scholarly journals Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Haematologica ◽  
2021 ◽  
Author(s):  
Leo Nicolai ◽  
Rainer Kaiser ◽  
Raphael Escaig ◽  
Marie-Louise Hoffknecht ◽  
Afra Anjum ◽  
...  
Keyword(s):  
Single Cell ◽  
Platelet Function ◽  
Cell Tracking ◽  
Mouse Strains ◽  
Single Cell Level ◽  
Cell Level ◽  
Reporter Mouse ◽  
Reporter Mice

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Credriven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of megakaryocytes in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to WT controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and megakaryocyte biology on a single cell level.

scholarly journals A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues

2021 ◽  
Vol 12 ◽  
Author(s):  
Christian Lohasz ◽  
Jacqueline Loretan ◽  
Dario Sterker ◽  
Ekkehard Görlach ◽  
Kasper Renggli ◽  
...  
Keyword(s):  
Single Cell ◽  
Drug Exposure ◽  
Dependent Manner ◽  
Single Cell Level ◽  
Cell Level ◽  
Treatment Regimens ◽  
Cell Culturing ◽  
Drug Concentrations

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo–like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

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 In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Min-Seok Oh ◽  
Seul-Gi Lee ◽  
Gwan-Ho Lee ◽  
C-Yoon Kim ◽  
Eun-Young Kim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Single Cell ◽  
Accelerator Mass Spectrometry ◽  
Cell Tracking ◽  
Liquid Scintillator ◽  
Single Cell Level ◽  
Cell Level

AbstractDespite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

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

Enhanced IFN-γ Production in Vitro by CD8+ T Cells in Hemophiliacs with AIDS as Demonstrated on the Single-Cell Level

1999 ◽  
Vol 92 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Y. Pae ◽  
H. Minagawa ◽  
J. Hayashi ◽  
S. Kashiwagi ◽  
Y. Yanagi
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Ifn Γ

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.

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