scholarly journals Single cell RNA-seq in regenerative and fibrotic biomaterial environments defines new macrophage subsets

2019 ◽  
Author(s):  
Sven D. Sommerfeld ◽  
Christopher Cherry ◽  
Remi M. Schwab ◽  
Liam Chung ◽  
David R Maestas ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Small Population ◽  
Foreign Body Response ◽  
Phenotypic Characterization ◽  
Sequencing Analysis ◽  
Environmental Signals ◽  
Macrophage Subset ◽  
Macrophage Subsets

SummaryMacrophages play diverse roles in the immune response to infection, cancer, and wound healing where they respond to local environmental signals, yet identification and phenotypic characterization of functional subsets in vivo remains limited. We performed single cell RNA sequencing analysis on differentiated macrophages sorted from a biologic matrix-induced regenerative environment versus a synthetic biomaterial foreign body response (FBR), characterized by TH2/interleukin (IL)-4 and TH17/IL-17, respectively. In the regenerative environment, unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle. In the FBR environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules characteristic of certain auto-immune responses that were virtually absent in mice lacking the IL-17 receptor. Surface marker combinations including CD9 and CD301b defined macrophage fibrotic and regenerative subsets enabling functional assessment and identification in human tissue. Application of the terminal macrophage subsets to train the SingleCellNet algorithm and comparison to human and mouse macrophages in tumor, lung, and liver suggest broad relevance of macrophage classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide new targets for potential therapeutic modulation of certain pathologic states and tissue repair.

scholarly journals Interleukin-36γ–producing macrophages drive IL-17–mediated fibrosis

2019 ◽  
Vol 4 (40) ◽  
pp. eaax4783 ◽  
Author(s):  
Sven D. Sommerfeld ◽  
Christopher Cherry ◽  
Remi M. Schwab ◽  
Liam Chung ◽  
David R. Maestas ◽  
...  
Keyword(s):  
Tissue Repair ◽  
Expression Profiles ◽  
Small Population ◽  
Foreign Body Response ◽  
Phenotypic Characterization ◽  
Sequencing Analysis ◽  
Tissue Samples ◽  
Urinary Bladder Matrix ◽  
Macrophage Subsets

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)–4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17–dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

scholarly journals The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

2019 ◽  
Vol 116 (47) ◽  
pp. 23618-23624 ◽  
Author(s):  
Audrey C. A. Cleuren ◽  
Martijn A. van der Ent ◽  
Hui Jiang ◽  
Kristina L. Hunker ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Profiles ◽  
Affinity Purification ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Vascular Beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

scholarly journals EWSR1-induced circNEIL3 promotes glioma progression and exosome-mediated macrophage immunosuppressive polarization via stabilizing IGF2BP3

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Ziwen Pan ◽  
Rongrong Zhao ◽  
Boyan Li ◽  
Yanhua Qi ◽  
Wei Qiu ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Expression Profiles ◽  
Tumour Microenvironment ◽  
Malignant Progression ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Glioma Progression

Abstract Background Gliomas are the most common malignant primary brain tumours with a highly immunosuppressive tumour microenvironment (TME) and poor prognosis. Circular RNAs (circRNA), a newly found type of endogenous noncoding RNA, characterized by high stability, abundance, conservation, have been shown to play an important role in the pathophysiological processes and TME remodelling of various tumours. Methods CircRNA sequencing analysis was performed to explore circRNA expression profiles in normal and glioma tissues. The biological function of a novel circRNA, namely, circNEIL3, in glioma development was confirmed both in vitro and in vivo. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation (RIP), luciferase reporter, and co-immunoprecipitation assays were conducted. Results We identified circNEIL3, which could be cyclized by EWS RNA-binding protein 1(EWSR1), to be upregulated in glioma tissues and to correlate positively with glioma malignant progression. Functionally, we confirmed that circNEIL3 promotes tumorigenesis and carcinogenic progression of glioma in vitro and in vivo. Mechanistically, circNEIL3 stabilizes IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) protein, a known oncogenic protein, by preventing HECTD4-mediated ubiquitination. Moreover, circNEIL3 overexpression glioma cells drives macrophage infiltration into the tumour microenvironment (TME). Finally, circNEIL3 is packaged into exosomes by hnRNPA2B1 and transmitted to infiltrated tumour associated macrophages (TAMs), enabling them to acquire immunosuppressive properties by stabilizing IGF2BP3 and in turn promoting glioma progression. Conclusions This work reveals that circNEIL3 plays a nonnegligible multifaceted role in promoting gliomagenesis, malignant progression and macrophage tumour-promoting phenotypes polarization, highlighting that circNEIL3 is a potential prognostic biomarker and therapeutic target in glioma.

Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Shuin Park ◽  
Sara Ranjbarvaziri ◽  
Fides Lay ◽  
Peng Zhao ◽  
Aldons J Lusis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Expression Profiles ◽  
Inbred Mouse ◽  
Gene Expression Profiles ◽  
Mouse Strains ◽  
Sequencing Analysis ◽  
Different Strains

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression in vivo in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.

scholarly journals Single-Cell Transcriptome Analysis Profile of Meniscal Tissue Macrophages in Human Osteoarthritis

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Jingbin Zhou ◽  
Zhihong Zhao ◽  
Chen He ◽  
Feng Gao ◽  
Yu Guo ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Vital Role ◽  
Low Grade ◽  
Sequencing Analysis ◽  
Human Knee ◽  
Meniscal Tissue ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Osteoarthritis (OA) has long been considered as a degenerative disease, but growing evidence suggests that inflammation plays a vital role in its pathogenesis. Unlike rheumatoid arthritis and other autoimmune diseases, inflammation in OA is chronic and, in relatively low grade, mainly mediated by the innate immune system, especially macrophages. However, due to its low abundance, there is a lack of systematic studies on macrophages in the OA condition. Here, we have used single-cell RNA sequencing analysis to gain insight into the heterogeneity and functional specialization of human knee macrophages. We also compared the gene expression profiles of macrophages in healthy people and OA patients and found the characteristic changes of special macrophages in the OA knee. We believe that this in-depth understanding of the basis of OA inflammation will bring hope for the development of new therapies.

scholarly journals The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

2019 ◽  
Author(s):  
Audrey C.A. Cleuren ◽  
Martijn A. van der Ent ◽  
Hui Jiang ◽  
Kristina L. Hunker ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Single Cell ◽  
Blood Vessels ◽  
Rna Sequencing ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Vascular Beds

AbstractEndothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression following EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single cell suspensions for fluorescence-activated cell sorting (FACS) or single cell RNA sequencing analysis. Comparison of our EC-TRAP to published single cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.SignificanceEndothelial cells (ECs), which line all vertebrate blood vessels, are highly heterogeneous across different tissues. The present study uses a genetic approach to specifically tag mRNAs within ECs of the mouse, thereby allowing recovery and sequence analysis to evaluate the EC-specific gene expression program directly from intact organs. Our findings demonstrate marked heterogeneity in EC gene expression across different vascular beds under both normal and disease conditions, with a more accurate picture than can be achieved using other methods. The data generated in these studies advance our understanding of EC function in different blood vessels and provide a valuable resource for future studies.

scholarly journals Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis

2018 ◽  
Vol 77 (9) ◽  
pp. 1362-1371 ◽  
Author(s):  
Christopher P Denton ◽  
Voon H Ong ◽  
Shiwen Xu ◽  
Haiyin Chen-Harris ◽  
Zora Modrusan ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Interleukin 6 ◽  
Transforming Growth Factor ◽  
Expression Profiles ◽  
Dermal Fibroblasts ◽  
Sequencing Analysis ◽  
Growth Factor Beta

ObjectivesSkin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGFβ) and interleukin-6 (IL-6) are key candidate mediators in SSc. Our aim was to elucidate the specific effect of IL-6 pathway blockade on the biology of SSc fibroblasts in vivo by using samples from a unique clinical experiment—the faSScinate study—in which patients with SSc were treated for 24 weeks with tocilizumab (TCZ), an IL-6 receptor-α inhibitor.MethodsWe analysed the molecular, functional and genomic characteristics of explant fibroblasts cultured from matched skin biopsy samples collected at baseline and at week 24 from 12 patients receiving placebo (n=6) or TCZ (n=6) and compared these with matched healthy control fibroblast strains.ResultsThe hallmark functional and molecular-activated phenotype was defined in SSc samples and was stable over 24 weeks in placebo-treated cases. RNA sequencing analysis robustly defined key dysregulated pathways likely to drive SSc fibroblast activation in vivo. Treatment with TCZ for 24 weeks profoundly altered the biological characteristics of explant dermal fibroblasts by normalising functional properties and reversing gene expression profiles dominated by TGFβ-regulated genes and molecular pathways.ConclusionsWe demonstrated the exceptional value of using explant dermal fibroblast cultures from a well-designed trial in SSc to provide a molecular framework linking IL-6 to key profibrotic pathways. The profound impact of IL-6R blockade on the activated fibroblast phenotype highlights the potential of IL-6 as a therapeutic target in SSc and other fibrotic diseases.Trial registration numberNCT01532869; Post-results.

scholarly journals Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

2021 ◽  
Author(s):  
Evan S Walsh ◽  
Tammy Tollison ◽  
Hayden Brochu ◽  
Brian Shaw ◽  
Kayliegh Diveley ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
High Throughput ◽  
Rhesus Macaques ◽  
Expression Profiles ◽  
Single Cells ◽  
Model Organisms ◽  
Sequencing Analysis ◽  
Immune Repertoire ◽  
Repertoire Sequencing

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light- chains of immunoglobulins (Ig) and VDJ- and VJ- chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

scholarly journals Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality

2016 ◽  
Vol 2 (11) ◽  
pp. e1600874 ◽  
Author(s):  
Zi Yin ◽  
Jia-jie Hu ◽  
Long Yang ◽  
Ze-Feng Zheng ◽  
Cheng-rui An ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Population ◽  
Single Cell Analysis ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Analysis ◽  
Nestin Expression

The repair of injured tendons remains a formidable clinical challenge because of our limited understanding of tendon stem cells and the regulation of tenogenesis. With single-cell analysis to characterize the gene expression profiles of individual cells isolated from tendon tissue, a subpopulation of nestin+ tendon stem/progenitor cells (TSPCs) was identified within the tendon cell population. Using Gene Expression Omnibus datasets and immunofluorescence assays, we found that nestin expression was activated at specific stages of tendon development. Moreover, isolated nestin+ TSPCs exhibited superior tenogenic capacity compared to nestin− TSPCs. Knockdown of nestin expression in TSPCs suppressed their clonogenic capacity and reduced their tenogenic potential significantly both in vitro and in vivo. Hence, these findings provide new insights into the identification of subpopulations of TSPCs and illustrate the crucial roles of nestin in TSPC fate decisions and phenotype maintenance, which may assist in future therapeutic strategies to treat tendon disease.

CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
Sonali Arora ◽  
Anca Mihalas ◽  
John Bassett ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Single Cell ◽  
Developmental Trajectories ◽  
Expression Patterns ◽  
Experimental Models ◽  
Tumor Formation ◽  
Sequencing Analysis ◽  
Rna Seq ◽  
Human Glioblastoma

Abstract Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.

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