Fig 1.
Description of the study and methodological design.
Fig 2.
Phenotypical characterization and differentiation potential of endMSCs.
(A) Flow cytometry analysis of endMSCs isolated from menstrual blood. A representative histogram together with the expression levels is shown. The expression of cell surface markers is represented as the percentage of positive cells (black lined histogram) above the negative control (grey lined histogram). (B) In vitro differentiation potential of endMSCs. The cells were in vitro cultured for 21 days with standard medium (Control) or with specific differentiation media for adipogenic, osteogenic and chondrogenic lineages (Induced). Differentiation towards adipocytes, osteocytes and chondrocytes was evidenced by Oil Red O, Alizarin Red and Alcian Blue 8GX respectively. Scale bar: 100 μm. (C) The adipogenic (above), osteogenic (middle) and chondrogenic (below) differentiation degree was quantified by determining the absorbance of the extracts at 490 nm (Oil Red O and Alizarin Red S staining) and at 600 nm (Alcian Blue 8GX). Four independent experiments using four different cell lines were performed and a Mann-Whitney U test was used. p-values are shown in the figure.
Fig 3.
Characterization of EV-endMSCs.
(A) Representative graph of nanoparticle tracking analysis to quantify size distribution and particle concentration of EV-endMSCs. (B) Flow cytometry analysis on EV-endMSCs for exosomal markers CD9 and CD63. A representative histogram together with the expression levels is shown. The expression of cell surface markers is represented as as the percentage of positive cells (black lined histogram) above the negative control (grey lined histogram).
Fig 4.
Total cell number of murine blastocysts cultured in presence or absence of EV-endMSCs.
Total cell number of the obtained expanded blastocysts was obtained after Hoechst 33342 staining and subsequent evaluation by fluorescence microscopy. For each treatment, the individual blastomere count is represented. Horizontal bars show the mean values. All the treatments differ statistically from the control (p<0.05). Representative micrographs of expanded blastocysts cultured with varying dosages of EV-endMSCs are shown.
Table 1.
Embryo development to the blastocyst stage and hatching rates.
Fig 5.
Quantification of VEGF and PDGF-AA secreted during embryo culture.
Soluble factors released by the developing embryos co-cultured with EV-endMSCS were quantified by the Luminex xMAP technology at the third day of embryo culture. (A) PDGF-AA secreted by blastocyst embryos. All the data were compared by Student t-test for paired comparisons with respect to control group. The mean (dotted line) ±SD from four independent experiments, as well as individual measures (rhombuses), are shown. (B) VEGF secreted by blastocyst embryos. All the data were compared by Student t-test for paired comparisons with respect to control group. The mean (dotted line) ±SD from four independent experiments, as well as individual measures (rhombuses), are shown. (C) Correlation between PDGF-AA and VEGF. Correlation line as well as individual measures (rhombuses) are represented. The Pearson correlation coefficient (r) together with its significance level (p) is shown.