Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells

Abstract

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.

Keywords: EV characterisation; EV isolation; equine; extracellular vesicles; mesenchymal stem cell.

Figure 1

Characterisation of the equine bone-marrow-derived cells showing trilineage differentiation capacity and canonical surface marker expression of equine MSCs. (a) Bone-marrow-derived cells were stained with the indicated cell surface antigens (dark grey plots) or Immunoglobulin (Ig) isotype controls (light grey plots) and analysed by flow cytometry (one representative FT-FC experiment is shown). Cells stained positive for CD90, CD44 and CD29 and negative for CD31 and Pan B, as well as IgG isotype controls. Displayed on the x-axis is either Phycoerythrin (PE) or Fluorescein isothiocyanate (FITC) conjugated to one the previous mentioned antibodies. (b–d) Bone-marrow-derived cells showing trilineage differentiation into the adipogenic ((b) stained with Oil red O, scale bar: 400 µm), chondrogenic ((c) stained with Alcian blue, scale bar: 400 µm and 100 µm for the control) and osteogenic ((d) stained with von Kossa stain, scale bar: 400 µm) lineage. The corresponding controls (cells grown in expansion medium) are shown in the insert in the top left corner of each micrograph.

Figure 2

Illustration of the experimental setup: MSCs derived from the bone marrow of 3 equine donors were cultured in serum-free DMEM for 48 h (n = 3 biological replicates, plus 3 technical replicates per donor). Then, 22 mL of cell-free supernatant was collected from 8 × 10⁶ cells per replicate and centrifuged in a 50 mL falcon tube with 3000× g for 20 min at 4 °C. In total, 20 mL of debris-free supernatant was recovered. Half of it was used to isolate EVs by the SEC columns, and the other half was filled into UC tubes.

Figure 3

Particle number and size distribution of equine MSC-EVs based on NTA measurements. (a) Significantly more particles per ml were found in the UC isolates (** p = 0.0021). Black dots correspond to the SEC isolated samples and black squares to the UC isolated samples. (b) However, both methods resulted in a similar size distribution pattern, with most particles present below 200 nm. (c) Statistical analysis of the size distribution of particles identified by NTA analysis showed no significant differences of particles isolated using the UC or SEC.

Figure 4

Flow-cytometry-based characterisation of equine MSC-EVs. (a) Representative image of fluorescence-triggered flow cytometry analysis of EV isolates enriched by either SEC (top row) or UC (bottom row). Data were collected by measuring the CD81 signal and CMG (CellMask Green) signal separately (n = 3 technical replicates). On the y-axis is the side scattering (SSC) and on the x-axis is FITC. (b) Significantly (**** p < 0.0001) more particles per millilitre stained positive for CMG in the UC isolates (188,752 ± 71,241 particles/mL) compared to the SEC isolates (35,811 ± 6729 particles/mL) (c). Significantly (**** p < 0.0001) more particles per millilitre stained positive for CD81 in the UC isolates (49,616 ± 22,588) compared to SEC (8470 ± 1751) (d). The size distribution of particles ≤200 nm (* p = 0.0446) and >500 nm (* p = 0.0105) identified by the FT-FC analysis was statistically significantly different between the two isolation methods.

Figure 5

Western blot of equine MSC cell isolates and UC isolated EVs: All isolates from all donors were positive for CD9 and CD63 in the whole-cell lysates and their respective EV isolates.

Figure 6

Transelectron Microscopy (TEM) images of the UC EV isolates in the left panels and the SEC EV isolates in the right panels taken at decreasing (top to bottom) magnifications: (a) 12,000× magnification gives an overview of the different sizes of the particles with a 1000 nm scale bar; (b) 50,000× magnification shows the different electron densities by different grey scales; (c) 85,000× magnification shows clearly the EVs identifiable by their lipid bilayer (arrows). In the UC isolates, the size distribution of the EVs is heterogeneous, ranging from large particle agglomerates to small EVs. In contrast, in the SEC isolates, particles are more uniformly sized without agglomerates.