Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†

Abstract

The supplementation of embryo culture medium with extracellular vesicles from endometrialderived mesenchymal stem cells increases the quality of aged embryos

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

“…Furthermore, CD9 and CD63 exosomal markers were positively expressed, in accordance with the Minimal information for studies of extracellular vesicles 2018 (MISEV2018) guidelines (Théry et al, 2018) (Supplementary Figure 1). An accurate explanation and the characterization of cells and vesicles can be found in our previous studies (Álvarez et al, 2018;Blázquez et al, 2018;Marinaro et al, 2019).…”

“…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.…”

“…For EV-endMSCs isolation, the standard culture medium (for control EV-endMSCs), or the culture medium containing IFNγ (for IFNγ/EV-endMSCs) were removed, and replaced by DMEM containing 1% insulintransferrin-selenium (ThermoFisher Scientific), after rinsing with PBS. After 4 days, the supernatants were collected and EVs isolated according to a previous optimized protocol (Álvarez et al, 2018;Marinaro et al, 2019). Briefly, supernatants were centrifuged at 1,000 × g for 10 min and 5,000 × g for 20 min at 4 • C to eliminate dead cells and debris.…”

“…The functional studies performed by TGF-β blockade demonstrated that this molecule is partially involved in the immunomodulatory effect of these vesicles (Álvarez et al, 2018). Apart from their immunomodulatory effects, EV-endMSCs have been used as coadjuvants to improve the in vitro fertilization outcomes in murine models (Blázquez et al, 2018), and the proteomic analysis of these EVs revealed an abundant expression of proteins involved in embryo development (Marinaro et al, 2019).…”