No subjects were recruited or consented for this study since we used discarded placenta from normal term, not-in-labor cesarean sections that were de-identified before they were received by lab staff, as described previously (Sheller et al., 2016). Placental samples obtained for this study were from the John Sealy Hospital at The University of Texas Medical Branch (UTMB) at Galveston, TX, USA. The collection of placenta was approved by the institutional review board at The University of Texas Medical Branch at Galveston in compliance with all applicable Federal regulations governing the protection of human subjects (#11-251 April 2013). This protocol allowed us to collect discarded placental specimens after normal term cesarean deliveries or vaginal deliveries as an exempt protocol that does not require subject’s consent.

All reagents and media were warmed to 37°C prior to use. The amniotic membrane was processed within 15 min after delivery as described previously (Lim et al., 2013; Menon et al., 2013; Sheller et al., 2016). Primary AECs (n = 4) were cultured in T75 flasks containing complete media consisting of Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 media (DMEM/F12; Mediatech Inc., Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 10% Penicillin/Streptomycin (Mediatech Inc.) and 100 μg/mL epidermal growth factor (EGF; Sigma-Aldrich) at 37°C, 5% CO2, and 95% air humidity to 60–65% confluence.

Culture media was removed and cells were serum starved for 1 h in DMEM/F12 with 5% pen/strep prior to treatment with exosome-depleted media (DMEM/F12, 5% pen/strep and 10% exosome-depleted FBS) for 48 h. FBS (Sigma-Aldrich) was depleted of exosomes by ultracentrifugation at 100,000 × g for 18 h then filter-sterilized with 0.22 μm filter (Millipore, MA, USA) (Soo et al., 2012; Kobayashi et al., 2014). Culture media were collected and stored at -80°C until exosome isolation. Media was thawed overnight then isolated using differential ultracentrifugation as described previously, (Sheller et al., 2016) with the following modifications. After the 2 h 100,000 × g centrifugation, the supernatant was removed and the exosome pellet was resuspended in PBS. The sample was then split: half was centrifuged for 1 h at 100,000 × g while the other half was labeled with DiR. The final pellets were resuspended in cold PBS and stored at -80°C.

To fluorescently label exosomes for in vivo imaging, we resuspended the pellet centrifuged at 100,000 × g for 2 h in 7.0 mL 7.5 μM DiR (Life Technologies, Carlsbad, CA, USA) in PBS. After mixing, the exosomes were incubated in the DiR/PBS solution for 15 min at room temperature in the dark and then ultracentrifuged at 100,000 × g for 1 h. The final pellet was resuspended in 50 μL PBS and stored at -80°C.

To show that exosomes isolated from primary AECs exhibit classic exosome shape and morphology, Transmission Electron Microscopy (TEM) studies were performed as described previously (Sheller et al., 2016), with the following modification: exosomes were fixed in 5% buffered formalin; then, 5 μL of exosome suspension were dropped onto the grid and left to dry at room temperature for 10 min. To show exosome and amnion cell markers, we performed a Western blot as described previously (Sheller et al., 2016).

All animal procedures were approved by the Animal Care and Use Committee of Johns Hopkins University. Timed-pregnant CD-1 mice, outbred mice reflecting diverse genetic backgrounds in humans, were purchased from Charles River Laboratories (Houston, TX, USA) and received on gestational day 9 (E9). Animals had access to food and water ad libitum freely during housing and the experimental period. To determine the biodistribution of exosomes in vivo, we anesthetized pregnant CD-1 mice on E16 (n = 9) and E17 (n = 9) with continuous isoflurane in oxygen and performed intrauterine injections of DiR-labeled exosomes or phosphate-buffered saline solution (PBS).

Mice were subjected to mini-laparotomy, as illustrated in Figure 1. Using a Hamilton syringe, saline (n = 3 per gestational day) or DiR-labeled exosomes in PBS (n = 6 per gestational day) were injected intra-amniotically into each gestational sac on the right side of the cervix (maximum of five injections). The left side of the uterus was not injected and served as an internal control. Surgical incisions were closed, and the dams recovered in individual cages.

After 24 h, animals were imaged under anesthesia on both the dorsal (after hair removal) and abdominal sides using the Bruker In Vivo MS FX PRO Imager (Bruker, Billerica, MA, USA). Upon completion of live imaging, the animals were sacrificed by carbon dioxide inhalation according to the IACUC and American Veterinary Medical Association guidelines. The fetus contained within the uterus was collected in 4% paraformaldehyde (Sigma-Aldrich) and analyzed by histology for the presence of exosomes. Uteruses were also removed from saline- and exosome-injected mice and imaged using IVIS 200 (PerkinElmer, Inc., Waltham, MA, USA). Any image modifications (brightness, contrast, and smoothing) were applied to the entire image using Image J (open source). Maternal plasma was collected for exosome isolation.

Embryos were removed and fixed in 4% PFA at 4°C overnight. The next day, specimens were washed with PBS extensively and immersed in 30% sucrose until saturation, followed by cryosection at a 20-μm thickness. All photographs were taken with Zeiss AxioPlan 2 Microscope System (Jena, Germany). Routine hematoxylin and eosin (H&E) histochemical staining were performed on the neighbor sections.

To determine whether exosomes injected into the amniotic cavity can reach the maternal side via the systemic route, we analyzed serum samples for fluorescently labeled exosomes in maternal serum. Due to the low volume of serum collected, samples were pooled prior to isolation. Exosomes were isolated from maternal plasma, as described above, and the final pellet was resuspended in 100 μL 1:1 glycerol/ethanol solution. To determine if the isolated exosomes contained fluorescence, we pipetted 50 μL of each sample and control in duplicate into a black 96-well plate (Corning) and imaged them on the Biotek Synergy H4 Hybrid (Biotek, Winooski, VT, USA). Wavelength was set for excitation at 745 nm and emission at 779 nm. A serial dilution of DiR in 1:1 glycerol/ethanol solution was used as the positive control, while the glycerol/ethanol solution was used as a negative control. Values were determined using relative fluorescence units (RFU). Upper and lower limits were established by DiR serial dilution (positive control, RFU > 50) and the glycerol/ethanol solution (negative control, RFU < 50). Statistical analysis was not performed as we analyzed pooled sample sets (n = 2 in each group) and therefore we report a fold change between the two groups.

Isolated exosomes were characterized using TEM and Western blot for exosome and amnion markers as described previously (Sheller et al., 2016). TEM analysis revealed vesicles with classic exosome size and morphology (Figure 2A), consistent with previously published reports for exosomes (Redman and Sargent, 2008; Kshirsagar et al., 2012; Salomon et al., 2014; Mitchell et al., 2015). Western blot analysis was performed to determine exosome-enriched markers HSC70, CD81, and HSP70, as well as embryonic stem cell marker Nanog (Figure 2B).

To determine the trafficking of exosomes injected into the amniotic cavity, we imaged animals 24 h after injection. As a negative control, saline was injected into the amniotic cavity and imaged after 24 h. Fluorescent signals could not be seen in saline-injected mice when imaged (data not shown).

Fluorescently labeled exosomes were injected into mice and imaged after 24 h. Regardless of the gestational day, exosome-injected mouse images showed fluorescence on the dorsal side (Figure 3A), although the signal remained on the injected side. When uteruses were removed and imaged (Figure 3B), fluorescent signals were seen only on the injected sides and not on the uninjected sides, confirming the dye stays contained within the exosomes and does not leak from the membrane of the exosomes. On gestational day 18, images of the maternal kidneys from saline- and exosome-injected mice were also taken (Figure 3C). Fluorescent signals were not seen in the saline-injected kidneys, but fluorescence was seen in the kidneys of the exosome-injected mice.

Histologic analyses of placental and uterine tissues were performed to observe exosome trafficking in the reproductive tissues. On E17, the collected exosome- and saline-injected mouse placentas showed no signals (Figure 4); however, on E18, signals emerging from exosomes can be seen on the maternal side of the placentas, whereas signals were not seen on the fetal side or in the saline-injected placentas (Figure 5). In vivo imaging of the uterine tissue showed saline-injected uterine tissue did not have fluorescent signals, whereas uterine tissues from the exosome-injected animals showed fluorescent signals (Figure 6). Fluorescent signals were validated using the Biotek Synergy H4 Hybrid, in which the exosome-injected uterine tissues on day 18 had sixfold higher RFU than the saline-injected tissues (data not shown).

To validate the observations above and trafficking of exosomes through a systemic route, we analyzed exosomes isolated from maternal serum for fluorescence. Exosome injection solution was also analyzed for fluorescence to ensure exosomes were successfully labeled. Serum from saline-injected mice on E17 and E18, as well as from mice injected with exosomes on E17, had an RFU below 50, similar to the negative control (glycerol/ethanol solution). The serum from the exosome-injected mice on day 18 had an RFU above 50, indicating the exosomes from the amniotic fluid can traffic through the maternal serum later in pregnancy and may be dependent on timing. We report a 2.3-fold higher RFU in the serum of animals injected with DiR labeled exosomes compared to saline controls.

In summary, we demonstrated that exosomes may diffuse to the placental side where they can reach maternal tissues through a systemic route.