scholarly journals The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

10.3791/56100 ◽  
2017 ◽  
Author(s):  
Nan Zhang ◽  
Liakot A Khan ◽  
Edward Membreno ◽  
Gholamali Jafari ◽  
Siyang Yan ◽  
...  
Keyword(s):  
Single Cell ◽  
Function Analysis ◽  
Single Cell Level ◽  
Membrane Biogenesis ◽  
Loss Of Function ◽  
Cell Level ◽  
C Elegans ◽  
Antibody Staining

scholarly journals Three-dimensional drug screen identifies HDAC inhibitors as therapeutic agents in mTORC1-driven lymphangioleiomyomatosis

2021 ◽  
Author(s):  
Adam Pietrobon ◽  
Julien Yockell-Lelièvre ◽  
Nicole Melong ◽  
Laura J. Smith ◽  
Sean P. Delaney ◽  
...  
Keyword(s):  
Single Cell ◽  
Hdac Inhibitors ◽  
Three Dimensional ◽  
Drug Screen ◽  
Single Cell Level ◽  
Loss Of Function ◽  
Cell Level ◽  
Mtorc1 Signaling

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring constitutive mTORC1 signaling. Rapamycin is the only clinically approved disease-modifying treatment, but its action is cytostatic and disease progresses upon its withdrawal. There is a critical need to identify novel agents that prevent the invasive phenotype and/or eradicate the neoplastic LAM cells. Here, we employed novel cellular and extracellular models to screen for candidate therapeutics in a physiologically relevant setting. We observed that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional tissue culture plastic. Leveraging our culture system, we conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. We identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. Unexpectedly, we observed that next generation ATP-competitive mTORC1/2 inhibitors potentiate invasion. We determined anti-invasive effects of HDAC inhibitors to be independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Drug performance was subsequently evaluated at the single cell level in zebrafish xenografts. We observed consistent therapeutic efficacy in vivo at equivalent concentrations to those used in vitro, substantiating HDAC inhibitors as potential therapeutic candidates for pursuit in patients with LAM.

scholarly journals A Mass-Ratiometry-Based CD45 Barcoding Method for Mass Cytometry Detection

2019 ◽  
Vol 24 (4) ◽  
pp. 408-419
Author(s):  
Hongu Meng ◽  
Antony Warden ◽  
Lulu Zhang ◽  
Ting Zhang ◽  
Yiyang Li ◽  
...  
Keyword(s):  
Data Acquisition ◽  
Sample Size ◽  
Single Cell ◽  
Large Sample Size ◽  
Single Cell Level ◽  
Mass Cytometry ◽  
Cell Level ◽  
Large Sample ◽  
Antibody Staining ◽  
Instrument Sensitivity

Mass cytometry (CyTOF) is a critical cell profiling tool in acquiring multiparameter proteome data at the single-cell level. A major challenge in CyTOF analysis is sample-to-sample variance arising from the pipetting process, staining variation, and instrument sensitivity. To reduce such variations, cell barcoding strategies that enable the combination of individual samples prior to antibody staining and data acquisition on CyTOF are often utilized. The most prevalent barcoding strategy is based on a binary scheme that cross-examines the existence or nonexistence of certain mass signals; however, it is limited by low barcoding efficiency and high cost, especially for large sample size. Herein, we present a novel barcoding method for CyTOF application based on mass ratiometry. Different mass tags with specific fixed ratios are used to label CD45 antibody to achieve sample barcoding. The presented method exponentially increases the number of possible barcoded samples with the same amount of mass tags compared with conventional methods. It also reduces the overall time for the labeling process to 40 min and avoids the need for expensive commercial barcoding buffer reagents. Moreover, unlike the conventional barcoding process, this strategy does not pre-permeabilize cells before the barcoding procedure, which offers additional benefits in preserving surface biomarker signals.

scholarly journals Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e36246 ◽  
Author(s):  
Rodolphe Suspène ◽  
Andreas Meyerhans
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Hiv 1

scholarly journals A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

2021 ◽  
Author(s):  
Sundeep Khosla ◽  
Dominik Saul ◽  
Robyn Laura Kosinsky ◽  
Elizabeth Atkinson ◽  
Madison Doolittle ◽  
...  
Keyword(s):  
Single Cell ◽  
Mesenchymal Cells ◽  
Senescent Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Gene Set ◽  
Age Related ◽  
Cell Clearance ◽  
Bone Biopsies

Abstract Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

scholarly journals The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Patrick Lenz ◽  
Fabienne Hilgers ◽  
Alina Burmeister ◽  
Leonie Zimmermann ◽  
Kristina Volkenborn ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Single Cell ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Online Monitoring ◽  
Protein Detection ◽  
Single Cell Level ◽  
Cell Level

Abstract Background Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. Results As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. Conclusion Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. Graphic abstract

scholarly journals Single-Cell Gene Expression Analysis and Evaluation of the Therapeutic Function of Murine Adipose-Derived Stromal Cells (ASCs) from the Subcutaneous and Visceral Compartment

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Dominik Pförringer ◽  
Matthias M. Aitzetmüller ◽  
Elizabeth A. Brett ◽  
Khosrow S. Houschyar ◽  
Richard Schäfer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Single Cell ◽  
Stromal Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Adipose Derived Stromal Cells ◽  
Wound Healing Model ◽  
Healing Model

Introduction. Adipose-derived stromal cells (ASCs) are a promising resource for wound healing and tissue regeneration because of their multipotent properties and cytokine secretion. ASCs are typically isolated from the subcutaneous fat compartment, but can also be obtained from visceral adipose tissue. The data on their equivalence diverges. The present study analyzes the cell-specific gene expression profiles and functional differences of ASCs derived from the subcutaneous (S-ASCs) and the visceral (V-ASCs) compartment. Material and Methods. Subcutaneous and visceral ASCs were obtained from mouse inguinal fat and omentum. The transcriptional profiles of the ASCs were compared on single-cell level. S-ASCs and V-ASCs were then compared in a murine wound healing model to evaluate their regenerative functionality. Results. On a single-cell level, S-ASCs and V-ASCs displayed distinct transcriptional profiles. Specifically, significant differences were detected in genes associated with neoangiogenesis and tissue remodeling (for example, Ccl2, Hif1α, Fgf7, and Igf). In addition, a different subpopulation ecology could be identified employing a cluster model. Nevertheless, both S-ASCs and V-ASCs induced accelerated healing rates and neoangiogenesis in a mouse wound healing model. Conclusion. With similar therapeutic potential in vivo, the significantly different gene expression patterns of ASCs from the subcutaneous and visceral compartments suggest different signaling pathways underlying their efficacy. This study clearly demonstrates that review of transcriptional results in vivo is advisable to confirm the tentative effect of cell therapies.

scholarly journals Dynamic Imaging of Marrow-Resident Granulocytes Interacting with Human Mesenchymal Stem Cells upon Systemic Lipopolysaccharide Challenge

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Jay T. Myers ◽  
Deborah S. Barkauskas ◽  
Alex Y. Huang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Single Cell ◽  
Human Mesenchymal Stem Cells ◽  
Dynamic Imaging ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Single Cell Level ◽  
Cell Level

Human mesenchymal stem cells (hMSCs) have gained intense research interest due to their immune-modulatory, tissue differentiating, and homing properties to sites of inflammation. Despite evidence demonstrating the biodistribution of infused hMSCs in target organs using static fluorescence imaging or whole-body imaging techniques, surprisingly little is known about how hMSCs behave dynamically within host tissues on a single-cell levelin vivo. Here, we infused fluorescently labeled clinical-grade hMSCs into immune-competent mice in which neutrophils and monocytes express a second fluorescent marker under the lysozyme M (LysM) promoter. Using intravital two-photon microscopy (TPM), we were able for the first time to capture dynamic interactions between hMSCs and LysM+granulocytes in the calvarium bone marrow of recipient mice during systemic LPS challenge in real time. Interestingly, many of the infused hMSCs remained intact despite repeated cellular contacts with host neutrophils. However, we were able to observe the destruction and subsequent phagocytosis of some hMSCs by surrounding granulocytes. Thus, our imaging platform provides opportunities to gain insight into the biology and therapeutic mechanisms of hMSCsin vivoat a single-cell level within live hosts.

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