Contributed equally
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)
Cell-derived extracellular vesicles (EVs) contribute to intercellular communication by transferring proteins, bioactive lipids and nucleic acids (
Acute kidney injury (AKI) involves the rapid loss of kidney function consequent to a number of causes, which represents one of the main causes of morbidity and mortality in hospitalized patients. Moreover, AKI frequently evolves into chronic renal dysfunction (
Previously, we found that EVs derived from human MSCs accelerated recovery following AKI in SCID mice in a manner comparable to the cells (
To evaluate whether MSC-derived EVs may represent a potential therapeutic tool for AKI, it is essential to investigate
The aim of this study was to use OI as a technique to visualize
The MSCs were supplied by Lonza (Lonza, Basel, Switzerland) and cultured in the presence of MSC basal medium (MSCBM; Lonza). MSC-derived EVs were collected from the supernatant of MSCs cultured overnight in RPMI-1640 (Lonza) supplemented with 0.5% of BSA (Sigma-Aldrich, St. Louis, MO, USA). The cell supernatant was centrifuged twice at 3,000 × g for 20 min to remove cell debris and then ultracentrifuged at 100,000 × g (Beckman Coulter Optima L-90K ultracentrifuge; Beckman Coulter, Brea, CA, USA) for 1 h at 4ºC. EVs were stored in serum-free RPMI-1640 supplement with 1% DMSO at −80ºC. EV protein content was quantified by the Bradford method (Bio-Rad, Hercules, CA, USA).
Two labeling protocols were used: i) Cells were stained in suspension and incubated with 5 μM Vybrant Cell Tracers DiD [excitation (Ex), 640 nm; emission (Em), 700 nm] or DiI (Ex, 530 nm; Em, 580 nm) (Molecular Probes, Eugene, OR, USA) solution without serum for 20 min at 37ºC. Cells were then washed in complete medium by centrifugation and cultivated for 24 h prior to supernatant collection. i) EVs were isolated by ultracentrifugation as previously described (LCD-EVs) (
EVs labeled with the two methods were characterized by cytofluorimetric analysis using FITC- or PE-conjugated antibody against CD44, CD105, CD90 and α5-integrin. FITC or PE mouse non-immune isotypic IgG (Miltenyi Biotec, Bergisch Gladbach, Germany) were used as the controls. Briefly, EVs (10 μg) were incubated for 15 min at 4ºC with antibodies in 100 μl and then diluted in 300 μl and immediately acquired. FACS analysis was performed using a guava easyCyte Flow Cytometer (Millipore, Billerica, MA, USA) and analyzed with InCyte software, as previously described (
The size distribution of the LCD-EVs and DL-EVs was analyzed using a NanoSight LM10 instrument (NanoSight Ltd., Amesbury, UK) equipped with the nanoparticle tracking analyses (NTA) 2.0 analytic software.
Human renal proximal tubular epitheial cells (PTECs) were labeled following the manufacturer’s instructions with the CFSE green dye (Vybrant CFDA SE Cell Tracer kit; Molecular Probes). Cells were incubated for 5 h at 37ºC with 50 μg/ml DL-EVs or LCD-EVs and after washing the cells were fixed in 3.5% paraformaldehyde containing 2% sucrose. Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal model confocal microscope (Carl Zeiss, Oberkochen, Germany). Hoechst 33258 dye (Sigma-Aldrich) was added for nuclear staining.
Studies were conducted in accordance with the national guidelines and regulations and were approved by the Ethics Committee of the University of Torino. Male CD1 nude mice (6–8 weeks old) (Charles River Laboratories, Lyon, France), were fed for 1 week with a special diet (AIN 79; Mucedola, Settimo Milanese, Italy) to reduce tissue autofluorescence. AKI was induced, as previously described (
The mice were anesthetized with 2.5% isoflurane (Merial, Lyon, France) and images were acquired in the prone and supine position after 15 min, 5 and 24 h post-EV injection. The mice with AKI and the healthy mice treated with PBS were used as blank controls for the fluorescence signal of EVs in the AKI and healthy groups, respectively. The fluorescence signal was quantified in the kidney region and in the abdominal area, in ROI draw freehand. The relative mean fluorescence intensity of each ROI was obtained by subtracting the mean fluorescence intensity of the corresponding ROI on the blank mouse from the measured mean fluorescent intensity, as previously described (
At the end of the experiments (5 or 24 h post-EV injection), the mice were sacrificed and dissected tissues (kidneys, spleen, liver and lungs) were imaged immediately. The mean fluorescence of each tissue sample was obtained by subtracting the fluorescence intensity of corresponding tissue from the blank mouse, as previously described (
Mice were sacrificed at 5 and 24 h and confocal microscopy analysis (Leica TSC SP5 II) was performed on frozen sections for localization of DiD-labeled EVs in the kidneys. Hoechst 33258 dye (Sigma-Aldrich) was added for nuclear staining. Images were analyzed using ImageJ software.
The results are generally expressed as the means ± SD. Statistical analysis was performed by ANOVA with Dunnet’s multi-comparison test or the Newman
The OI of EVs obtained with the two following methods was compared: i) DL-EVs were labeled with DiD after their production and purification; ii) LCD-EVs were obtained by the supernatant of MSCs previously labeled with DiD and then cultured for 24 h prior to EV collection. OI images were acquired after EV dilution, ranging from 15 to 100 μg of EV proteins, in 100 μl of PBS and the average intensity within the entire circle area of each well was calculated. The fluorescence signal correlated linearly with the EV concentration for both labeling methods. DL-EVs were brighter compared with LCD-EVs (
EVs labeled with two methods showed the same phenotype as unlabeled EVs. Cytofluorimetric analyses showed their fluorescent signal in the NIR region (
To evaluate the ability of labeled EVs to be incorporated by PTECs, 50 μg/ml of EVs labeled with the red dye, DiI, following the same procedure described above, were added to the cells. DL-EVs and LCD-EVs were equally incorporated within PTECs, as observed by confocal microscopy after 5 h of incubation (
The ability of labeled EVs to be visualized by OI on the whole body of live mice was assessed using an IVIS 200 system in a model of AKI induced by an intramuscular glycerol injection, as previously described (
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
The fluorescence signal of DL-EVs was also detected in the spleen and particularly in the liver with high variability. The fluorescence signal of DL-EVs was of low intensity in the lungs of both the AKI and healthy groups. LCD-EVs were detectable only in the injured kidneys.
The presence of LCD-EVs and of DL-EVs within injured kidneys was confirmed by confocal analysis using the appropriate wavelength (
The results of the present study demonstrate that it is possible to analyze the biodistribution of EVs either by direct labeling or by the production of labeled EVs from MSCs. In particular, labeled MSC-derived EVs were found to localize within the injured kidneys.
The imaging of EVs
The use of small-molecule fluorophores and, in particular, NIR molecules, is a powerful tool to track EVs for non-invasive visualization. These dyes present strong and stable fluorescence in the EV membrane (
Previous publications addressing the biodistribution of EVs, have used the
In this study, we compared the efficiency and sensibility of two labeling methods to visualize EVs in living animals. DiD-labeled EVs were obtained by direct labeling after their production or from the supernatant of MSCs previously incubated with DiD. Labeled EVs were administered to a mouse model of AKI induced by a glycerol injection and compared with healthy controls, to observe their biodistribution. EVs derived from human MSCs have been shown to accelerate the recovery of AKI in different mouse models (
In conclusion, both these labeling methods were found to be suitable for the
We thank Federica Antico for providing precious technical support. This study was supported by the Stem Kidney grant of Fresenius Medical Care and by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award no. UH2TR000880. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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.
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.
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.