Abstract
Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell communication and typically reflect the phenotype of the cell of origin. MSC-EVs could resemble MSC functions, and are being contemplated as an improved alternative to the MSC-based immunomodulatory therapy. In the present work, we focused on the factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were isolated from MSC-conditioned medium (CM) using size-exclusion chromatography (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell proliferation was compared to that of the whole MSCs. Co-culture with MSC or unfractionated CM induced naïve and CD24hiCD38hi, IL-10 producing (Breg) phenotypes on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, inducing a naïve phenotype, and even though they did not induce the shift toward a CD24hiCD38hi population, MSC-PF fostered IL-10 production by B cells. Conversely, MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs induced CD24hiCD38hi B cells to a similar extent of that of MSC, but not bona fide Bregs since they did not produce IL-10. Our results show that B cell modulation by MSC is partially mediated by soluble factors other than EVs.
Highlights
Mesenchymal stem or stromal (MSC) are immunomodulatory toward numerous immune cell types in vitro as well as in vivo [1,2,3]
Fractions eluted from size-exclusion chromatography (SEC) were evaluated for CD9 and CD90, which are known extracellular vesicles (EVs) [8] and Mesenchymal stem or stromal cells (MSC)-EV markers [17], respectively (Figure 1A)
As previously described by our group [11], we consistently observed that SEC EV-enriched fractions were free from the bulk of soluble proteins, that eluted in later fractions
Summary
Mesenchymal stem or stromal (MSC) are immunomodulatory toward numerous immune cell types in vitro as well as in vivo [1,2,3]. We recently showed their ability to induce regulatory (Breg) and naïve B cells while reducing activated and memory B cells [4]. Parameters related to the EV isolation method -including purity- are key to downstream analyses Used techniques such as ultracentrifugation (UC) or precipitating agents-based methods cause the co-precipitation of EVs with other potentially confusing soluble molecules [12], whilst sizeexclusion chromatography (SEC) is being considered the method of choice to highly enrich functional EVs [13]
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