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Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging.

Grange C, Tapparo M, Bruno S, Chatterjee D, Quesenberry PJ, Tetta C, Camussi G - Int. J. Mol. Med. (2014)

Bottom Line: The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells.The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells.The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, University of Torino, Torino, Italy.

ABSTRACT
Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

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Ex vivo optical imaging (OI) analysis of dissected organs. (A) Representative OI images of dissected organs of mice with acute kidney injury (AKI) and healthy mice treated with LCD- and DL-EVs sacrificed at 5 and 24 h after injection. (B) Fluorescence quantification of kidneys, expressed as the average radiance ± standard deviation (SD). ANOVA with Newman-Keuls multi-comparison test was performed. *p<0.05 AKI EVs vs. healthy EVs. (C) Fluorescence quantification, expressed as the average radiance ± SD, of lungs, liver and spleen. AKI mice treated with LCD-EVs sacrificed at 5 h (n=9) and at 24 h (n=7); AKI mice treated with DL-EVs sacrificed at 5 h (n=6) and at 24 h (n=5); healthy mice treated with LCD-EVs sacrificed at 5 h (n=6) and at 24 h (n=6); healthy mice treated with DL-EVs sacrificed at 5 h (n=3) and at 24 h (n=3). LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.
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f6-ijmm-33-05-1055: Ex vivo optical imaging (OI) analysis of dissected organs. (A) Representative OI images of dissected organs of mice with acute kidney injury (AKI) and healthy mice treated with LCD- and DL-EVs sacrificed at 5 and 24 h after injection. (B) Fluorescence quantification of kidneys, expressed as the average radiance ± standard deviation (SD). ANOVA with Newman-Keuls multi-comparison test was performed. *p<0.05 AKI EVs vs. healthy EVs. (C) Fluorescence quantification, expressed as the average radiance ± SD, of lungs, liver and spleen. AKI mice treated with LCD-EVs sacrificed at 5 h (n=9) and at 24 h (n=7); AKI mice treated with DL-EVs sacrificed at 5 h (n=6) and at 24 h (n=5); healthy mice treated with LCD-EVs sacrificed at 5 h (n=6) and at 24 h (n=6); healthy mice treated with DL-EVs sacrificed at 5 h (n=3) and at 24 h (n=3). LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Mentions: For each experimental group, the mice were sacrificed at 5 and 24 h after the EV injection and the fluorescent signal from freshly dissected tissues was quantified immediately by OI. The fluorescence intensity of the kidneys of mice with AKI treated with LCD-EVs and DL-EVs was significantly higher after 5 h compared with the kidneys of mice with AKI treated with PBS [AKI CTL (control)], as shown in Fig 6. Nevertheless, the increase in fluorescence was significantly maintained for 24 h following treatment in the DL-EV group, whereas it decreased in the LCD-EV group (Fig. 6A). No signal was detected in the kidneys of healthy mice treated with LCD-EVs and DL-EVs, suggesting a specific accumulation of EVs at the site of injury.


Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging.

Grange C, Tapparo M, Bruno S, Chatterjee D, Quesenberry PJ, Tetta C, Camussi G - Int. J. Mol. Med. (2014)

Ex vivo optical imaging (OI) analysis of dissected organs. (A) Representative OI images of dissected organs of mice with acute kidney injury (AKI) and healthy mice treated with LCD- and DL-EVs sacrificed at 5 and 24 h after injection. (B) Fluorescence quantification of kidneys, expressed as the average radiance ± standard deviation (SD). ANOVA with Newman-Keuls multi-comparison test was performed. *p<0.05 AKI EVs vs. healthy EVs. (C) Fluorescence quantification, expressed as the average radiance ± SD, of lungs, liver and spleen. AKI mice treated with LCD-EVs sacrificed at 5 h (n=9) and at 24 h (n=7); AKI mice treated with DL-EVs sacrificed at 5 h (n=6) and at 24 h (n=5); healthy mice treated with LCD-EVs sacrificed at 5 h (n=6) and at 24 h (n=6); healthy mice treated with DL-EVs sacrificed at 5 h (n=3) and at 24 h (n=3). LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020482&req=5

f6-ijmm-33-05-1055: Ex vivo optical imaging (OI) analysis of dissected organs. (A) Representative OI images of dissected organs of mice with acute kidney injury (AKI) and healthy mice treated with LCD- and DL-EVs sacrificed at 5 and 24 h after injection. (B) Fluorescence quantification of kidneys, expressed as the average radiance ± standard deviation (SD). ANOVA with Newman-Keuls multi-comparison test was performed. *p<0.05 AKI EVs vs. healthy EVs. (C) Fluorescence quantification, expressed as the average radiance ± SD, of lungs, liver and spleen. AKI mice treated with LCD-EVs sacrificed at 5 h (n=9) and at 24 h (n=7); AKI mice treated with DL-EVs sacrificed at 5 h (n=6) and at 24 h (n=5); healthy mice treated with LCD-EVs sacrificed at 5 h (n=6) and at 24 h (n=6); healthy mice treated with DL-EVs sacrificed at 5 h (n=3) and at 24 h (n=3). LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.
Mentions: For each experimental group, the mice were sacrificed at 5 and 24 h after the EV injection and the fluorescent signal from freshly dissected tissues was quantified immediately by OI. The fluorescence intensity of the kidneys of mice with AKI treated with LCD-EVs and DL-EVs was significantly higher after 5 h compared with the kidneys of mice with AKI treated with PBS [AKI CTL (control)], as shown in Fig 6. Nevertheless, the increase in fluorescence was significantly maintained for 24 h following treatment in the DL-EV group, whereas it decreased in the LCD-EV group (Fig. 6A). No signal was detected in the kidneys of healthy mice treated with LCD-EVs and DL-EVs, suggesting a specific accumulation of EVs at the site of injury.

Bottom Line: The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells.The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells.The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, University of Torino, Torino, Italy.

ABSTRACT
Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

Show MeSH
Related in: MedlinePlus