Figure 4: MVs from ES cells are transferred to trophoblasts and promote implantation.

(a) Description of experiments (Experiment 1 and 2) conducted to demonstrate that MV cargo is transferred to trophoblasts. (b) Experiment 1; MVs from mock-transfected ES cells (Control) or ES cells ectopically expressing PM-GFP were isolated and then either lysed or resuspended in medium. Lysates of the transfectants (ES cell WCL, lanes 1 and 2), and their MVs (ES cell MVs, lanes 3 and 4), were immunoblotted for GFP, the MV marker flotillin-2, the cytosolic-specific marker FAK, and β-actin, as a loading control. The MVs resuspended in medium were added to trophoblasts for 3 h, at which time the cells were extensively washed, lysed, and immunoblotted as indicated (HTR8/SVneo WCL, lanes 5 and 6). (c) Trophoblasts treated for 3 h with basal medium (Control) or ES cell CM treated with FM1-43fx plasma membrane dye before being subjected to the MV isolation procedure were fixed and visualized by brightfield and fluorescence microscopy. The scale bar is 50 μm. (d) Experiment 2; PBS (Control, top panels), or MVs derived from either ES cells (middle panels) or HTR8/SVneo trophoblasts (bottom panels) ectopically expressing PM-GFP were injected into E3.5 blastocysts. Three hours later bright field (left panels), fluorescence (GFP, centre panels), and merged images (right panels) of the blastocysts were taken. The ICM and trophectoderm (Troph.) in the blastocysts are labelled, as is the GFP signal detected within the blastocysts (arrowheads). Scale bar, 50 μm. (e) The ratio of blastocysts in d with detected levels of GFP signal in their trophectoderm. (f) E3.5 blastocysts were injected with either a PBS vehicle control or MVs from ES cells. The vehicle alone-injected blastocysts were surgically placed into the right uterine horn of a surrogate mouse, while the blastocysts injected with MVs were placed in the left uterine horn of the same mouse. Three days later, the uteri were harvested and the rate of implantation for each condition/mouse was determined and plotted as color-coded pairs. A total of 168 blastocysts and 12 surrogate mice were used in four independent experiments. Differences were analyzed using Wilcoxon signed-rank test; *P≤0.05.