scholarly journals A single cell transcriptional atlas of early synovial joint development

Development ◽  
2020 ◽  
Vol 147 (14) ◽  
pp. dev185777 ◽  
Author(s):  
Qin Bian ◽  
Yu-Hao Cheng ◽  
Jordan P. Wilson ◽  
Emily Y. Su ◽  
Dong Won Kim ◽  
...  
Keyword(s):  
Knee Joint ◽  
Single Cell ◽  
Lineage Tracing ◽  
Rna Seq ◽  
Synovial Joint ◽  
Isolated Populations ◽  
Joint Development

ABSTRACTSynovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

scholarly journals A single cell transcriptional atlas of early synovial joint development

2019 ◽  
Author(s):  
Qin Bian ◽  
Yu-Hao Cheng ◽  
Jordan P Wilson ◽  
Dong Won Kim ◽  
Hong Wang ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Knee Joint ◽  
Single Cell ◽  
Lineage Tracing ◽  
Transcriptional Networks ◽  
Rna Seq ◽  
Synovial Joint ◽  
Isolated Populations ◽  
Joint Development

SUMMARYSynovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 6,202 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization, and functional analysis of prospectively isolated populations, we have inferred the underlying transcriptional networks of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

scholarly journals CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data

2019 ◽  
Author(s):  
Rebekka Wegmann ◽  
Marilisa Neri ◽  
Sven Schuierer ◽  
Bilada Bilican ◽  
Huyen Hartkopf ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Populations ◽  
Rna Seq ◽  
Differentiation Protocol ◽  
Layer V ◽  
Retzius Cells ◽  
Rare Cells ◽  
Cell Cycle Status

AbstractComprehensive benchmarking of computational methods for single-cell RNA sequencing (scRNA-seq) analysis is scarce. Using a modular workflow and a large dataset with known cell composition, we benchmarked feature selection and clustering methodologies for scRNA-seq data. Results highlighted a methodology gap for rare cell population identification for which we developed CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS outperformed existing approaches, enabled the identification of rare cell populations and, in contrast to other methods, simultaneously revealed transcriptomic signatures indicative of the rare cells’ function. We exemplified the use of our workflow and CellSIUS for the characterization of a human pluripotent cell 3D spheroid differentiation protocol recapitulating deep-layer corticogenesis in vitro. Results revealed lineage bifurcation between Cajal-Retzius cells and layer V/VI neurons as well as rare cell populations that differ by migratory, metabolic, or cell cycle status, including a choroid plexus neuroepithelial subgroup, revealing previously unrecognized complexity in human stem cell-derived cellular populations.

scholarly journals Single-cell RNA-seq profiling of mouse endothelial cells in response to pulmonary arterial hypertension

2021 ◽  
Author(s):  
Julie Rodor ◽  
Shiau-Haln Chen ◽  
Jessica P Scanlon ◽  
João P Monteiro ◽  
Axelle Caudrillier ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Single Cell ◽  
Lineage Tracing ◽  
Rna Seq ◽  
Cell Dysfunction ◽  
Pulmonary Arterial ◽  
Transcriptomic Changes

Abstract Aims Endothelial cell dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution. Methods and Results We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/Hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. Conclusions This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

In Vitro Assay for Single-cell Characterization of Impaired Deformability in Red Blood Cells under Recurrent Episodes of Hypoxia

Lab on a Chip ◽  
2021 ◽  
Author(s):  
YUHAO QIANG ◽  
Jia Liu ◽  
Ming Dao ◽  
E Du
Keyword(s):  
Shear Stress ◽  
Red Blood Cells ◽  
Single Cell ◽  
Blood Cells ◽  
Blood Circulation ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Cyclic Shear

Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that...

scholarly journals Identification of Potential Key lncRNAs in the Context of Mouse Myeloid Differentiation by Systematic Transcriptomics Analysis

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 630
Author(s):  
Yongqing Lan ◽  
Meng Li ◽  
Shuangli Mi
Keyword(s):  
Transcription Factor ◽  
Cell Model ◽  
Myeloid Differentiation ◽  
Rna Seq ◽  
Hematopoietic Differentiation ◽  
Granulocytic Differentiation ◽  
Expression Of Genes ◽  
Non Coding Rnas

Hematopoietic differentiation is a well-orchestrated process by many regulators such as transcription factor and long non-coding RNAs (lncRNAs). However, due to the large number of lncRNAs and the difficulty in determining their roles, the study of lncRNAs is a considerable challenge in hematopoietic differentiation. Here, through gene co-expression network analysis over RNA-seq data generated from representative types of mouse myeloid cells, we obtained a catalog of potential key lncRNAs in the context of mouse myeloid differentiation. Then, employing a widely used in vitro cell model, we screened a novel lncRNA, named Gdal1 (Granulocytic differentiation associated lncRNA 1), from this list and demonstrated that Gdal1 was required for granulocytic differentiation. Furthermore, knockdown of Cebpe, a principal transcription factor of granulocytic differentiation regulation, led to down-regulation of Gdal1, but not vice versa. In addition, expression of genes involved in myeloid differentiation and its regulation, such as Cebpa, were influenced in Gdal1 knockdown cells with differentiation blockage. We thus systematically identified myeloid differentiation associated lncRNAs and substantiated the identification by investigation of one of these lncRNAs on cellular phenotype and gene regulation levels. This study promotes our understanding of the regulation of myeloid differentiation and the characterization of roles of lncRNAs in hematopoietic system.

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

scholarly journals Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lars Velten ◽  
Benjamin A. Story ◽  
Pablo Hernández-Malmierca ◽  
Simon Raffel ◽  
Daniel R. Leonce ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Single Cell ◽  
Lineage Tracing ◽  
Specific Gene ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mutational Status

AbstractCancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

scholarly journals Isolation and characterization of adrenocortical progenitors involved in the adaptation to stress

2018 ◽  
Vol 115 (51) ◽  
pp. 12997-13002 ◽  
Author(s):  
Charlotte Steenblock ◽  
Maria F. Rubin de Celis ◽  
Luis F. Delgadillo Silva ◽  
Verena Pawolski ◽  
Ana Brennand ◽  
...  
Keyword(s):  
Lineage Tracing ◽  
Differentiated Cells ◽  
Isolation And Characterization ◽  
Proliferation And Differentiation ◽  
Response To Stress ◽  
Steroidogenic Cells ◽  
High Degree

The adrenal gland is a master regulator of the human body during response to stress. This organ shows constant replacement of senescent cells by newly differentiated cells. A high degree of plasticity is critical to sustain homeostasis under different physiological demands. This is achieved in part through proliferation and differentiation of adult adrenal progenitors. Here, we report the isolation and characterization of a Nestin+ population of adrenocortical progenitors located under the adrenal capsule and scattered throughout the cortex. These cells are interconnected with progenitors in the medulla. In vivo lineage tracing revealed that, under basal conditions, this population is noncommitted and slowly migrates centripetally. Under stress, this migration is greatly enhanced, and the cells differentiate into steroidogenic cells. Nestin+ cells cultured in vitro also show multipotency, as they differentiate into mineralocorticoid and glucocorticoid-producing cells, which can be further influenced by the exposure to Angiotensin II, adrenocorticotropic hormone, and the agonist of luteinizing hormone-releasing hormone, triptorelin. Taken together, Nestin+ cells in the adult adrenal cortex exhibit the features of adrenocortical progenitor cells. Our study provides evidence for a role of Nestin+ cells in organ homeostasis and emphasizes their role under stress. This cell population might be a potential source of cell replacement for the treatment of adrenal insufficiency.

scholarly journals Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

2020 ◽  
Author(s):  
Silvia Llonch ◽  
Montserrat Barragán ◽  
Paula Nieto ◽  
Anna Mallol ◽  
Marc Elosua-Bayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Chromosome Segregation ◽  
Maternal Age ◽  
Maturation Stage ◽  
P Value ◽  
Rna Seq ◽  
Transcript Levels ◽  
Human Oocytes ◽  
The Impact

AbstractStudy questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)?Summary answerWhile the oocytes’ transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age.Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32).Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2™ medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant.Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age.Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes.The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts.Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes.Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”.The authors have no conflict of interest to declare.

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