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

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.

Figure 1

In vitro fluorescence assay. (A) Representative in vitro fluorescence images of extracellular vesicle (EV) dilutions from 15 to 100 μg of protein in 100 μl of phosphate-buffered saline (PBS). (B) Quantification of fluorescence signal calculated in the entire circle area of each well. Data are expressed as average radiance ± standard deviation (SD) of three different experiments. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Figure 2

Evaluation of fluorescence of labeled cell-derived extracellular vesicles (EVs) by a Guava cytofluorimeter. (A and B). Representative dot plots and histogram plots of the fluorescent signal of labeled EVs. (A) DL-EVs show a higher fluorescence compared with (B) LCD-EVs. (C) Representative cytofluorimeter analyses of DL-EVs showing the expression of CD44, CD105, CD90 and α5-integrin. EVs produced with the two staining procedures displayed analogous expression patterns of surface markers (data not shown). White filled histograms indicate the isotypic controls. Three different EV preparations were analyzed with similar results. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Figure 3

Size analysis and incorporation by renal tubular epithelial cells. (A) Representative cell-derived extracellular vesicle (EV) sizes analyzed by measurement with NanoSight. Three different preparation were analyzed with similar results. LCD-EVs maintain the same size distribution than the non-labeled EVs (180±73 nm). DL-EVs show a size of 250±89 nm with a second larger peak. (B) Representative micrographs of EV incorporation (5 h at 37ºC) in renal proximal tubular epithelial cells (PTECs). The EVs (red) produced with the two labeling procedures are equally incorporated by PTEC cells (green). Three experiments were performed with similar results. Nuclei were counterstained with DAPI (blue). Original magnification, ×630. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Figure 4

In vivo cell-derived extracellular vesicle (EV) biodistribution in kidney region by optical imaging (OI). (A) Representative OI images, acquired in the posterior position following the induction of acute kidney injury (AKI) in mice and in healthy mice treated intravenously with 200 μg of LCD-EVs or DL-EVs or with an equal volume of phosphate-buffered saline (PBS) (CTL). (B) Quantification of fluorescence intensity in regions-of-interest (ROI) draw free hand in the region of kidneys, expressed as the average radiance ± standard deviation (SD). Sixteen AKI mice were treated with LCD-EVs, 11 AKI mice were treated with DL-EVs; healthy mice received the same amount of LCD- and DL-EVs (n=12 for LCD-EVs and n=6 for DL-EVs). ANOVA with Newman-Keuls multi-comparison test was performed. ^*p<0.01 AKI DL-EV vs. all the other groups. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Figure 5

In vivo cell-derived extracellular vesicle (EV) bio-distribution in abdominal area by optical imaging (OI). (A) Representative OI images, acquired the supine position following the induction of acute kidney injury (AKI) in mice and in healthy mice treated intravenously with 200 μg of LCD-EVs or DL-EVs or with an equal volume of phosphate-buffered saline (PBS) (CTL). (B) Quantification of fluorescence intensity in regions-of-interest (ROI) draw free hand in the abdominal area, expressed as the average radiance ± standard deviation (SD). Sixteen AKI mice were treated with LCD-EVs, 11 AKI mice were treated with DL-EVs; healthy mice received the same amount of LCD- and of DL-EVs (n=12 for LCD-EVs and n=6 for DL-EVs). ANOVA with Newman-Keuls multi-comparison test was performed. ^*p<0.01 AKI DL-EV vs. all the other groups. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.

Figure 6

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.

Figure 7

Confocal microscopy of fluorescent cell-derived extracellular vesicles (EVs) in kidneys. Representative micrographs of kidney sections of mice with acute kidney injury (AKI) and healthy mice sacrificed at 5 and 24 h after EV injection (red). Nuclei were counterstained with Hoechst dye. Two kidney specimens were analyzed for each experimental point. Original magnification, ×630. LCD-EV, labeled EVs produced by donor cells; DL-EV, directly labeled EVs.