Isolation of HUCMSCs
Human umbilical cord tissues were collected from full-term, healthy cesarean births and were donated by consenting mothers at the Department of Obstetrics of Shanghai First Maternity and Infant Hospital from 2018 to 2019. The demographic and clinical characteristics of the participants in this study were reported in our previous study[11]. Umbilical arteries and veins were removed, and the remaining Wharton’s jelly was transferred to sterile, ice-cold Hank's balanced salt solution (HBSS, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing antibiotics (streptomycin/penicillin, Gibco). The tissues were washed multiple times with HBSS to remove excess blood and were subsequently cut into small pieces. The pieces were transferred to 10 cm2 dishes with 5 mL of minimum Eagle’s medium alpha (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin/penicillin and were incubated at 37 °C in 5% CO2. The explants were left undisturbed for 7-10 days to allow cell migration, and the medium was changed every three days. After 2 weeks, the HUCMSCs (passage 0) were grown to 60-70% confluence and passaged by trypsinization. Sample collection was approved by the Research and Ethics Committee of Shanghai First Maternity and Infant Hospital.
Identification of HUCMSCs by flow cytometry and osteogenic differentiation
MSC surface marker detection by a Fluorescence-activated cell sorting (FACS) Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) was previously shown[11]. Briefly, 1×106 passage 3 cells were trypsinized and suspended in 100 µL of phosphate buffer saline (PBS). Then, the cells were incubated for 30 min with the following monoclonal antibodies against HUCMSC surface markers: CD45-PE, CD73-APC, CD90-FITC and CD105-PE-Cy7 (BD Biosciences, San Jose, CA, USA).
For osteogenic differentiation, passage 3 cells were cultured in differentiation medium (Gibco) for 3 weeks in six-well plates. After the cells were fixed with 4% paraformaldehyde (PFA) and stained with alizarin red S (Sigma-Aldrich, Merck, Darmstadt, Germany), the cells were observed by microscopy (Nikon Eclipse Ti, Tokyo, Japan).
Isolation and characteristics of HUCMSC-exos
HUCMSCs at passages 3-6 were cultured in FBS-free medium with 2% bovine serum albumin (BSA, Sigma-Aldrich) for 48 h, and then 500 mL of the supernatant was centrifuged at 3,000 g for 10 min to remove cell debris and passed through a 0.22-µm filter. The cleared supernatant was ultracentrifuged at 110,000 g for 70 min and washed in PBS using the same ultracentrifugation conditions to isolate exosomes. Exosome pellets were suspended in 5 mL of PBS and stored at -80 °C.
The size and morphology of the exosomes were examined using a transmission electron microscope (TEM) at the Laboratory of Electron Microscopy (Chinese Academy of Sciences,Shanghai, China). The size distribution of HUCMSC-exos was determined using nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix, Meerbusch, Germany). These procedures were performed as previously described[8].
Mice
All mouse experiments were approved by the Department of Laboratory Animal Science, Tongji University. Adult male (n=40) and female (n=80) CD-1 (ICR) mice were purchased from Jackson Laboratories. Animals were housed in a temperature- and humidity-regulated environment with a 12-h light cycle. For a consistent and accurate assessment of the gestational age of mouse embryos, male and female (1:2) mice were pair-housed for one night. Embryonic day 0.5 (E0.5) was designed as the first morning at which a vaginal plug was noted.
Pregnant mice (n=32) were randomly divided into four groups (n=8 per group) and injected with either Cytomegalovirus (CMV)-null adenovirus or murine sFlt-1 adenovirus on E8.5 and HUCMSC-exos (EXO, 100 µg/dam) or sterile saline (NS, 100 µL) on E6.5, E9.5, E12.5 and E15.5 via the tail vein. The groups were designated as follows: (i) control (CTL): pregnant mice administered CMV-null adenovirus and NS; (ii) EXO: pregnant mice administered CMV-null adenovirus and HUCMSC-exos; (iii) sFlt-1: pregnant mice administered sFlt-1 adenovirus and NS; and (iv) sFlt-1+EXO: pregnant mice administered sFlt-1 adenovirus and HUCMSC-exos. In total, there were 4 treatment groups with respect to sFlt-1 overexpression and HUCMSC-exos administration. Blood pressure was noninvasively measured on E18.5 by determining the tail blood volume with a volume pressure recording sensor (CODA System, Kent Scientific, Torrington, CT, USA) that was averaged over a 10-min period. Embryos were harvested on E18.5, and serum, tissues, and urine samples were obtained before euthanasia as described elsewhere[12].
Tissue preparation for histological analysis
Placental and kidney tissues were fixed with 4% PFA for 48 h and processed by conventional procedures. Sections 3-5 μm in thickness were cut from the paraffin-embedded tissues, mounted on poly-L-lysine-coated slides, deparaffinized in xylene, dehydrated in alcohol and then stained with hematoxylin and eosin (H&E) or periodic acid-methenamine silver (PAS) stain.
Immunohistochemical (IHC) analysis of mouse placental tissues
For IHC analysis, paraffin sections were deparaffinized and incubated with citrate buffer for antigen retrieval. The slides were then incubated with rabbit anti-CD31 polyclonal antibody (1:100, Abcam, Cambridge, MA, USA) overnight at 4 °C and developed using the ImmPRESS horseradish peroxidase (HRP) anti-rabbit IgG polymer detection kit (Weiao, Shanghai, China).
Morphological analysis
Using CD31 as an endothelial marker, the densities, diameters, and areas of fetal blood vessels were analyzed as previously described[13]. Four images per tissue section were taken using a Nikon inverted microscope with a 40× objective (vascular density) or 100× objective (vascular diameter). For each image, the number of capillaries was counted, and the lumen area was measured using ImageJ imaging analysis software (NIH, Bethesda, MD). Five capillaries per image were randomly selected for diameter measurements by CaseViewer software (3DHISTECH, Ltd., Hungary).
ELISAs
ELISAs for mouse sFlt-1 and sEndoglin were performed according to the manufacturer’s instructions (R&D Systems). Urine albumin/creatinine ratio was measured using Urinary Albumin and Creatinine Assay kits (Abnova, Taipei City, Taiwan, China). Briefly, the various samples were diluted 1:2 in dilutions and were incubated in a 96-well plate pre-coated with capture antibodies. The wells were washed and incubated with a secondary antibody conjugated to horseradish peroxidase. Then substrate solution was added, and optical density was determined at 450 nm. The concentrations were calculated using a standard curve of the respective recombinant proteins.
Western blot
Total protein concentration was measured using the bicinchoninic acid assay (Pierce®, Thermo Fisher Scientific, Bonn, Germany). Western blot for expressions in exosomes were performed using CD63 (1:1000, SBI), CD81 (1:1000, SBI), CD9 (1:1000, SBI), sFlt-1 (1:1000, Abcam), Flag (1:2000, Sigma), eNOS (1:1000, CST), Versican (1:1000, Abcam), α-Tublin (1:2000, Abmart, Shanghai, China), Flotillin-1 (1:1000, CST) and GAPDH (1:5000, Abmart, Shanghai, China) antibodies by previously described methodology[8].
Cell culture
The HUVECs were isolated from 3 individual donors by a standard collagenase enzyme digestion method and cultured steadily in Endothelial Cell Medium (ECM, ScienCell, San Diego, CA) containing 5% FBS, 1% P/S and 1% ECGS.
Tet-One induced sFlt-1 expression in HUVECs
The human sFlt-1 (ID: NM_001159920.1) cDNA was cloned into Lenti-X Tet-One System expression vector (Clontech, Moutain View, CA) as described previously. The recombinant lentivirus tet-sFlt-1 and the negative control lentivirus (NC-lentivirus; Hanyin Co. Shanghai, China) were prepared and titered to 109 TU/ml (transfection unit). HUVECs were infected with lentiviruses expressing Tet-on-sFlt-1 to obtain cells overexpressing sFlt-1 only at the time when tetracycline existing. HUVECs expressed the sFlt-1 in the cellular background with doxycycline (Dox) and the efficiency of overexpression was examined by western blot analysis.
Exosome labelling
Exosomes were labeled with the fluorescent dye 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (Dil, red, Sigma) by addition to PBS and incubated for 15 min according to the manufacturer's protocol [14]. The labeled exosome suspensions were filtered using a 100-kDa MWCO hollow fiber membrane (Thermo Fisher Scientific) to remove the excess dye. HUVECs were seeded in 6-well plates and incubated with the Dil-labeled exosomes (100 μg/mL) for 24 h. Then the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with phalloidin-FITC (Sigma). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). The cellular uptake of exosomes was tested by a confocal microscopy (TCS SP8; Leica, Wetzlar, Germany).
Cell proliferation and migration assays
Cell proliferation and migration ability was performed using modified systems as described previously[15]. In brief, 3 x 103 cells/well were plated in 96-well plates, later Dox or the vehicle were added to induce overexpression of sFlt-1. After 24 h, HUCMSC-exos with different doses were added. Then cell numbers were assessed using the cell counting kit-8 (CCK-8) at 450 nm. For migration, HUVECs were plated in 6-well plates and treated with Dox or the vehicle to induce overexpression of sFlt-1. Then 3 x 104 cells were seeded into the upper chambers and exosomes (100 µg/mL) or NS were added into the lower chambers. Following incubation for 16 h, fluorescent stain (calcein-AM) was added to each lower chamber. The migrated cells were counted by fluorescence analysis (Nikon, Tokyo, Japan).
Proteomic analysis
The HUCMSCs-exo and HUCMSCs samples were processed for tandem mass tag (TMT)-based quantitative proteomic analysis by Lu-Ming Biotech Co., Ltd. (Shanghai, China). High-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) was used to compare the proteomic content of HUCMSCs-exo and cells. Differentially expressed proteins were identified with a cutoff of absolute fold change ≥ 2 and P-value < 0.05
Statistics
All data were expressed as the mean ± the standard error of the mean (S.E.M) and analyzed using the SPSS 23.0 statistical analysis software (SPSS Inc., Chicago, IL, USA). Statistical significance was determined by performing paired Student’s t-test, one-way analysis of variance (ANOVA) and Dunett’s post-hot test. A p-value < 0.05 was considered statistically significant.