The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta

Abstract

Endometrial mesenchymal stem cells (endMSCs) reside in the basal and functional layer of human endometrium and participate in tissue remodelling, which is required for maintaining the regenerative capacity of the endometrium. The endMSCs are multipotent stem cells and exhibit immunomodulatory effects. This paper aimed to evaluate the regulatory effects of extracellular vesicles derived from endMSCs (EV‐endMSCs) in the setting of T cell activation. In vitro stimulations of lymphocytes were performed in the presence of EV‐endMSCs. These in vitro‐stimulated lymphocytes were functionally and phenotypically characterized to distinguish CD4+ and CD8+ T cell differentiation subsets. Moreover, the inhibition of TGFβ was performed with neutralizing antibodies. The phenotype and nanoparticle tracking analysis of the EV‐endMSCs demonstrated that they are similar in terms of size distribution to other mesenchymal stem cells‐derived exosomes. The in vitro assays showed an immunomodulatory potential of these vesicles to counteract the differentiation of CD4+ T cells. The quantification of active TGFβ in EV‐endMSCs was found to be very high when compared with extracellular vesicles‐free concentrated supernatants. Finally, the neutralization of TGFβ significantly attenuated the immunomodulatory activity of EV‐endMSCs. In summary, this is the first report demonstrating that EV‐endMSCs exhibit a potent inhibitory effect against CD4+ T cell activation, which is partially mediated by TGFβ signalling.

Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications

“…The endMSCs were obtained from menstrual blood collected by four healthy pre-menopausal women (30-34 years of age). Cells were isolated according to previously described protocols (Álvarez et al, 2018;Marinaro et al, 2019). Briefly, the menstrual blood was diluted 1:2 in phosphate buffered saline (PBS) and centrifuged at 450 × g for 10 min.…”

“…The adherent endMSCs were cultured to 80% confluency, then detached using PBS containing 0.25% trypsin (Lonza, Gaithersburg, MD, USA) and seeded again into 175 cm 2 culture flasks at a density of 5,000 cells/cm 2 , changing the cell culture medium every 4 days. endMSCs characterization was carried out by flow cytometry and differentiation assay, as previously mentioned (Álvarez et al, 2018;Marinaro et al, 2019). Briefly, the phenotypic analysis by flow cytometry was performed on 2 × 10 5 cells (passages 3-4), stained with human monoclonal antibodies against CD14, CD20, CD34, CD44, CD45, CD73, CD80, CD90, CD117, and HLA-DR, using the isotype-matched antibodies as negative controls.…”

Immunoregulatory Effects of Stem Cell-Derived Extracellular Vesicles on Immune Cells

“…Emerging evidence indicates that double-stranded DNA, DNA-binding histones, and certain miRNAs are not associated with small EVs such as exosomes (9). These inclusions are attached to EV membranes or included within the vesicles (10)(11)(12). The components of EVs vary with environmental conditions, cell origin, and cell activation conditions.…”

Cellular therapies for the endometrium: An update

“…Extracellular vesicles and associated microRNAs have been isolated from uterine fluid, supporting their role in endometrial cell‐cell and endometrial cell‐embryo crosstalk . These findings have been supported by the discovery that eSCs produce eVs with immunomodulatory effects on T‐cells . Moreover, eSC eVs increase blastomere count and VEGF release in co‐culture with murine embryos .…”

“…40 These findings have been supported by the discovery that eSCs produce eVs with immunomodulatory effects on T-cells. 41 Moreover, eSC eVs increase blastomere count and VEGF release in co-culture with murine embryos. 42 One approach to this has been the use of platelet-rich plasma in clinical trials for intrauterine infusion.…”