accuracy ◽  
Cell Types ◽  
Superior Performance ◽  
Marker Genes ◽  
Data Sets ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing

Abstract Single-cell RNA sequencing (scRNA-seq) has been rapidly developing and widely applied in biological and medical research. Identification of cell types in scRNA-seq data sets is an essential step before in-depth investigations of their functional and pathological roles. However, the conventional workflow based on clustering and marker genes is not scalable for an increasingly large number of scRNA-seq data sets due to complicated procedures and manual annotation. Therefore, a number of tools have been developed recently to predict cell types in new data sets using reference data sets. These methods have not been generally adapted due to a lack of tool benchmarking and user guidance. In this article, we performed a comprehensive and impartial evaluation of nine classification software tools specifically designed for scRNA-seq data sets. Results showed that Seurat based on random forest, SingleR based on correlation analysis and CaSTLe based on XGBoost performed better than others. A simple ensemble voting of all tools can improve the predictive accuracy. Under nonideal situations, such as small-sized and class-imbalanced reference data sets, tools based on cluster-level similarities have superior performance. However, even with the function of assigning ‘unassigned’ labels, it is still challenging to catch novel cell types by solely using any of the single-cell classifiers. This article provides a guideline for researchers to select and apply suitable classification tools in their analysis workflows and sheds some lights on potential direction of future improvement on classification tools.

scholarly journals Single-Cell RNA Sequencing Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells

Circulation ◽  
2020 ◽  
Vol 142 (19) ◽  
pp. 1848-1862 ◽  
Author(s):  
David T. Paik ◽  
Lei Tian ◽  
Ian M. Williams ◽  
Siyeon Rhee ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Transcriptional Networks ◽  
Sequencing Data ◽  
Tissue Specific ◽  
Functional Relationships ◽  
Single Cell Rna Sequencing

Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. Whereas these functional differences are presumably imprinted in the transcriptome, the pathways and networks that sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA sequencing data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We used a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from single-cell RNA sequencing data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either single-cell RNA sequencing or bulk RNA sequencing, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (eg, liver, brain), whereas others (ie, adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. We found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. We identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: We used single-cell RNA sequencing data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

scholarly journals Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

2019 ◽  
Vol 116 (48) ◽  
pp. 24100-24107 ◽  
Author(s):  
Andrew P. Voigt ◽  
Kelly Mulfaul ◽  
Nathaniel K. Mullin ◽  
Miles J. Flamme-Wiese ◽  
Joseph C. Giacalone ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Retinal Pigment Epithelium ◽  
Macular Degeneration ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Age Related ◽  
Single Cell Rna Sequencing

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

scholarly journals Unveiling the heterogeneity of NKT cells in the liver through single cell RNA sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hao Shen ◽  
Chan Gu ◽  
Tao Liang ◽  
Haifeng Liu ◽  
Fan Guo ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Subset ◽  
Nkt Cells ◽  
Organ Distribution ◽  
Marker Genes ◽  
Type I ◽  
Nkt Cell ◽  
Single Cell Rna Sequencing

Abstract CD1d-dependent type I NKT cells, which are activated by lipid antigen, are known to play important roles in innate and adaptive immunity, as are a portion of type II NKT cells. However, the heterogeneity of NKT cells, especially NKT-like cells, remains largely unknown. Here, we report the profiling of NKT (NK1.1+CD3e+) cells in livers from wild type (WT), Jα18-deficient and CD1d-deficient mice by single-cell RNA sequencing. Unbiased transcriptional clustering revealed distinct cell subsets. The transcriptomic profiles identified the well-known CD1d-dependent NKT cells and defined two CD1d-independent NKT cell subsets. In addition, validation of marker genes revealed the differential organ distribution and landscape of NKT cell subsets during liver tumor progression. More importantly, we found that CD1d-independent Sca-1−CD62L+ NKT cells showed a strong ability to secrete IFN-γ after costimulation with IL-2, IL-12 and IL-18 in vitro. Collectively, our findings provide a comprehensive characterization of NKT cell heterogeneity and unveil a previously undefined functional NKT cell subset.

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