scholarly journals Single-Cell RNA Sequencing Reveals Novel Populations of Fibroblasts and Transcriptomic Changes Within Abdominal Skeletal Muscle in a Mouse Model of Aromatase-Induced Inguinal Hernia

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A816-A816
Author(s):  
Matthew Joseph Taylor ◽  
Tanvi Potluri ◽  
Hong Zhao ◽  
Serdar Ekrem Bulun
Keyword(s):  
Inguinal Hernia ◽  
Mouse Model ◽  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Pathological State ◽  
Suspension Cells ◽  
Estrogen Exposure ◽  
Single Cell Rna Sequencing

Abstract Introduction: Inguinal hernia is a highly prevalent condition in men, of which the only currently available treatment is invasive surgical repair. An inguinal hernia often results from a protrusion of the intra-abdominal contents through a weakened region of the lower abdominal wall, but the etiology is unknown. One potential cause is aging-related steroid hormonal changes, which coincide with an increased incidence of hernia in aged men. Our group previously developed the first mouse model of inguinal hernia (AromHum) that is generated via the humanized expression of the enzyme aromatase, which converts androgens to estrogens. In the lower abdominal muscle (LAM), an aromatase-mediated increase in tissue estrogen causes fibroblast proliferation, fibrosis, and myocyte atrophy, resulting in hernias. However, the molecular mechanism of this phenotype remains unclear. In this study, we aimed to find genome-wide transcriptomic differences in AromHum compared to WT mice at a single-cell resolution. We hypothesized that in relation to WT mice, AromHum mice would have distinct fibroblast signatures that arise from the increased estrogen exposure to LAM tissue. Methods: LAM was harvested from 9-10-week-old male WT and AromHum mice (n=3 each) and digested into a single-cell suspension. Cells were processed via the 10X Genomics Chromium platform for single-cell RNA sequencing. The 6 samples combined yielded a total of ~63,000 cells. Data was analyzed using Cell Ranger v3, Seurat v3, Slingshot, and PROGENy R packages. Results: UMAP visualization of WT and AromHum LAM tissue revealed 22 cell clusters, which we grouped into 10 broad cell types through known marker gene expression. AromHum LAM contained a significantly higher proportion of fibroblasts than WT (44% vs. 27% of total analyzed cells), and AromHum fibroblasts expressed more pro-fibrotic genes, such as Timp1, Spon2, and Postn. In AromHum and WT combined, we found 6 clusters of fibroblast-like cells. Two of these clusters (clusters 2 and 3) were heavily represented by cells derived from AromHum mice (85-90% of cells in each cluster), which we termed “hernia-associated fibroblasts” (HAFs). Cluster 3 HAFs expressed high levels of Esr1 (gene encoding ERα), as well as estrogen-responsive genes such as Pgr and Greb1, and was enriched for estrogen, hypoxia, and TGFβ signaling pathways. Cluster 2 HAFs expressed genes associated with a pathological state, such as Lbp, Cthrc1, Mmp3, and Il33, and was enriched for the NF-κB and TNF-α signaling pathways. Conclusions: We found that LAM fibrosis in AromHum may result from the expansion of two distinct populations of HAFs - one is estrogen-responsive, and another is pathologic. Further in vitro / in vivo experiments are required to determine the relative contributions of these sub-populations of HAFs to fibrosis and inguinal hernias, leading to developing novel intervention strategies.

scholarly journals Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dandan Cao ◽  
Rachel W. S. Chan ◽  
Ernest H. Y. Ng ◽  
Kristina Gemzell-Danielsson ◽  
William S. B. Yeung
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Cellular Heterogeneity ◽  
Stromal Fibroblast ◽  
Perivascular Cells ◽  
Endometrial Cells ◽  
Single Cell Rna Sequencing

Abstract Background Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. Methods In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. Results Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. Conclusion In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

scholarly journals Identification of EMT signaling cross-talk and gene regulatory networks by single-cell RNA sequencing

2021 ◽  
Vol 118 (19) ◽  
pp. e2102050118
Author(s):  
Abhijeet P. Deshmukh ◽  
Suhas V. Vasaikar ◽  
Katarzyna Tomczak ◽  
Shubham Tripathi ◽  
Petra den Hollander ◽  
...  
Keyword(s):  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Cancer Progression ◽  
Tyrosine Kinases ◽  
Regulatory Networks ◽  
Expression Profiles ◽  
Critical Role ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing

The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP, Wnt–β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β–induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.

scholarly journals SMaSH: A scalable, general marker gene identification framework for single-cell RNA sequencing and Spatial Transcriptomics

2021 ◽  
Author(s):  
Michael E Nelson ◽  
Simone G Riva ◽  
Ann Cvejic
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Marker Genes ◽  
Sequencing Data ◽  
Computational Framework ◽  
Data Set ◽  
Spatially Resolved ◽  
Single Cell Rna Sequencing ◽  
The Given

Spatial transcriptomics is revolutionising the study of single-cell RNA and tissue-wide cell heterogeneity, but few robust methods connecting spatially resolved cells to so-called marker genes from single-cell RNA sequencing, which generate significant insight gleaned from spatial methods, exist. Here we present SMaSH, a general computational framework for extracting key marker genes from single-cell RNA sequencing data for spatial transcriptomics approaches. SMaSH extracts robust and biologically well-motivated marker genes, which characterise the given data-set better than existing and limited computational approaches for global marker gene calculation.

scholarly journals Single Cell RNA Sequencing of Blood Outgrowth Endothelial Cells from Patients with Low Von Willebrand Factor Reveal Multiple Novel Signaling Pathways

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 982-982
Author(s):  
Christopher J. Ng ◽  
Alice Liu ◽  
Katrina J. Ashworth ◽  
Kenneth L. Jones ◽  
Jorge Di Paola
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Mixed Model ◽  
Transcriptional Profiling ◽  
Bioinformatic Analysis ◽  
Single Cell Rna Sequencing ◽  
Age Range

Abstract Background The mechanisms that determine low VWF levels in patients with VWF levels between 30-50 IU/dL and no mutations in VWF are poorly understood. Hypothesis/Objective We hypothesize that the study of blood outgrowth endothelial cells (BOECs) from individuals with low VWF levels may reveal unique transcriptional profiles that contribute to the low VWF levels seen in these patients. Methods BOEC Derivation: Patients with low VWF levels and mucocutaneous bleeding (MCB) (30-50 IU/dL) were enrolled in an IRB-approved study. The mononuclear layer from whole blood was isolated and plated onto collagen coated plates. After extended incubation, BOECs were validated by visual inspection and flow cytometry. Endothelial Transcriptional Characterization: A total of 9 cells lines including those from individuals with low VWF and HUVEC and BOECs from individuals with normal VWF levels as control were assayed via single cell RNA sequencing. Bioinformatic analysis included generalized transcriptional expression, Ingenuity Pathway Analysis (IPA), and expression of VWF. RNA-sequencing expression data was filtered according to a standardized algorithm. Cells that were defined as monocytes (TYROBP expression > 2 copies) were excluded. Following monocyte exclusions, cells were determined to be of endothelial origin if they demonstrated the presence of transcripts of PECAM1, CDH5, ROBO4, ESAM, TIE1, or NOTCH4 as previously described by Butler et al. (Cell Reports 2016). Results BOEC Derivation: A total of eight BOEC lines were generated, 6 from individuals with MCB and VWF levels between 30-50 IU/dL (5:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years) with normal VWF levels and no symptoms of MCB. Transcriptional Profiling of single endothelial cells from Low VWF Individuals: A 3D T-SNE plot that assesses unbiased differences in gene expression profiling was generated with each cell line represented by a different color (Figure 1A) demonstrating that individual cell lines have significant differences in their underlying transcriptional profiling. VWF Expression in Low VWF Samples: Overall expression of VWF was significantly decreased in low VWF BOEC samples (5.341 transcripts/cell) vs. control (9.076 transcripts/cell) ECs (figure 1B), P<0.0001. Further, histogram and mixed model (multiple gaussian) analysis of VWF expression revealed changes in generalized expression of VWF in Low VWF BOECs compatible with multiple populations of VWF-expressing BOECs, demonstrating cell mosaicism within each sample. IPA Analysis of Low VWF vs Control BOECs: IPA analysis demonstrated 64 pathways with a z-score difference >1 in Low VWF BOECs when compared to control BOECs (table 1), including multiple signaling pathways such as PI3kinase and AKT as well as several cytoskeleton pathways. Conclusions Single cell RNA sequencing of Low VWF BOECs reveal significant differences in transcriptional profiling when compared to control endothelial cell lines (control BOECs + HUVEC). BOECs from individuals with Low VWF levels demonstrate significantly lower VWF transcript expression than the control endothelial cells. Interestingly, BOECs from low VWF patients show significant differences in VWF transcript number within cells from the same individual demonstrating a degree of mosaicism previously described in murine endothelial cells. Finally, there are multiple cellular pathways that are differentially regulated in Low VWF BOECs as compared to control endothelial cells. Disclosures Ng: CSL Behring: Consultancy; Shire: Consultancy.

scholarly journals A critical assessment of single-cell transcriptomes sampled following patch-clamp electrophysiology

2018 ◽  
Author(s):  
Shreejoy J. Tripathy ◽  
Lilah Toker ◽  
Claire Bomkamp ◽  
B. Ogan Mancarci ◽  
Manuel Belmadani ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Critical Assessment ◽  
Cell Type ◽  
High Quality ◽  
Single Cell Rna Sequencing ◽  
Patch Pipette ◽  
Patch Clamp Electrophysiology

AbstractPatch-seq, combining patch-clamp electrophysiology with single-cell RNA-sequencing (scRNAseq), enables unprecedented single-cell access to a neuron’s transcriptomic, electrophysiological, and morphological features. Here, we present a systematic review and re-analysis of scRNAseq profiles from 4 recent patch-seq datasets, benchmarking these against analogous profiles from cellular-dissociation based scRNAseq. We found an increased likelihood for off-target cell-type mRNA contamination in patch-seq, likely due to the passage of the patch-pipette through the processes of adjacent cells. We also observed that patch-seq samples varied considerably in the amount of mRNA that could be extracted from each cell, strongly biasing the numbers of detectable genes. We present a straightforward marker gene-based approach for controlling for these artifacts and show that our method improves the correspondence between gene expression and electrophysiological features. Our analysis suggests that these technical confounds likely limit the interpretability of patch-seq based single-cell transcriptomes. However, we provide concrete recommendations for quality control steps that can be performed prior to costly RNA-sequencing to optimize the yield of high quality samples.

scholarly journals Neurodevelopmental deficits and cell-type-specific transcriptomic perturbations in a mouse model of HNRNPU haploinsufficiency

2020 ◽  
Author(s):  
Sarah A. Dugger ◽  
Ryan S. Dhindsa ◽  
Gabriela De Almeida Sampaio ◽  
Elizabeth E. Rafikian ◽  
Sabrina Petri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Single Cell ◽  
Rna Sequencing ◽  
De Novo ◽  
Global Developmental Delay ◽  
Cell Type ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific ◽  
Insight Into

AbstractHeterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated neuroanatomical abnormalities, global developmental delay and impaired ultrasonic vocalizations, and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum—a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlighted the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms, and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.

scholarly journals Single-cell RNA Sequencing Reveals Transcriptional Signatures and Cell-cell Crosstalk in Patients With Hypertensive Nephropathy

2021 ◽  
Author(s):  
Rong Tang ◽  
Wei Lin ◽  
Chanjuan Shen ◽  
Ting Meng ◽  
Joshua D Ooi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Distinct Cell ◽  
Single Cell Rna Sequencing ◽  
Cell Crosstalk ◽  
Cell Cell

Abstract BackgroundHypertensive nephropathy (HTN) is one of the leading causes of end-stage renal disease, yet the precise mechanisms and cell-specific gene expression changes are still unknown. This study used single-cell RNA sequencing (scRNA-seq) to explore novel molecular mechanisms and gene targets for HTN for the first time. Methods: The gene expression profiles of renal biopsy samples obtained from HTN patients and healthy living donor controls were determined by scRNA-seq technology. Distinct cell clusters, differential gene expression, cell-cell interaction and potential signaling pathways involved in HTN were determined. Results18 distinct cell clusters were identified in kidney from HTN and control subjects. Endothelial cells overexpressed LRG1 , a pleiotropic factor linked to apoptosis and inflammation, providing a potential novel molecular target. HTN endothelium also overexpressed genes linked to cellular adhesion, extracellular matrix accumulation and inflammation. In HTN patients, mesangial cells highly expressed proliferation related signatures ( MGST1 , TMSB10, EPS8 and IER2 ) not detected in renal diseases before. The upregulated genes in tubules of HTN were mainly participating in inflammatory signatures including IFN-γ signature, IL-17 signaling and TLR signaling. Specific gene expression of kidney-resident CD8 + T cells exhibited a proinflammatory, chemotactic and cytotoxic phenotype. Furthermore, receptor-ligand interaction analysis indicated cell-cell crosstalk in kidney contributes to recruitment and infiltration of inflammatory cells into kidneys, and fibrotic process in hypertensive renal injury. ConclusionsIn summary, our data identifies a distinct cell-specific gene expression profile, pathogenic signaling pathways and potential cell-cell communications in the pathogenesis of HTN. These findings will provide a promising novel landscape for mechanisms and treatment of HTN.

scholarly journals Single-Cell RNA Sequencing Reveals the Migration of Osteoclasts in Giant Cell Tumor of Bone

2021 ◽  
Vol 11 ◽  
Author(s):  
Wenyu Feng ◽  
Mingwei He ◽  
Xiaohong Jiang ◽  
Huijiang Liu ◽  
Tianyu Xie ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Signaling Pathways ◽  
Giant Cell Tumor ◽  
Giant Cell ◽  
Rna Sequencing ◽  
Bone Destruction ◽  
Cell Types ◽  
Cell Tumor ◽  
Single Cell Rna Sequencing

Giant cell tumor of bone (GCTB) is benign tumor that can cause significant osteolysis and bone destruction at the epiphysis of long bones. Osteoclasts are thought to be highly associated with osteolysis in GCTB. However, the migration of osteoclasts in GCTB remains unclear. A deeper understanding of the complex tumor microenvironment is required in order to delineate the migration of osteoclasts in GCTB. In this study, samples were isolated from one patient diagnosed with GCTB. Single-cell RNA sequencing (scRNA-seq) was used to detect the heterogeneity of GCTB. Multiplex immunofluorescence staining was used to evaluate the cell subtypes identified by scRNA-seq. A total of 8,033 cells were obtained from one patient diagnosed with GCTB, which were divided into eight major cell types as depicted by a single-cell transcriptional map. The osteoclasts were divided into three subsets, and their differentiation trajectory and migration status were further analyzed. Osteoclast migration may be regulated via a series of genes associated with cell migration. Furthermore, four signaling pathways (RANKL, PARs, CD137 and SMEA3 signaling pathway) were found to be highly associated with osteoclast migration. This comprehensive single-cell transcriptome analysis of GCTB identified a series of genes associated with cell migration as well as four major signaling pathways that were highly related to the migration of osteoclasts in GCTB. Our findings broaden the understanding of GCTB bionetworks and provides a theoretical basis for anti-osteolysis therapy against GCTB in the future.

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