Static magnetic field enhances synthesis and secretion of membrane-derived microvesicles (MVs) rich in VEGF and BMP-2 in equine adipose-derived stromal cells (EqASCs)-a new approach in veterinary regenerative medicine

Abstract

The aim of this work study was to evaluate the cytophysiological activity of equine adipose-derived stem cells (ASCs) cultured under conditions of static magnetic field. Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. In order to investigate the effects of magnetic field on stem cell signaling, the localization and density and content of microvesicles (MVs) as well as morphology, ultrastructure, and proliferation rate of equine ASCs were evaluated. Results showed that potential of equine adipose-derived mesenchymal stem cells was accelerated when magnetic field was applied. Resazurin-based assay indicated that the cells cultured in the magnetic field reached the population doubling time earlier and colony-forming potential of equine ASCs was higher when cells were cultured under magnetic field conditions. Morphological and ultrastructural examination of equine ASCs showed that the exposure to magnetic field did not cause any significant changes in cell morphology whereas the polarity of the cells was observed under the magnetic field conditions in ultrastructural examinations. Exposition to MF resulted in a considerable increase in the number of secreted MVs-we have clearly observed the differences between the numbers of MVs shed from the cells cultured under MF in comparison to the control culture and were rich in growth factors. Microvesicles derived from ASCs cultured in the MF condition might be utilized in the stem cell-based treatment of equine musculoskeletal disorders and tendon injuries.

Figure 1.

Analysis of ASC phenotypes using specific immunofluorescence staining. The population obtained expressed the following markers: CD29 (A), CD73b (B), CD105 (C), CD44 (D), CD 45 (E). Appropriate scale bars are indicated in each panel.

Figure 2.

Comparison of equine ASC morphology nuclei distribution (A, B) and ultrastructure (C, D) after exposure to a magnetic field (A, C) and under control conditions (B, D). Red asterisks indicate displacement of the nuclei to the peripheral part of the cells. Appropriate scale bars are indicated in each panel.

Figure 3.

SEM images of equine ASC cultures exposed to magnetic field (A) and the control culture (B); MVs secreted from the cells cultured under magnetic field (C) and control conditions (D). MVs diameters and appropriate scale bars are indicated.

Figure 4.

Comparison of the number of MVs on the cell surface after exposure to magnetic field (A, C, E, G) and control conditions (B, D, F, H). In the figure panels C and D, specific indication was used to visualized MVs (green dots) and boundaries of cells (red line). The total number of microvesicles per cell—differences between cells cultured in the presence of MF and in control conditions (I).

Figure 5.

Population doubling time of equine ASCs cultured in control conditions and in the presence of magnetic field (A). Analysis of colony-forming efficiency (B, C, D) under MF (B, C) and control conditions (B, D).

Figure 6.

Quantitative ELISA results of BMP-2 (A), VEGF (B), TNF-α (C), and p53 content in MVs from magnetic field and control conditions. EDX analysis of the elements content in samples after magnetic field treatment (E) and control (F) in particular calcium concentration (G).