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Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Di Trapani M, Bassi G, Midolo M, Gatti A, Kamga PT, Cassaro A, Carusone R, Adamo A, Krampera M - Sci Rep (2016)

Bottom Line: Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease.We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division.Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy.

ABSTRACT
Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus

Characterization of MSC-derived EVs.(a) Size distribution of resting and primed EVs obtained by NTA. (b) Immunoblot analysis of Giantin, LAMP1, Alix, GRP78, HSP70 and CD9 expression in resting and primed MSCs and purified EVs. This blot is representative of three independent experiments showing the same trends. (c) Representative plots of the immunophenotypic analysis of MSC-derived EVs showing the expression profile of a specific exosome marker (CD63), mesenchymal stromal cell markers (CD73, CD90 and CD105), adhesion molecules (ICAM-1 and VCAM-1) and MHC class I and II (HLA-ABC and HLA-DR, respectively). The histograms display the isotopic controls (dotted curve) and specific markers of resting (filled curve) and primed (black curve) EVs. (d) Graph showing quantitative differences of EV release between resting and primed MSCs. Data represented as mean ± SEM of ratio between number of EVs and number of cells of origin obtained from 33 independent experiments. ***P < 0.001.
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f4: Characterization of MSC-derived EVs.(a) Size distribution of resting and primed EVs obtained by NTA. (b) Immunoblot analysis of Giantin, LAMP1, Alix, GRP78, HSP70 and CD9 expression in resting and primed MSCs and purified EVs. This blot is representative of three independent experiments showing the same trends. (c) Representative plots of the immunophenotypic analysis of MSC-derived EVs showing the expression profile of a specific exosome marker (CD63), mesenchymal stromal cell markers (CD73, CD90 and CD105), adhesion molecules (ICAM-1 and VCAM-1) and MHC class I and II (HLA-ABC and HLA-DR, respectively). The histograms display the isotopic controls (dotted curve) and specific markers of resting (filled curve) and primed (black curve) EVs. (d) Graph showing quantitative differences of EV release between resting and primed MSCs. Data represented as mean ± SEM of ratio between number of EVs and number of cells of origin obtained from 33 independent experiments. ***P < 0.001.

Mentions: Next, we performed a qualitative and quantitative analysis of MSC-derived EVs. NTA showed that the size of isolated EVs ranged between 60 nm and 150 nm, corresponding to exosomes, while only a few peaks ranged from 200 nm and 400 nm, corresponding to larger EVs (Fig. 4a).


Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Di Trapani M, Bassi G, Midolo M, Gatti A, Kamga PT, Cassaro A, Carusone R, Adamo A, Krampera M - Sci Rep (2016)

Characterization of MSC-derived EVs.(a) Size distribution of resting and primed EVs obtained by NTA. (b) Immunoblot analysis of Giantin, LAMP1, Alix, GRP78, HSP70 and CD9 expression in resting and primed MSCs and purified EVs. This blot is representative of three independent experiments showing the same trends. (c) Representative plots of the immunophenotypic analysis of MSC-derived EVs showing the expression profile of a specific exosome marker (CD63), mesenchymal stromal cell markers (CD73, CD90 and CD105), adhesion molecules (ICAM-1 and VCAM-1) and MHC class I and II (HLA-ABC and HLA-DR, respectively). The histograms display the isotopic controls (dotted curve) and specific markers of resting (filled curve) and primed (black curve) EVs. (d) Graph showing quantitative differences of EV release between resting and primed MSCs. Data represented as mean ± SEM of ratio between number of EVs and number of cells of origin obtained from 33 independent experiments. ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829861&req=5

f4: Characterization of MSC-derived EVs.(a) Size distribution of resting and primed EVs obtained by NTA. (b) Immunoblot analysis of Giantin, LAMP1, Alix, GRP78, HSP70 and CD9 expression in resting and primed MSCs and purified EVs. This blot is representative of three independent experiments showing the same trends. (c) Representative plots of the immunophenotypic analysis of MSC-derived EVs showing the expression profile of a specific exosome marker (CD63), mesenchymal stromal cell markers (CD73, CD90 and CD105), adhesion molecules (ICAM-1 and VCAM-1) and MHC class I and II (HLA-ABC and HLA-DR, respectively). The histograms display the isotopic controls (dotted curve) and specific markers of resting (filled curve) and primed (black curve) EVs. (d) Graph showing quantitative differences of EV release between resting and primed MSCs. Data represented as mean ± SEM of ratio between number of EVs and number of cells of origin obtained from 33 independent experiments. ***P < 0.001.
Mentions: Next, we performed a qualitative and quantitative analysis of MSC-derived EVs. NTA showed that the size of isolated EVs ranged between 60 nm and 150 nm, corresponding to exosomes, while only a few peaks ranged from 200 nm and 400 nm, corresponding to larger EVs (Fig. 4a).

Bottom Line: Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease.We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division.Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy.

ABSTRACT
Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus