scholarly journals Adipose Stem Cell-Derived Extracellular Vesicles Induce Proliferation of Schwann Cells via Internalization

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 163 ◽  
Author(s):  
Maximilian Haertinger ◽  
Tamara Weiss ◽  
Anda Mann ◽  
Annette Tabi ◽  
Victoria Brandel ◽  
...  
Keyword(s):  
Image Analysis ◽  
Stem Cell ◽  
Extracellular Vesicles ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Automated Image Analysis ◽  
Dependent Manner ◽  
Adipose Stem Cell ◽  
Silica Beads

Recent studies showed a beneficial effect of adipose stem cell-derived extracellular vesicles (ADSC-EVs) on sciatic nerve repair, presumably through Schwann cell (SC) modulation. However, it has not yet been elucidated whether ADSC-EVs exert this supportive effect on SCs by extracellular receptor binding, fusion to the SC membrane, or endocytosis mediated internalization. ADSCs, ADSC-EVs, and SCs were isolated from rats and characterized according to associated marker expression and properties. The proliferation rate of SCs in response to ADSC-EVs was determined using a multicolor immunofluorescence staining panel followed by automated image analysis. SCs treated with ADSC-EVs and silica beads were further investigated by 3-D high resolution confocal microscopy and live cell imaging. Our findings demonstrated that ADSC-EVs significantly enhanced the proliferation of SCs in a time- and dose-dependent manner. 3-D image analysis revealed a perinuclear location of ADSC-EVs and their accumulation in vesicular-like structures within the SC cytoplasm. Upon comparing intracellular localization patterns of silica beads and ADSC-EVs in SCs, we found striking resemblance in size and distribution. Live cell imaging visualized that the uptake of ADSC-EVs preferentially took place at the SC processes from which the EVs were transported towards the nucleus. This study provided first evidence for an endocytosis mediated internalization of ADSC-EVs by SCs and underlines the therapeutic potential of ADSC-EVs in future approaches for nerve regeneration.

scholarly journals Ex vivo Inducing Apoptotic Mesenchymal Stem Cell by High Hydrostatic Pressure

2020 ◽  
Author(s):  
Tien Minh Le ◽  
Naoki Morimoto ◽  
Nhung Thi My Ly ◽  
Toshihito Mitsui ◽  
Sharon Claudia Notodihardjo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hydrostatic Pressure ◽  
Mesenchymal Stem Cell ◽  
High Hydrostatic Pressure ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Morphological Changes ◽  
Fluid Pressure ◽  
Live Cell ◽  
Tunel Staining

Abstract Background: Apoptosis was reported to take crucial role in mesenchymal stem cell (MSC)-mediated immunomodulation, in which apoptotic MSCs were shown to be superior compared to living MSCs. Furthermore, extracellular vesicles (Evs) derived from MSCs were revealed more specific advantages for patient safety such as lower propensity to trigger innate and adaptive immune responses. As a safety and simple operation, high hydrostatic pressure (HHP), a physical technique that uses only fluid pressure to inactivate cells or tissues, has been developed and applied in a lot of field of biosciences, including biotechnology, biomaterials, or tissue engineering. Methods: MSCs isolated from human bone marrow were suspended cultured in appropriate medium and subjected to pressurization at 50 MPa for 36 h. Then cells were collected and investigated apoptotic pathway by transmission electron microscopy (TEM), phosphatidylserine membrane translocations, cleaved caspase-3/7 and terminal deoxy-nucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Besides, viability assays and live cell imaging were also used for assessement of cell survival after pressurization. Results: We found that HHP at 50 MPa for ≥36 h completely induced MSC death by Live/Dead assay, live cell imaging and WST-8 assay up to 7 days after pressurization. The large amount of apoptotic MSCs death was found based on morphological changes in TEM, phosphatidylserine exposure, caspase activation and detection of DNA fragmentations via TUNEL staining. Conclusions: In the current study, our data revealed that HHP treatment was convenient processing which safety and effectively induced MSCs undergo apoptosis. Especially, by capable of manufacture expanding, this technique might provide numbers of manipulated products using for industrial cell-based therapies.

scholarly journals Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging

2017 ◽  
Vol 19 (8) ◽  
pp. 1109-1118 ◽  
Author(s):  
Xin Mei ◽  
Yin-Sheng Chen ◽  
Fu-Rong Chen ◽  
Shao-Yan Xi ◽  
Zhong-Ping Chen
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Stem Cell Differentiation ◽  
Live Cell ◽  
Glioblastoma Stem Cell

scholarly journals A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1193
Author(s):  
Jorge L. Arias-Arias ◽  
Eugenia Corrales-Aguilar ◽  
Rodrigo A. Mora-Rodríguez
Keyword(s):  
Image Analysis ◽  
Real Time ◽  
Crystal Violet ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cytopathic Effect ◽  
Plaque Assay ◽  
Live Cell ◽  
Cell Permeability ◽  
Cytotoxic Activities

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Quantitative Assessment of P-Glycoprotein Expression and Function Using Confocal Image Analysis

2014 ◽  
Vol 20 (5) ◽  
pp. 1329-1339 ◽  
Author(s):  
Zahra Hamrang ◽  
Yamini Arthanari ◽  
David Clarke ◽  
Alain Pluen
Keyword(s):  
Image Analysis ◽  
Cell Lines ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Microscopic Analysis ◽  
Live Cell ◽  
Distribution Analysis ◽  
Spatial Intensity Distribution ◽  
P Glycoprotein

AbstractP-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at “proof of concept” level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin–Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose–response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

scholarly journals KV1.5–KVβ1.3 Recycling Is PKC-Dependent

2021 ◽  
Vol 22 (3) ◽  
pp. 1336
Author(s):  
Alvaro Macias ◽  
Alicia de la Cruz ◽  
Diego A. Peraza ◽  
Angela de Benito-Bueno ◽  
Teresa Gonzalez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Fast Inactivation ◽  
Patch Clamp Technique ◽  
Relevant Issue ◽  
Regulatory Subunits

KV1.5 channel function is modified by different regulatory subunits. KVβ1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVβ1.3-induced fast inactivation, decreases the amplitude of the current KV1.5–KVβ1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5–KVβ1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5–KVβ1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5–KVβ1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5–KVβ1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.

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