Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

Sylwia Dabrowska; Andrea Del Fattore; Elzbieta Karnas; Malgorzata Frontczak-Baniewicz; Hanna Kozlowska; Maurizio Muraca; Miroslaw Janowski; Barbara Lukomska

doi:10.2147/IJN.S159404

Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

Sylwia Dabrowska, Andrea Del Fattore, Elzbieta Karnas, Malgorzata Frontczak-Baniewicz, Hanna Kozlowska, Maurizio Muraca, Miroslaw Janowski, Barbara Lukomska

School of Medicine

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

Abstract

Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs. Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles. Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs. Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI.

Original language	English (US)
Pages (from-to)	1653-1664
Number of pages	12
Journal	International journal of nanomedicine
Volume	13
DOIs	https://doi.org/10.2147/IJN.S159404
State	Published - Mar 19 2018

Keywords

Cell tracking
Extracellular vesicles
Fluorescent dye
Iron oxide
MRI
Mesenchymal stem cells

ASJC Scopus subject areas

Biophysics
Bioengineering
Biomaterials
Pharmaceutical Science
Drug Discovery
Organic Chemistry

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10.2147/IJN.S159404

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@article{7808e1a7c6174e288644c837168f6420,

title = "Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels",

abstract = "Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs. Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B{\texttrademark}) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles. Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs. Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI.",

keywords = "Cell tracking, Extracellular vesicles, Fluorescent dye, Iron oxide, MRI, Mesenchymal stem cells",

author = "Sylwia Dabrowska and {Del Fattore}, Andrea and Elzbieta Karnas and Malgorzata Frontczak-Baniewicz and Hanna Kozlowska and Maurizio Muraca and Miroslaw Janowski and Barbara Lukomska",

note = "Funding Information: We thank Jaroslaw Orzel for his help in performing MRI experiments. This work was supported by the Polish Ministry of Scientific Research and Higher Education grant, KNOW 06 project, STRATEGMED1/235773/19/ NCBR/2016 “EXPLORE ME” project (SD, MJ, and BL) and R01 NS091110 project (MJ). MM gratefully acknowledges the financial support of “Fondazione Citt{\`a} dell{\`a} Speranza” (Padova, Italy). Ultracentrifuge Series Thermo Fisher Scientific used in these studies was sponsored in part by the Centre for Preclinical Research and Technology (CePT), Funding Information: a project co-sponsored by The Innovative Economy, The National Cohesion Strategy of Poland. MRI experiments carried out with the use of the CePT infrastructure were financed by the European Union – the European Regional Development Fund in the Operational Programme “Innovative Economy” for 2007–2013. Publisher Copyright: {\textcopyright} 2018 Dabrowska et al.",

year = "2018",

month = mar,

day = "19",

doi = "10.2147/IJN.S159404",

language = "English (US)",

volume = "13",

pages = "1653--1664",

journal = "International journal of nanomedicine",

issn = "1176-9114",

publisher = "Dove Medical Press Ltd.",

}

TY - JOUR

T1 - Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

AU - Dabrowska, Sylwia

AU - Del Fattore, Andrea

AU - Karnas, Elzbieta

AU - Frontczak-Baniewicz, Malgorzata

AU - Kozlowska, Hanna

AU - Muraca, Maurizio

AU - Janowski, Miroslaw

AU - Lukomska, Barbara

N1 - Funding Information: We thank Jaroslaw Orzel for his help in performing MRI experiments. This work was supported by the Polish Ministry of Scientific Research and Higher Education grant, KNOW 06 project, STRATEGMED1/235773/19/ NCBR/2016 “EXPLORE ME” project (SD, MJ, and BL) and R01 NS091110 project (MJ). MM gratefully acknowledges the financial support of “Fondazione Città dellà Speranza” (Padova, Italy). Ultracentrifuge Series Thermo Fisher Scientific used in these studies was sponsored in part by the Centre for Preclinical Research and Technology (CePT), Funding Information: a project co-sponsored by The Innovative Economy, The National Cohesion Strategy of Poland. MRI experiments carried out with the use of the CePT infrastructure were financed by the European Union – the European Regional Development Fund in the Operational Programme “Innovative Economy” for 2007–2013. Publisher Copyright: © 2018 Dabrowska et al.

PY - 2018/3/19

Y1 - 2018/3/19

N2 - Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs. Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles. Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs. Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI.

AB - Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs. Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles. Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs. Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI.

KW - Cell tracking

KW - Extracellular vesicles

KW - Fluorescent dye

KW - Iron oxide

KW - MRI

KW - Mesenchymal stem cells

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VL - 13

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EP - 1664

JO - International journal of nanomedicine

JF - International journal of nanomedicine

ER -

Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

Abstract

Keywords

ASJC Scopus subject areas

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