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1. Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Author

Lozano-Andrés, Estefanía, Van Den Broeck, Tina, Wang, Lili, Mehrpouyan, Majid, Tian, Ye, Yan, Xiaomei, Arkesteijn, Ger J A, Wauben, Marca H M, Lozano-Andrés, Estefanía, Van Den Broeck, Tina, Wang, Lili, Mehrpouyan, Majid, Tian, Ye, Yan, Xiaomei, Arkesteijn, Ger J A, and Wauben, Marca H M

Abstract

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

Published
2024

2. Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Author

IOV Facs, Celbiologie, Cell Biology, Metabolism & Cancer - Cellbiology, Research Department OH - Biomolecular Health Sciences, Lozano-Andrés, Estefanía, Van Den Broeck, Tina, Wang, Lili, Mehrpouyan, Majid, Tian, Ye, Yan, Xiaomei, Arkesteijn, Ger J A, Wauben, Marca H M, IOV Facs, Celbiologie, Cell Biology, Metabolism & Cancer - Cellbiology, Research Department OH - Biomolecular Health Sciences, Lozano-Andrés, Estefanía, Van Den Broeck, Tina, Wang, Lili, Mehrpouyan, Majid, Tian, Ye, Yan, Xiaomei, Arkesteijn, Ger J A, and Wauben, Marca H M

Published
2024

3. Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Author

Lozano‐Andrés, Estefanía, primary, Enciso‐Martinez, Agustin, additional, Gijsbers, Abril, additional, Ridolfi, Andrea, additional, Van Niel, Guillaume, additional, Libregts, Sten F. W. M., additional, Pinheiro, Cláudio, additional, van Herwijnen, Martijn J. C., additional, Hendrix, An, additional, Brucale, Marco, additional, Valle, Francesco, additional, Peters, Peter J., additional, Otto, Cees, additional, Arkesteijn, Ger J. A., additional, and Wauben, Marca H. M., additional

Published
2023
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4. Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Author

Lozano-Andrés, Estefanía, Enciso-Martinez, Agustin, Gijsbers, Abril, Ridolfi, Andrea, Van Niel, Guillaume, Libregts, Sten F W M, Pinheiro, Cláudio, van Herwijnen, Martijn J C, Hendrix, An, Brucale, Marco, Valle, Francesco, Peters, Peter J, Otto, Cees, Arkesteijn, Ger J A, Wauben, Marca H M, Lozano-Andrés, Estefanía, Enciso-Martinez, Agustin, Gijsbers, Abril, Ridolfi, Andrea, Van Niel, Guillaume, Libregts, Sten F W M, Pinheiro, Cláudio, van Herwijnen, Martijn J C, Hendrix, An, Brucale, Marco, Valle, Francesco, Peters, Peter J, Otto, Cees, Arkesteijn, Ger J A, and Wauben, Marca H M

Abstract

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling.

Published
2023

5. A compendium of single extracellular vesicle flow cytometry

Author

Welsh, Joshua A, Arkesteijn, Ger J A, Bremer, Michel, Cimorelli, Michael, Dignat-George, Françoise, Giebel, Bernd, Görgens, André, Hendrix, An, Kuiper, Martine, Lacroix, Romaric, Lannigan, Joanne, van Leeuwen, Ton G, Lozano-Andrés, Estefanía, Rao, Shoaib, Robert, Stéphane, de Rond, Leonie, Tang, Vera A, Tertel, Tobias, Yan, Xiaomei, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, Nieuwland, Rienk, van der Pol, Edwin, Welsh, Joshua A, Arkesteijn, Ger J A, Bremer, Michel, Cimorelli, Michael, Dignat-George, Françoise, Giebel, Bernd, Görgens, André, Hendrix, An, Kuiper, Martine, Lacroix, Romaric, Lannigan, Joanne, van Leeuwen, Ton G, Lozano-Andrés, Estefanía, Rao, Shoaib, Robert, Stéphane, de Rond, Leonie, Tang, Vera A, Tertel, Tobias, Yan, Xiaomei, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, Nieuwland, Rienk, and van der Pol, Edwin

Abstract

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

Published
2023

6. A compendium of single extracellular vesicle flow cytometry

Author

IOV Facs, Celbiologie, Welsh, Joshua A, Arkesteijn, Ger J A, Bremer, Michel, Cimorelli, Michael, Dignat-George, Françoise, Giebel, Bernd, Görgens, André, Hendrix, An, Kuiper, Martine, Lacroix, Romaric, Lannigan, Joanne, van Leeuwen, Ton G, Lozano-Andrés, Estefanía, Rao, Shoaib, Robert, Stéphane, de Rond, Leonie, Tang, Vera A, Tertel, Tobias, Yan, Xiaomei, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, Nieuwland, Rienk, van der Pol, Edwin, IOV Facs, Celbiologie, Welsh, Joshua A, Arkesteijn, Ger J A, Bremer, Michel, Cimorelli, Michael, Dignat-George, Françoise, Giebel, Bernd, Görgens, André, Hendrix, An, Kuiper, Martine, Lacroix, Romaric, Lannigan, Joanne, van Leeuwen, Ton G, Lozano-Andrés, Estefanía, Rao, Shoaib, Robert, Stéphane, de Rond, Leonie, Tang, Vera A, Tertel, Tobias, Yan, Xiaomei, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, Nieuwland, Rienk, and van der Pol, Edwin

Published
2023

7. Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Author

IOV Facs, Celbiologie, Cell Biology, Metabolism & Cancer - Cellbiology, Research Department OH - Biomolecular Health Sciences, Lozano-Andrés, Estefanía, Enciso-Martinez, Agustin, Gijsbers, Abril, Ridolfi, Andrea, Van Niel, Guillaume, Libregts, Sten F W M, Pinheiro, Cláudio, van Herwijnen, Martijn J C, Hendrix, An, Brucale, Marco, Valle, Francesco, Peters, Peter J, Otto, Cees, Arkesteijn, Ger J A, Wauben, Marca H M, IOV Facs, Celbiologie, Cell Biology, Metabolism & Cancer - Cellbiology, Research Department OH - Biomolecular Health Sciences, Lozano-Andrés, Estefanía, Enciso-Martinez, Agustin, Gijsbers, Abril, Ridolfi, Andrea, Van Niel, Guillaume, Libregts, Sten F W M, Pinheiro, Cláudio, van Herwijnen, Martijn J C, Hendrix, An, Brucale, Marco, Valle, Francesco, Peters, Peter J, Otto, Cees, Arkesteijn, Ger J A, and Wauben, Marca H M

Published
2023

8. A compendium of single extracellular vesicle flow cytometry

Author

Welsh, Joshua A., Arkesteijn, Ger J. A., Bremer, Michel, Cimorelli, Michael, Dignat-George, Françoise, Giebel, Bernd, Görgens, André, Hendrix, An, Kuiper, Martine, Lacroix, Romaric, Lannigan, Joanne, van Leeuwen, Ton G., Lozano-Andrés, Estefanía, Rao, Shoaib, Robert, Stéphane, de Rond, Leonie, Tang, Vera A., Tertel, Tobias, Yan, Xiaomei, Wauben, Marca H. M., Nolan, John P., Jones, Jennifer C., Nieuwland, Rienk, and van der Pol, Edwin

Subjects
Medizin
Abstract

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses. CA extern

Published
2023

9. A compendium of single extracellular vesicle flow cytometry

Author

Welsh, Joshua A., primary, Arkesteijn, Ger J. A., additional, Bremer, Michel, additional, Cimorelli, Michael, additional, Dignat‐George, Françoise, additional, Giebel, Bernd, additional, Görgens, André, additional, Hendrix, An, additional, Kuiper, Martine, additional, Lacroix, Romaric, additional, Lannigan, Joanne, additional, van Leeuwen, Ton G., additional, Lozano‐Andrés, Estefanía, additional, Rao, Shoaib, additional, Robert, Stéphane, additional, de Rond, Leonie, additional, Tang, Vera A., additional, Tertel, Tobias, additional, Yan, Xiaomei, additional, Wauben, Marca H. M., additional, Nolan, John P., additional, Jones, Jennifer C., additional, Nieuwland, Rienk, additional, and van der Pol, Edwin, additional

Published
2023
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10. Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Author

Deddens, Janine C., Vrijsen, Krijn R., Colijn, Johanna M., Oerlemans, Martinus I., Metz, Corina H. G., van der Vlist, Els J., Nolte-’t Hoen, Esther N. M., den Ouden, Krista, Jansen Of Lorkeers, Sanne J., van der Spoel, Tycho I. G., Koudstaal, Stefan, Arkesteijn, Ger J., Wauben, Marca H. M., van Laake, Linda W., Doevendans, Pieter A., Chamuleau, Steven A. J., and Sluijter, Joost P. G.

Published
2016
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12. Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores

Author

Suiker, Inge M, Arkesteijn, Ger J A, Zeegers, Petra J, Wösten, Han A B, Suiker, Inge M, Arkesteijn, Ger J A, Zeegers, Petra J, and Wösten, Han A B

Abstract

Saccharomyces cerevisiae sub-species diastaticus (S. diastaticus) is the main fungal cause of spoilage of carbonated fermented beverages in the brewing industry. Here, prevalence of S. diastaticus in nature and breweries was assessed as well as the spoilage capacity of its vegetative cells and spores. S. diastaticus could only be enriched from 1 out of 136 bark and soil samples from the Netherlands, being the first described natural isolate of this yeast outside South America. On the other hand, it was identified by PCR and selective enrichment in 25 and 21 out of 54 biofilm samples from beer filling halls in Asia, Africa, Europe and North America. ITS sequencing revealed that S. cerevisiae (including S. diastaticus) represented <0.05% of fungal DNA in 17 out of 20 samples, while it represented 0.1, 2 and 32% in samples VH6, VH1 and VH3 respectively. Next, vegetative cells and ascospores of the natural S. diastaticus isolate MB523 were inoculated in a variety of beer products containing 0.0–5.0% alcohol (v/v). Ascospores spoiled all beer products, while vegetative cells did not grow in Radler lemon 0.0, Radler lime mint 0.0 and Radler lemon lime 0.0. Notably, vegetative cells could spoil these Radlers when they first had been grown in alcohol free beer either or not mixed with Radler lemon lime 0.0. Conversely, vegetative cells that had been grown in Radler lemon lime lost their spoilage potential of this beer product when they had grown in YPD medium for more than 24 h. In addition, it was shown that cells grown in alcohol free beer were more heat resistant than cells grown in YPD (D52 40 min and ≤ 10.3 min, respectively). Together, these data show that S. diastaticus is a less prevalent variant of S. cerevisiae in nature, while it accumulates in breweries in mixed biofilms. Data also show that both vegetative cells and spores can spoil all tested beer products, the latter cell type irrespective of its environmental history.

Published
2021

14. Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Author

Maas, Sybren L. N., primary, van Solinge, Thomas S., additional, Schnoor, Rosalie, additional, Yekula, Anudeep, additional, Senders, Joeky T., additional, de Vrij, Jeroen, additional, Robe, Pierre, additional, Carter, Bob S., additional, Balaj, Leonora, additional, Arkesteijn, Ger J. A., additional, Nolte-’t Hoen, Esther N. M., additional, and Broekman, Marike L. D., additional

Published
2020
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17. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments

Author

Welsh, Joshua A, Van Der Pol, Edwin, Arkesteijn, Ger J A, Bremer, Michel, Brisson, Alain, Coumans, Frank, Dignat-George, Françoise, Duggan, Erika, Ghiran, Ionita, Giebel, Bernd, Görgens, André, Hendrix, An, Lacroix, Romaric, Lannigan, Joanne, Libregts, Sten F W M, Lozano-Andrés, Estefanía, Morales-Kastresana, Aizea, Robert, Stephane, De Rond, Leonie, Tertel, Tobias, Tigges, John, De Wever, Olivier, Yan, Xiaomei, Nieuwland, Rienk, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, Welsh, Joshua A, Van Der Pol, Edwin, Arkesteijn, Ger J A, Bremer, Michel, Brisson, Alain, Coumans, Frank, Dignat-George, Françoise, Duggan, Erika, Ghiran, Ionita, Giebel, Bernd, Görgens, André, Hendrix, An, Lacroix, Romaric, Lannigan, Joanne, Libregts, Sten F W M, Lozano-Andrés, Estefanía, Morales-Kastresana, Aizea, Robert, Stephane, De Rond, Leonie, Tertel, Tobias, Tigges, John, De Wever, Olivier, Yan, Xiaomei, Nieuwland, Rienk, Wauben, Marca H M, Nolan, John P, and Jones, Jennifer C

Abstract

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratori

Published
2020

18. Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Author

Maas, Sybren L N, van Solinge, Thomas S, Schnoor, Rosalie, Yekula, Anudeep, Senders, Joeky T, de Vrij, Jeroen, Robe, Pierre, Carter, Bob S, Balaj, Leonora, Arkesteijn, Ger J A, Nolte-'t Hoen, Esther N M, Broekman, Marike L D, Maas, Sybren L N, van Solinge, Thomas S, Schnoor, Rosalie, Yekula, Anudeep, Senders, Joeky T, de Vrij, Jeroen, Robe, Pierre, Carter, Bob S, Balaj, Leonora, Arkesteijn, Ger J A, Nolte-'t Hoen, Esther N M, and Broekman, Marike L D

Abstract

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

Published
2020

19. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments

Author

LS Celbiologie-Algemeen, Celbiologie, dB&C I&I, Welsh, Joshua A, Van Der Pol, Edwin, Arkesteijn, Ger J A, Bremer, Michel, Brisson, Alain, Coumans, Frank, Dignat-George, Françoise, Duggan, Erika, Ghiran, Ionita, Giebel, Bernd, Görgens, André, Hendrix, An, Lacroix, Romaric, Lannigan, Joanne, Libregts, Sten F W M, Lozano-Andrés, Estefanía, Morales-Kastresana, Aizea, Robert, Stephane, De Rond, Leonie, Tertel, Tobias, Tigges, John, De Wever, Olivier, Yan, Xiaomei, Nieuwland, Rienk, Wauben, Marca H M, Nolan, John P, Jones, Jennifer C, LS Celbiologie-Algemeen, Celbiologie, dB&C I&I, Welsh, Joshua A, Van Der Pol, Edwin, Arkesteijn, Ger J A, Bremer, Michel, Brisson, Alain, Coumans, Frank, Dignat-George, Françoise, Duggan, Erika, Ghiran, Ionita, Giebel, Bernd, Görgens, André, Hendrix, An, Lacroix, Romaric, Lannigan, Joanne, Libregts, Sten F W M, Lozano-Andrés, Estefanía, Morales-Kastresana, Aizea, Robert, Stephane, De Rond, Leonie, Tertel, Tobias, Tigges, John, De Wever, Olivier, Yan, Xiaomei, Nieuwland, Rienk, Wauben, Marca H M, Nolan, John P, and Jones, Jennifer C

Published
2020

22. Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential

Author

van der Grein, Susanne G, Defourny, Kyra A Y, Rabouw, Huib H, Galiveti, Chenna R, Langereis, Martijn A, Wauben, Marca H M, Arkesteijn, Ger J A, van Kuppeveld, Frank J M, Nolte-'t Hoen, Esther N M, van der Grein, Susanne G, Defourny, Kyra A Y, Rabouw, Huib H, Galiveti, Chenna R, Langereis, Martijn A, Wauben, Marca H M, Arkesteijn, Ger J A, van Kuppeveld, Frank J M, and Nolte-'t Hoen, Esther N M

Abstract

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.

Published
2019

23. Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential

Author

dB&C I&I, LS Celbiologie-Algemeen, LS Virologie, dI&I I&I-1, van der Grein, Susanne G, Defourny, Kyra A Y, Rabouw, Huib H, Galiveti, Chenna R, Langereis, Martijn A, Wauben, Marca H M, Arkesteijn, Ger J A, van Kuppeveld, Frank J M, Nolte-'t Hoen, Esther N M, dB&C I&I, LS Celbiologie-Algemeen, LS Virologie, dI&I I&I-1, van der Grein, Susanne G, Defourny, Kyra A Y, Rabouw, Huib H, Galiveti, Chenna R, Langereis, Martijn A, Wauben, Marca H M, Arkesteijn, Ger J A, van Kuppeveld, Frank J M, and Nolte-'t Hoen, Esther N M

Published
2019

24. Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential

Author

van der Grein, Susanne G., primary, Defourny, Kyra A. Y., additional, Rabouw, Huib H., additional, Galiveti, Chenna R., additional, Langereis, Martijn A., additional, Wauben, Marca H. M., additional, Arkesteijn, Ger J. A., additional, van Kuppeveld, Frank J. M., additional, and Nolte-‘t Hoen, Esther N. M., additional

Published
2019
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25. Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases

Author

Groot Kormelink, Tom, Arkesteijn, Ger J A, van de Lest, Chris H A, Geerts, Willie J C, Goerdayal, Soenita, Altelaar, Maarten A F, Redegeld, Frank A, Nolte-'t Hoen, Esther N M, Wauben, Marca H M, Pharmacology, Biomolecular Mass Spectrometry and Proteomics, Cryo-EM, dES RMSC, dB&C I&I, dB&C FR-RMSC FR, Dep Infectieziekten Immunologie, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, Sub Cryo - EM, Sub Biomol.Mass Spect. and Proteomics, Sub Immunopharmacology, LS Celbiologie-Algemeen, Pharmacology, Biomolecular Mass Spectrometry and Proteomics, Cryo-EM, dES RMSC, dB&C I&I, dB&C FR-RMSC FR, Dep Infectieziekten Immunologie, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, Sub Cryo - EM, Sub Biomol.Mass Spect. and Proteomics, Sub Immunopharmacology, and LS Celbiologie-Algemeen

Subjects
0301 basic medicine, Proteases, Cell Degranulation, Immunology, Biology, Extracellular Vesicles, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Taverne, medicine, Animals, Immunology and Allergy, Mast Cells, Phosphatidylinositol, Lipid bilayer, Cells, Cultured, Degranulation, Phosphatidic acid, Mast cell, Cell biology, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, chemistry, 030220 oncology & carcinogenesis, Intracellular, Peptide Hydrolases
Abstract

Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue–type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9+ EV, degranulation was accompanied by the release of CD63+ EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63+ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9+ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63+ EV from connective tissue–type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.

Published
2016

26. Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Author

Boere, Janneke, van de Lest, Chris H A, Libregts, Sten F W M, Arkesteijn, Ger J A, Geerts, Willie J C, Nolte-'t Hoen, Esther N M, Malda, Jos, van Weeren, P René, Wauben, Marca H M, dB&C I&I, Cryo-EM, dB&C FR-RMSC FR, dES RMSC, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, LS Celbiologie-Algemeen, Sub Cryo - EM, and Dep Gezondheidszorg Paard

Subjects
rapid test, lateral flow immunoassay, ELISA, exosomes, in vitro diagnostics
Abstract

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

Published
2016

27. Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

Author

Buishand, Floryne O, Arkesteijn, Ger J A, Feenstra, Laurien R, Oorsprong, Claire W D, Mestemaker, Margiet, Starke, Achim, Speel, Ernst-Jan M, Kirpensteijn, Jolle, Mol, Jan A, Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, dCSCA AVR, dCSCA RMSC-2, Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, dCSCA AVR, dCSCA RMSC-2, RS: GROW - R2 - Basic and Translational Cancer Biology, and Pathologie

Subjects
0301 basic medicine, Embryo, Nonmammalian, Carcinogenesis, medicine.drug_class, Mice, Nude, Biology, medicine.disease_cause, Monoclonal antibody, Bioinformatics, 03 medical and health sciences, Original Research Reports, Cancer stem cell, Cell Line, Tumor, Gangliosides, Biomarkers, Tumor, medicine, Animals, Humans, CD90, Molecular Targeted Therapy, Insulinoma, Zebrafish, Cell Biology, Hematology, Cell sorting, Flow Cytometry, medicine.disease, Immunohistochemistry, Xenograft Model Antitumor Assays, 030104 developmental biology, Gene Expression Regulation, Doxorubicin, Cell culture, embryonic structures, Neoplastic Stem Cells, Cancer research, Thy-1 Antigens, Developmental Biology
Abstract

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment.

Published
2016

28. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry

Author

Kormelink, Tom Groot, Arkesteijn, Ger J A, Nauwelaers, Frans A, van den Engh, Ger, Nolte - t Hoen, Esther, Wauben, Marca H M, Fertility & Reproduction, LS Celbiologie-Algemeen, LS Immunologie, and dB&C I&I

Abstract

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is

Published
2015

29. The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes

Author

Bach, Frances C, Rutten, Kirsten, Hendriks, Kristyanne, Riemers, Frank M, Cornelissen, Peter, de Bruin, Alain, Arkesteijn, Ger J, Wubbolts, Richard, Horton, William A, Penning, Louis C, Tryfonidou, Marianna A, Sub Neuro/Tandheelkunde, CSCA TR1, Biochemisch laboratorium, LS Pathobiologie, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Onderzoek, CSCA AVM, Advances in Veterinary Medicine, and Tissue Repair

Subjects
musculoskeletal diseases, Feedback, Physiological, Parathyroid Hormone-Related Protein, Gene Expression Regulation, Developmental, Cell Differentiation, Mice, Chondrocytes, Paracrine Communication, Animals, Humans, Growth Plate, hormones, hormone substitutes, and hormone antagonists, Cholecalciferol, Receptor, Parathyroid Hormone, Type 1
Abstract

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Published
2014

30. Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Author

Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Martinus I, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen Of Lorkeers, Sanne J, van der Spoel, Tycho I G, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, Sluijter, Joost P G, Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Martinus I, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen Of Lorkeers, Sanne J, van der Spoel, Tycho I G, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, and Sluijter, Joost P G

Abstract

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

Published
2016

31. Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Author

dB&C I&I, Cryo-EM, dB&C FR-RMSC FR, dES RMSC, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, LS Celbiologie-Algemeen, Sub Cryo - EM, Dep Gezondheidszorg Paard, Boere, Janneke, van de Lest, Chris H A, Libregts, Sten F W M, Arkesteijn, Ger J A, Geerts, Willie J C, Nolte-'t Hoen, Esther N M, Malda, Jos, van Weeren, P René, Wauben, Marca H M, dB&C I&I, Cryo-EM, dB&C FR-RMSC FR, dES RMSC, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, LS Celbiologie-Algemeen, Sub Cryo - EM, Dep Gezondheidszorg Paard, Boere, Janneke, van de Lest, Chris H A, Libregts, Sten F W M, Arkesteijn, Ger J A, Geerts, Willie J C, Nolte-'t Hoen, Esther N M, Malda, Jos, van Weeren, P René, and Wauben, Marca H M

Published
2016

32. Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Author

Sub Immunopharmacology, LS Celbiologie-Algemeen, Pharmacology, dB&C I&I, Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Martinus I, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen Of Lorkeers, Sanne J, van der Spoel, Tycho I G, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, Sluijter, Joost P G, Sub Immunopharmacology, LS Celbiologie-Algemeen, Pharmacology, dB&C I&I, Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Martinus I, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen Of Lorkeers, Sanne J, van der Spoel, Tycho I G, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, and Sluijter, Joost P G

Published
2016

33. Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases

Author

Pharmacology, Biomolecular Mass Spectrometry and Proteomics, Cryo-EM, dES RMSC, dB&C I&I, dB&C FR-RMSC FR, Dep Infectieziekten Immunologie, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, Sub Cryo - EM, Sub Biomol.Mass Spect. and Proteomics, Sub Immunopharmacology, LS Celbiologie-Algemeen, Groot Kormelink, Tom, Arkesteijn, Ger J A, van de Lest, Chris H A, Geerts, Willie J C, Goerdayal, Soenita, Altelaar, Maarten A F, Redegeld, Frank A, Nolte-'t Hoen, Esther N M, Wauben, Marca H M, Pharmacology, Biomolecular Mass Spectrometry and Proteomics, Cryo-EM, dES RMSC, dB&C I&I, dB&C FR-RMSC FR, Dep Infectieziekten Immunologie, LS Equine Muscoskeletal Biology, LS Veterinaire biochemie, Sub Cryo - EM, Sub Biomol.Mass Spect. and Proteomics, Sub Immunopharmacology, LS Celbiologie-Algemeen, Groot Kormelink, Tom, Arkesteijn, Ger J A, van de Lest, Chris H A, Geerts, Willie J C, Goerdayal, Soenita, Altelaar, Maarten A F, Redegeld, Frank A, Nolte-'t Hoen, Esther N M, and Wauben, Marca H M

Published
2016

34. Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

Author

Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, dCSCA AVR, dCSCA RMSC-2, Buishand, Floryne O, Arkesteijn, Ger J A, Feenstra, Laurien R, Oorsprong, Claire W D, Mestemaker, Margiet, Starke, Achim, Speel, Ernst-Jan M, Kirpensteijn, Jolle, Mol, Jan A, Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, dCSCA AVR, dCSCA RMSC-2, Buishand, Floryne O, Arkesteijn, Ger J A, Feenstra, Laurien R, Oorsprong, Claire W D, Mestemaker, Margiet, Starke, Achim, Speel, Ernst-Jan M, Kirpensteijn, Jolle, and Mol, Jan A

Published
2016

35. Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Author

Cardiologie Arts-onderzoekers, Aios en Stafsecr. Cardiologie, Circulatory Health, Cardiologie, MS Nefrologie, Regenerative Medicine and Stem Cells, Onderzoek Vrouw Hart & Vaatziekten, Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Marish, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen of Lorkeers, SJ, van der Spoel, TIG, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, Sluijter, Joost P G, Cardiologie Arts-onderzoekers, Aios en Stafsecr. Cardiologie, Circulatory Health, Cardiologie, MS Nefrologie, Regenerative Medicine and Stem Cells, Onderzoek Vrouw Hart & Vaatziekten, Deddens, Janine C, Vrijsen, Krijn R, Colijn, Johanna M, Oerlemans, Marish, Metz, Corina H G, van der Vlist, Els J, Nolte-'t Hoen, Esther N M, den Ouden, Krista, Jansen of Lorkeers, SJ, van der Spoel, TIG, Koudstaal, Stefan, Arkesteijn, Ger J, Wauben, Marca H M, van Laake, Linda W, Doevendans, Pieter A, Chamuleau, Steven A J, and Sluijter, Joost P G

Published
2016

36. Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

Author

dCSCA AVR, dCSCA RMSC-2, Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, Buishand, Floryne O, Arkesteijn, Ger J A, Feenstra, Laurien R, Oorsprong, Claire W D, Mestemaker, Margiet, Starke, Achim, Speel, Ernst-Jan M, Kirpensteijn, Jolle, Mol, Jan A, dCSCA AVR, dCSCA RMSC-2, Opleiding chirurgie, LS Algemene chirurgie, Onderzoek, Buishand, Floryne O, Arkesteijn, Ger J A, Feenstra, Laurien R, Oorsprong, Claire W D, Mestemaker, Margiet, Starke, Achim, Speel, Ernst-Jan M, Kirpensteijn, Jolle, and Mol, Jan A

Published
2016

37. Alpha 6 integrin is important for myogenic stem cell differentiation

Author

Wilschut, Karlijn J, van Tol, Helena T A, Arkesteijn, Ger J A, Haagsman, Henk P, Roelen, Bernard A J, LS Moleculaire Afweer, Sub Celbiologisch lab., LS Voortplanting Inwendige Ziekten, I&I SIB3, Strategic Infection Biology, LS Moleculaire Afweer, Sub Celbiologisch lab., LS Voortplanting Inwendige Ziekten, I&I SIB3, and Strategic Infection Biology

Subjects
Medicine(all), Cultured, Myogenesis, Swine, Cellular differentiation, Cells, Muscles, Stem Cells, Cell Differentiation, General Medicine, Cell Biology, Biology, Integrin alpha6, Molecular biology, Collagen receptor, Endothelial stem cell, Animals, Humans, MYF5, Stem cell, Progenitor cell, Cells, Cultured, Adult stem cell, Developmental Biology
Abstract

A muscle progenitor cell population, other than muscle satellite cells, can be isolated and purified from porcine muscle tissue. We show the presence of at least two types of stem cells in porcine muscle: those that express α6 integrin and those that lack expression of this integrin type. By flow cytometry, we could select for myogenic stem cell populations expressing the neural cell adhesion molecule in the presence and absence of α6 integrin. The expression of α6 integrin showed an advantage in the formation of myotubes, possibly by an improved cell fusion capacity. This notion was strengthened by qRT-PCR analysis showing sustained PAX7, MYF5 and DESMIN expression and a strong myogenic differentiation capacity of this stem cell population. Selective inhibition of α6 integrin function, both by blocking antibodies and RNA interference, showed the importance of α6 integrin in myogenic differentiation of muscle stem cells. It is concluded that α6 integrin expression can be used as biomarker to select for highly myogenic cell populations in muscle tissue.

Published
2011

38. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry

Author

Fertility & Reproduction, LS Celbiologie-Algemeen, LS Immunologie, dB&C I&I, Kormelink, Tom Groot, Arkesteijn, Ger J A, Nauwelaers, Frans A, van den Engh, Ger, Nolte - t Hoen, Esther, Wauben, Marca H M, Fertility & Reproduction, LS Celbiologie-Algemeen, LS Immunologie, dB&C I&I, Kormelink, Tom Groot, Arkesteijn, Ger J A, Nauwelaers, Frans A, van den Engh, Ger, Nolte - t Hoen, Esther, and Wauben, Marca H M

Published
2015

40. The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes

Author

Sub Neuro/Tandheelkunde, CSCA TR1, Biochemisch laboratorium, LS Pathobiologie, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Onderzoek, CSCA AVM, Advances in Veterinary Medicine, Tissue Repair, Bach, Frances C, Rutten, Kirsten, Hendriks, Kristyanne, Riemers, Frank M, Cornelissen, Peter, de Bruin, Alain, Arkesteijn, Ger J, Wubbolts, Richard, Horton, William A, Penning, Louis C, Tryfonidou, Marianna A, Sub Neuro/Tandheelkunde, CSCA TR1, Biochemisch laboratorium, LS Pathobiologie, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Onderzoek, CSCA AVM, Advances in Veterinary Medicine, Tissue Repair, Bach, Frances C, Rutten, Kirsten, Hendriks, Kristyanne, Riemers, Frank M, Cornelissen, Peter, de Bruin, Alain, Arkesteijn, Ger J, Wubbolts, Richard, Horton, William A, Penning, Louis C, and Tryfonidou, Marianna A

Published
2014

41. The Paracrine Feedback Loop Between Vitamin D3(1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

Author

Bach, Frances C., primary, Rutten, Kirsten, additional, Hendriks, Kristyanne, additional, Riemers, Frank M., additional, Cornelissen, Peter, additional, de Bruin, Alain, additional, Arkesteijn, Ger J., additional, Wubbolts, Richard, additional, Horton, William A., additional, Penning, Louis C., additional, and Tryfonidou, Marianna A., additional

Published
2014
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42. Alpha 6 integrin is important for myogenic stem cell differentiation

Author

LS Moleculaire Afweer, Sub Celbiologisch lab., LS Voortplanting Inwendige Ziekten, I&I SIB3, Strategic Infection Biology, Wilschut, Karlijn J, van Tol, Helena T A, Arkesteijn, Ger J A, Haagsman, Henk P, Roelen, Bernard A J, LS Moleculaire Afweer, Sub Celbiologisch lab., LS Voortplanting Inwendige Ziekten, I&I SIB3, Strategic Infection Biology, Wilschut, Karlijn J, van Tol, Helena T A, Arkesteijn, Ger J A, Haagsman, Henk P, and Roelen, Bernard A J

Published
2011

46. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

Author

Kormelink, Tom Groot, Arkesteijn, Ger J. A., Nauwelaers, Frans A., van den Engh, Ger, Nolte‐'t Hoen, Esther N. M., and Wauben, Marca H. M.

Abstract

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. © 2015 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Published
2016
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48. The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes.

Author

Bach, Frances C., Rutten, Kirsten, Hendriks, Kristyanne, Riemers, Frank M., Cornelissen, Peter, de Bruin, Alain, Arkesteijn, Ger J., Wubbolts, Richard, Horton, William A., Penning, Louis C., and Tryfonidou, Marianna A.

Subjects
PARACRINE mechanisms, VITAMIN D, PARATHYROID hormone-related protein, CARTILAGE cells, WESTERN immunoblotting, POLYMERASE chain reaction, IMMUNOFLUORESCENCE
Abstract

The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999-2014, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Published
2014
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49. Dynamics of dendritic cell‐derived vesicles: high‐resolution flow cytometric analysis of extracellular vesicle quantity and quality

Author

Nolte‐‘t Hoen, Esther N. M., Vlist, Els J., Boer‐Brouwer, Mieke, Arkesteijn, Ger J. A., Stoorvogel, Willem, and Wauben, Marca H. M.

Abstract

Application of a described high‐resolution flow cytometrybased analysis of individual extracellular vesicles, to study the dynamics of dendritic cell‐derived vesicle secretion and composition. Nano‐sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC‐derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC‐derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed high‐resolution flow cytometry‐based method, we can perform a high‐throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC‐derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS‐stimulated DC and CD4+T cells affects both the quantity and quality of LPS DC‐derived EVs present in the culture supernatant. These data indicate that flow cytometry‐based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell‐derived EVs.

Published
2013
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50. Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134

Author

Nolte‐’t Hoen, Esther N. M., Boot, Elmieke P. J., Wagenaar‐Hilbers, Josée P. A., Bilsen, Jolanda H. M., Arkesteijn, Ger J. A., Storm, Gert, Everse, Linda A., Eden, Willem, and Wauben, Marca H. M.

Abstract

Major problems in the analysis of CD4+effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease‐associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis.

Published
2008
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