Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy

Exosomes were isolated as previously described [3, 4, 7, 14]. Briefly, plasma from each patient was utilised to isolate exosomes. Plasma (2.5 ml) was diluted with equal volume of PBS (pH 7.4) and exosomes were isolated through differential centrifugation, microfiltration and buoyant density ultracentrifugation. Centrifugation was initially performed at 2,000 × g at 4°C for 30 min (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, high speed microcentrifuge, fixed rotor angle: 90°) followed by 12,000 × g at 4°C for 45 min to sediment cell nuclei, mitochondria and debris. The supernatant fluid (~5 ml) was transferred to an ultracentrifuge tube (Ultracrimp tubes, Thermo Fisher Scientific Ins., Asheville, NC, USA) and was centrifuged at 200,000 × g at 4°C for 2 h (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, T-8100, fixed angle ultracentrifuge rotor). The pellet was suspended in PBS (5 ml) and filtered through a 0.22 μm filter (SteritopTM, Millipore, Billerica, MA, USA). The filtrate was centrifuged at 200,000 × g at 4°C for 70 min (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, T-8100, fixed angle ultracentrifuge rotor) and the pellet resuspended in 2.5 M sucrose (4 ml).

The resuspended 200,000 g pellet in 2.5 M sucrose was added at the bottom of an ultracentrifuge tube. A continuous sucrose gradient (26 ml; 0.25-2.5 M) was made above 4 ml of exosome suspension using a Hoefer SG30 gradient maker (GE Healthcare, NSW, Australia) and centrifuged at 110,000 g for 20 h (Sorvall, SureSpin™ 630/360, Swinging-Bucket ultracentrifuge rotor). Fractions (10 in total, 3 ml each) were collected automatically using a Pulse-Free Flow Peristaltic Pump with a flow rate range of 3 ml per min (GILSON Miniplus® model 3) and the Fraction Collector (GILSON FC 203B model). The density of each fraction was determined using the refraction index with OPTi digital refractometer (Bellingham + Stanley Inc., Lawrenceville, GA, USA). The coefficient of variation (CV) was less than 8% for the density of each fraction. Fractions (3 ml each) were diluted in PBS (60 ml) and then centrifuged at 200,000 × g for 70 min. The 200,000 g pellet was resuspended in 50 μl PBS and stored at −80°C. Exosomal protein concentrations were determined by a colorimetric assay (DC™ Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA) [4].

NTA measurements were performed using a NanoSight NS500 instrument (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version Build 0033) following the manufacturer’s instructions. The NanoSight NS500 instrument measured the rate of Brownian motion of nanoparticles in a light scattering system that provides a reproducible platform for specific and general nanoparticle characterization (NanoSight Ltd., Amesbury, United Kingdom). Samples were processed in duplicates and diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per image (optimal ~50 particles x image) before analysing with NTA system. The samples were mixed before introducting into the chamber (temperature: 25°C and viscosity: 0.89 cP) and the camera level set to obtain image that has sufficient contrast to clearly identify particles while minimizing background noise a video recording (camera level: 10 and capture duration: 60 s). The captured videos (2 videos per sample) were then processed and analysed. A combination of high shutter speed (450) and gain (250) followed by manual focusing enabled optimum visualization of a maximum number of vesicles. We included a minimum of 200 tracks completed per video in duplicates. NTA post acquisition settings were optimized and kept constant between samples (Frames Processed: 1496 of 1496, Frames per Second: 30, camera shutter: 20 ms; Calibration: 139 nm/pixel, Blur: 3×3; Detection Threshold: 10; Min Track Length: Auto; Min Expected Size: Auto), and each video was then analyzed to give the mean, mode, and median particles size together with an estimate of the number of particles. An Excel spreadsheet (Microsoft Corp., Redmond, Washington) was also automatically generated, showing the concentration at each particle size.

For the TEM analysis, exosome pellets (as described above, 30 μg protein) were fixed in 3% (w/v) glutaraldehyde and 2% paraformaldehyde in cacodylate buffer, pH 7.3. Exosome samples were then applied to a continuous carbon grid and negatively stained with 2% uranyl acetate. The samples were examined in an FEI Tecnai 12 transmission electron microscope (FEI™, Hillsboro, Oregon, USA) in the Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology (QUT) (see Acknowledgements).

For placental cell-derived exosomes, the concentration of exosomal PLAP was quantified using a commercial ELISA kit (MYBioSource MBS701995, San Diego, CA, USA) according to manufacturer’s instructions (detection range: 84–2000 pg/ml; sensitivity: 34 pg/ml; intra-assay precision within an assay: CV% < 10%; inter-assay between assays: CV% < 15%) Briefly, 10 μg of exosomal protein was added to each well of a 96-well microtitre plate and incubated at 37°C for 30 min. Plates were washed three times while shaking for 20 s and 50 μl of HRP-conjugate was added to each well and incubated at 37°C for 20 min. Plates were washed and incubated with 50 μl of substrate A and 50 μl of substrate B at 37°C for 15 min. The incubation was terminated using 50 μl of stop solution at RT for 2 min under agitation. Absorbance was measured at 450 nm. Exosomal PLAP was expressed as pg PLAP /ml plasma.

To determine the stability of the exosomes during freeze-thaw cycles, fresh plasma (5.0 ml) from healthy women were obtained and divided into two 2.5 ml samples (A and B). Exosomes were immediately isolated from the first aliquot (A: fresh plasma) by differential and buoyant density centrifugation and then characterised by the number of exosome particles using an ELISA kit (ExoELISA™, System Biosciences, Mountain View, CA), morphologically by electron microscope, microRNA content by real time PCR and protein profiling by mass-spectrometry. Sample B plasma was stored at −80°C for 2 months (B: frozen plasma), prior to exosome isolation and characterisation. miRNA isolation: miRNA were isolated from exosome particles as we have previously described [14]. Ambion mirVana PARIS Kit (Invitrogen, USA) was used to extract exosomal total RNA from fresh and frozen plasma by following the manufacturer’s procedure. Exosomes were first lysed by adding cell disruption buffer and vortexed or pipetted vigorously. Denaturing solution was added to samples and incubated on ice for 5 min. The first two steps stabilize RNA and inactivate RNases. The lysate is then subjected to Acid-Phenol:Chloroform extraction by adding Acid-Phenol:Chloroform, vortexed and centrifuged at 10,000 × g for 5 min. Recovery of the aqueous phase obtains semi-pure RNA samples, removing most of the other cellular components. 100% ethanol was mixed and passed through a filter cartilage. The filter was washed three times and the RNA was eluted with nuclease-free water. Real-time PCR: Reverse transcription was performed using the miScript Reverse Transcription Kit (QIAGEN, Valencia, CA, USA) in a total volume of 20 μl. cDNA was synthesised from the maximum volume of exosomal RNA (12 μl) using the BIO-RAD T100™ Thermal Cycler (USA) running for 60 min at 37°C, 5 min for 95°C and 60 min for 37°C. As the control, RNase-free water was added as the RNA template. Real-time PCR was performed with miScript SYBR Green Kit (QIAGEN, Valencia, CA, USA). Forward primers (miScript primer assays, QIAGEN, Valencia, CA, USA) designed to detect the housekeeping gene, human RNU6-2 (RNU6B) was used. The reactions were performed in triplicate using the BIO-RAD iQ™5 Multicolor Real-Time PCR Detection System (USA) with the following conditions: 94°C for 3 min, 35 amplification cycles of 94°C for 45 s, 55°C for 30 s and 72°C for 30 s, 72°C for 10 min, 12°C for ∞ min. Proteomic analysis of exosomes by mass spectrometry (MS): We utilised a Liquid Chromatography (LC) and Mass Spectrometry (MS) LC/MS/MS instrumentation available within the University of Queensland Centre for Clinical Research (5500qTRAP and 5600 Triple TOF) to undertake in depth quantitative proteomic analysis of the exosome samples (isolated from fresh and frozen plasma) to determine the proteome of exosomes as we have previously published [4]. Briefly, exosomes were adjusted to 8 M urea in 50 mM ammonium bicarbonate, pH 8.5, and reduced with tris (2-carboxyethyl) phosphine (5 mM) at room temperature for 1 h. Proteins were then alkylated in 10 mM IAA for 1 h in the dark. The sample was diluted 1:10 with 50 mM ammonium bicarbonate and then digested with trypsin (20 μg) at 37°C for 18 h. The samples were dried by centrifugal evaporation to remove the acetonitrile and then redissolved in Solvent A. The digested protein samples were analysed using a 5600 Triple TOF mass spectrometer (ABSciex) to obtain initial high mass accuracy survey MS/MS data, identifing the peptides present in the samples. The in depth proteomic analysis was performed using the Information Dependent Acquisition (IDA) experiments on the 5600 Triple TOF MS and utilized an enhanced MS survey scan (m/z350–1500) followed by 50 data-dependent product ion scans of the 50 most intense precursor ions. The MS data was analysed with the Markerview software package using Principal Components Analysis (PCA) or PCA-Discriminate Analysis (PCA-DA) which compares data across multiple samples, groupings the data sets, and graphically showing the groups in a Scores plot. The Loadings plot provides valuable insight into variables that lead to sample clustering and illustrates which biomarkers are up- or down-regulated. All mass spectra were analysed using the Mascot and Protein Pilot search engines against the Swissprot-swissprot database with the species set as human (scores greater than 30). False discovery rate (FDR) was estimated using a reversed sequence database. Finally, proteins identified were submitted to bioinformatic pathway analysis (Ingenuity Pathway Analysis [IPA]; Ingenuity Systems, Mountain View, CA; http://​www.​ingenuity.​com).

Maternal plasma exosomes isolated by differential and sucrose density gradient centrifugation were characterised by a buoyant density of 1.122 to 1.197 g/ml (fractions 4 to 7) (Figure 1A-D). Nanoparticle tracking analysis showed a particle size distribution of 200,000 × g pellet (Figure 1A) ranging from 30 to 300 nm in diameter corresponding to microsomal fraction (including exosomes particles) with an average of 147 ± 71 nm (mean ± SD) (Figure 1B). After the sucrose continuous gradient, we mixed the enriched exosomal fractions (1.122 to 1.197 g/ml) (Figure 1C) and obtained a particle size distribution ranged from 50 to 140 nm in diameter, with an average of 98 ± 39 nm (mean ± SD) (Figure 1D). Electron microscopy revealed the presence of spherical vesicles, with a typical cup-shape and diameters ranging from 30 to 120 nm (Figure 1D, insert).

The stability of exosomes after a freeze and thaw cycle was evaluated using fresh and frozen plasma. No significant difference was observed using fresh or frozen plasma in exosome quantification, exosomal marker expression, microRNA expression or protein content (Figure 2A-D, Table 1).

Pooled exosome-containing fractions (i.e. fractions 4 to 7) were further characterised by determining the number of exosome (NEP) and exosomal PLAP concentration in the serial samples of maternal plasma obtained during first trimester of pregnancy (i.e. 6–12 weeks).

The gestational age variation in plasma exosome number was analysed by two-way ANOVA with the variance partitioned between gestational age and subject. A significantly effect of gestational age was identified (n = 69, one missing value, p < 0.005). A post-hoc multiple range test was used to identify statistically significant (p <0.05) differences between pairwise comparisons (Figure 3A). In addition, a significant effect of subject was identified (n = 69, one missing value, p < 0.05) (Figure 3B). In addition, NEP and gestational age (i.e. 6–12 weeks) displayed a significant positive linear relationship (r² = 0.202, p < 0.001, n = 69, one missing value).

To assess gestational variation in placenta-derived exosomes, exosomal immunoreative (IR) PLAP was quantified using a commercial ELISA kit (see Methods). IR exosomal PLAP concentrations were analysed by two-way ANOVA with the variance partitioned between gestational age and subject. A significant effect of gestational age was identified (p < 0.0001, n = 69, one missing value) (Figure 3C). A post-hoc multiple range test was used to identify statistically significant (p <0.05) differences between pairwise comparisons (Figure 3D). No significant effect of patient on exosomal PLAP concentration was identified (p = 0.123). Immunoreactive exosomal PLAP concentration and gestational age displayed a significant positive linear linear relationship (r² = 0.711, p < 0.001, n = 69, one missing value).

Exosomal PLAP concentration and exosome number were subjected to linear regression analysis. The fitted linear model was described by the following equation: plasma exosomal PLAP pg/ml = 85.6 + 5.47 × 10⁻¹¹ × exosome number/ml (p < 0.006, n = 69, one missing pair). The coefficient of determination (r²) was 10.8 (Figure 4A).

To estimate changes in the relative contribution of placental exosomes within the total exosomes present in maternal plasma and identify changes over the gestational age, the apparent PLAP content per 10⁹ exosome (PLAP ratio) was determined. Overall PLAP ratio averaged 2.01 ± 0.33 × 10⁻⁹ exosomal PLAP (pg) per exosome. The effects of gestational age on PLAP ratio were assessed by Kruskal-Wallis one-way ANOVA. No significant effect of gestational age on PLAP ratio was identified (p = 0.06) (Figure 4B).