Abstract
BackgroundMesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays.ResultsIt was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 108 (ultracentrifugation), 2.02 × 109 (precipitation) and 52.5 × 109 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens.ConclusionExosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation.
Highlights
Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects
Detection of exosome markers in adipose tissue derived stem cells (ASC) In order to get a first impression whether cell-to-cell communication in stem cells involve exosomes we looked for an expression of the appropriate markers from the tetraspanin family in ASC by immunocytochemical analysis
Expression of all three tetraspanins CD8, CD63 and CD81 were observed in ASC after cultivation in cell culture dishes showing a spot like pattern in the cytoplasm (Fig. 1)
Summary
Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. For several diseases it has been shown that the application of conditioned media of MSC is potent enough to reduce various disease states [9, 10] This therapeutic action can most likely be attributed to the release of cytokines into the culture medium qualifying MSC as bioreactors synthesizing the appropriate factors relevant for tissue regeneration [3]. One of the advantages using exosomes as the therapeutic agents is that these extracellular vesicles can be characterized by the expression of specific marker proteins from the tetraspanin superfamily such as CD9, CD63 and CD81 [18] These markers were commonly expressed on the membrane surface of exosomes and were important for the formation and transportation within the cell as well as for the recognition of target cells. In order to pave the way for a later clinical usage of exosomes, the aim of the presented study was to isolate exosomes from supernatants of equine adipose tissue derived stem cells (ASC) and to characterize these exosomes by immunohistochemistry, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunoelectron microscopy as well as Western blot analysis
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