Figure 1

Characterization of exosomes isolated from maternal plasma. (A) A representative cryo-electron microscopy image showing classic exosome characteristics of double-membrane vesicles from early gestation (E9) and late gestation (E18). The arrow indicates exosomes, and the scale bar represents 50 nm. (B) Exosomes from maternal plasma were lysed and analyzed for exosome markers using western blot analysis. Regardless of gestation day or pregnancy status, there was consistent expression of exosome markers CD9, CD81, and ALIX. Full-length blots are presented in Supplemental Fig. 2. (C) Representative flow cytometry histograms for maternal plasma exosome marker CD63 from E9 and E18 exosomes. X-axis, FITC intensity; y-axis, count or the number of beads positive for exosomes. Green represents the negative control; pink represents beads containing exosomes positive for CD63. (D) Exosome size was determined using nanoparticle tracking analysis and was not significantly different between gestation days. (E) Nanoparticle tracking analysis was performed to determine concentration of plasma exosomes throughout gestation. The average exosome concentration increased significantly throughout gestation (E5 through E19) compared to the NP state. The maximum number of exosomes were seen on day 18. (F) The average exosome concentration for each gestation day was normalized to the average number of pups per mouse to determine if the increase in exosome concentration throughout gestation was dependent on the number of pups each mouse was carrying. Exosome concentration per pup on E18 was still the maximum concentration observed. Exosome concentration per pup was significantly higher on E18 compared to E9 and E13 when adjusted for the number of pups.