MiRNA Profiles of Extracellular Vesicles Secreted by Mesenchymal Stromal Cells-Can They Predict Potential Off-Target Effects?

Abstract

The cardioprotective properties of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are currently being investigated in preclinical studies. Although microRNAs (miRNAs) encapsulated in EVs have been identified as one component responsible for the cardioprotective effect of MSCs, their potential off-target effects have not been sufficiently characterized. In the present study, we aimed to investigate the miRNA profile of EVs isolated from MSCs that were derived from cord blood (CB) and adipose tissue (AT). The identified miRNAs were then compared to known targets from the literature to discover possible adverse effects prior to clinical use. Our data show that while many cardioprotective miRNAs such as miR-22-3p, miR-26a-5p, miR-29c-3p, and miR-125b-5p were present in CB- and AT-MSC-derived EVs, a large number of known oncogenic and tumor suppressor miRNAs such as miR-16-5p, miR-23a-3p, and miR-191-5p were also detected. These findings highlight the importance of quality assessment for therapeutically applied EV preparations.

Keywords: adipose tissue; cardioprotection; cord blood; extracellular vesicles; mesenchymal stromal cells; microRNA; oncomiR; tumor suppressor.

Figure 1

Cord blood (CB)- and adipose tissue mesenchymal stromal cells (AT-MSCs) maintain their spindle-shaped morphology under extracellular vesicles (EV) biogenesis conditions. MSCs were expanded to a confluence of about 80%, washed with Dulbecco’s phosphate-buffered saline and cultivated for 48 h in exosome-depleted medium. Then, the cells were switched to starvation medium for 24 h to derive the conditioned medium for EV isolation. Representative bright-field images of cell morphology of CB-MSCs (A) and AT-MSCs (B) were taken by phase-contrast microscopy at the time of EV isolation. Bars, 200 µm.

Figure 2

Particle number, protein amount and size distribution of EVs isolated from CB- and AT-MSCs. Particle concentration (A) and size distribution (C) of EV preparations were measured by nanoparticle tracking analysis. Protein content (B) was determined by the bicinchoninic acid assay. In (A–C), the results are mean values ± standard deviation (SD) obtained from four different donors per cell type.

Figure 3

Identification of EV-like structures via transmission electron microscopy. CB- and AT-MSC-derived EVs shown in (A,B) were isolated using the Qiagen exoEasy Maxi Kit, and CB- and AT-MSC-derived EVs shown in (C,D) were isolated using sequential ultracentrifugation. All EVs exhibit the expected cup-like shape, an artefact of the fixation method. In (A,B), EVs are covered with phosphate-rich matter, and red triangles indicate structures in which covered EVs were detected. In (C,D), exemplary EVs are indicated by blue triangles. In (A–D), enlarged regions of selected EVs are shown on that top right.

Figure 4

CB- and AT-MSC-derived EVs display a distinct surface marker profile. Detection of the surface marker proteins CD9, CD63, CD73, CD81, HLA-ABC, and HLA-DR using flow cytometry on EV preparations. The data are presented as means ± SD of normalized mean fluorescence intensities (MFIs), which were calculated as the ratio of the geometric MFI of EV samples (beads + EVs + antibodies) to control samples (beads + antibodies). Statistical analysis was performed by the Mann-Whitney test with * p < 0.05. ND indicates not detected. EVs from four different donors per cell type were included.

Figure 5

Heatmap and dendrograms of all microRNAs (miRNAs) detected with certainty according to the guidelines of the Qiagen-Exiqon miRCURY LNA Universal RT microRNA PCR system. Sample IDs are shown on the x-axis. Samples with similar miRNA expression are clustered together. The heatmap was generated by RStudio and 2^dCT was used for data input. Z-scores of more than zero indicate a higher expression of miRNAs in one sample compared to the others; Z-scores of less than zero indicate the opposite.

Figure 6

Venn diagram of miRNAs found in CB- and AT-MSC-derived EVs. In total, 752 miRNAs were analyzed and categorized according to mean CTcorr values. High miRNA expression means CTcorr value ≤ 29.99; low miRNA expression means CTcorr value = 30.00–32.99. Five hundred and forty-seven miRNAs were not detected in EVs from CB-MSCs or in EVs from AT-MSCs (mean CTcorr value ≥ 33.00).

Figure 7

Heatmap and dendrograms of miRNAs that were significantly changed in AT-MSC-derived EVs compared to CB-MSC-derived EVs. Sample IDs are shown on the x-axis. Samples with similar miRNA expression are clustered together. The heatmap was generated by RStudio and 2^dCT was used for data input. Z-scores of more than zero indicate a higher expression of miRNAs in one sample compared to the others; Z-scores of less than zero indicate the opposite.

Figure 8

Diagram of literature search rules applied for all miRNAs with a low mean CTcorr value (≤ 29.99) in both CB- and AT-MSC-derived EVs. Search terms were “name of miRNA”, “name of miRNA” AND “heart”, “name of miRNA” AND “cancer”, “name of miRNA” AND “fibrosis”, “name of miRNA” AND “endothelial cells”, “name of miRNA” AND “angiogenesis”, “name of miRNA” AND “immunomodulation”, “name of miRNA” AND “macrophages”, “name of miRNA” AND “t-cells”, and “name of miRNA” AND “immune cells”.

Figure 9

Venn diagram of selected miRNAs based on their function. Gray, tumor suppressor miRNAs; yellow, oncogenic miRNAs; red, cardioprotective miRNAs. With the exception of miR-1260a, all miRNAs with a low mean CTcorr value (≤29.99) in both CB- and AT-MSC-derived EVs were included. MiR-1260a could not be included, as no targets were described in the literature so far. Further details on these miRNAs are given in Table A1, Table A2 and Table A3.