Placentas were obtained from full‑term births after caesarean section and age-matched placentas from severe preeclampsia after caesarean section with parental permission. All CV-MSCs were used at passages 3-6 in this study. In line with the ethical protocols of the Affiliated Hospital of Qingdao University, China, every procedure was conducted accordingly. In brief, the foetal portion of the placenta was minced into approximately 1-mm3 slices and then rinsed in PBS; the aim of that is to remove all blood. The tissues were treated with trypsin (0.25%) and collagenase (0.1%) (type I; Sigma-Aldrich, St. Louis, MO). Afterward, they were subjected to incubation at a temperature of thirty-seven degrees Celsius for thirty minutes. After filtration through a 100-μm nylon filter and centrifugation, we plated the cells on culture plates in a stem cell culture medium (SCCM), which contained Stem Cell Basic Medium (Dakewe Biotech Co., Guangzhou, China) and 5% UltraGROTM (Helios, USA). An incubator with thirty-seven degrees Celsius and 5% CO2 was used to culture the primary cells.
The obtained JAR, JEG-3, and HTR-8 cells from the Type Culture Collection China Centre were subjected to culture and then used for experiments. DMEM/F12 containing 10% FBS was used to culture all three trophoblast lines in an incubator with thirty-seven degrees Celsius and 5% CO2. The medium was changed when the confluency reached 50%. The cells were subjected to incubation for a set time at a temperature of thirty-seven degrees Celsius; the humidified atmosphere of the incubator contains 93% N2, 5% CO2, and 2% O2 (Invivo2 Hypoxia Workstation, Ruskinn Technology, Leeds, West Yorkshire, UK). For each experiment, these cells were subjected to culture triplicately.
The flow cytometry (with obtained antibodies from eBioscience, San Diego, CA, including CD34, CD105, CD73, CD90, CD44, CD45, IG1 and HLA-DR) was used to examine the expression of cellular markers in CV-MSCs (passage 3); these markers consist of positive markers (CD44, CD73, CD90 and CD105) and negative markers (CD34, CD45, CD146, IG1 and HLA-DR).
Moreover, CV-MSCs from normal placenta or severe preeclampsia placenta are able to change into osteoblasts as well as adipocytes by differentiation; therefore, we assessed this ability. CV-MSCs cultured in 6-well plates were grown to approximately 70-80% confluence. Then, the CV-MSCs were subjected to culture in a differentiation medium (osteogenic or adipogenic) (Gibco, Carlsbad, CA) for three weeks. Alizarin red S was used to stain the CV-MSCs to verify osteoblast differentiation; for adipocyte differentiation, oil red O was selected.
When CV-MSCs isolated from the placentas reached 80% confluence, we replaced the medium with DMEM/F12 (Gibco, Carlsbad, CA) lacking FBS and cultured the cells for an additional 24 h. After a twelve-minute centrifugation (1,200 × g), the medium underwent a filtration through a filter (0.22 µm) (Millipore, Billerica, MA). Then, it was collected for subsequent experiments. The DMEM/F12 medium lacking FBS were used as control CM.
Antibodies as well as reagents
We bought anti-microtubule-associated protein LC3 and BECN1 (beclin1, an initiator of autophagosomes) antibodies from Sigma-Aldrich Corporation, which is located in St. Louis, USA. Cell Signalling Technology (Danvers, MA, USA) provided the following antibodies: anti-STAT3, anti-pSTAT3, anti-JAK2, anti-pJAK2, anti-P62, anti-MMP2, and anti-MMP9. For cryptotanshinone (a STAT3 inhibitor), 3-methyladenine (an autophagy inhibitor) (3MA, 10 mM) and bafilomycin A1 (Baf A1, an inhibitor of autophagosome-lysosome fusion), they were purchased from MedChem Express (NH, USA). A STAT3 plasmid was purchased from Shanghai Genechem Co., Ltd. Recombinant human interleukin-6 (IL-6) and IL-6 antibodies, were bought from R&D Systems.
RNA-seq and gene set enrichment analysis (GSEA)
TRIzol Reagent (1 ml) (Thermo Fisher Scientific) was used to collect samples; these samples were then placed at a temperature of eighty degrees Celsius below zero. We prepared libraries based on the instructions for an Illumina TruSeq RNA Sample Prep Kit and carried out sequencing on a MiSeq instrument. RSEM software was used to analyze the experimental data from Annoroad Gene Technology Co., Ltd. (Beijing, China). Labelled as no. SRR9943697–no. SRR9943702, all these data (RNA-seq) can be accessed at Sequence Read Archive (SRA).
c2.cp.v5.1.symbols of the gene set database were used to carry out GSEA of the RNA-seq data when the fold change data were input into GSEA software (Broad Institute) from the analysis of differential expression.
Transmission electron microscopy
7.4-pH PBS with glutaraldehyde (2.5%) was used to fix the trophoblasts; then it was stored at 4 °C for more than two hours. 1% OsO4 was used to post-fix the cells for two hours; then, they were dehydrated in ethyl alcohol and propanone. Thereafter, we imbedded the cells in epoxy resin. After cutting ultrathin sections in a size of 50–60 nm, these sections were positioned on uncoated copper grids; 3% lead citrate-uranyl acetate was used to stain ultrathin sections. JEM-1200EX transmission electron microscope was used to obtain the images (JEOL, Tokyo, Japan).
Quantitative real-time PCR (RT-PCR)
CV-MSC CM was used to treat JAR, JEG-3, and HTR-8 cells for 24 h. Subsequently, we extracted total RNA from the trophoblasts using TRIzol Reagent (Takara, Japan). Then, we used a reverse transcription kit (Invitrogen) to synthesize complementary DNA. Gene-specific TaqMan probes (Applied Biosystems), as well as master mix (Thermo Fisher Scientific), were used to carry out quantitative RT-PCR; the operations obeyed the instructions made by the manufacturer. The expression of target gene was normalized to GAPDH expression. We used the TaqMan probes, including probes for IL6 (Hs00985639_m1), STAT3 (Hs00374280_m1), GAPDH (Hs02786624_g1), and leukaemia inhibitory factor (LIF; Hs01055668_m1). Three separate reactions were performed for each marker.
Trophoblast invasion assay
Cells from the three lines of trophoblasts (5×105 cells) were seeded in Transwell chambers in 24-well plates (Corning, NY, USA). 50 μl of a 1:10 dilution of Matrigel™ matrix was used to cover the pore membranes (8 μm) in the twenty four-well plates. The trophoblasts were cultured on the membranes for 12 h. One of two types of the medium was added into the lower chamber: CV-MSC CM supplemented with 10% FBS or normal culture medium supplemented with 10% FBS (control). Transwell assays were performed while culturing the trophoblasts without FBS for 24 h. Next, 4% paraformaldehyde was used to fix all cells. Trophoblasts that penetrated through the membranes were analysed. The cells that migrated through the membrane were counted in high-magnification fields. Every experiment was performed three times.
Cell proliferation analysis
We added cells from the three trophoblast lines to ninety six-well plates (density: 5,000 cells per well), then cultured these cells, and measured trophoblast proliferation daily via CCK-8 reagent (Thermo Fisher Scientific, MA, USA). We added the CCK-8 reagent to each well, and the trophoblasts were cultured for an additional 1.5 h. Then, colorimetric assays were performed by measuring the absorbance (optical density [OD] value) of each well in a microplate reader (wavelength: 450 nm). Growth curves were determined in three independent experiments.
Trophoblasts were lysed on ice for 12 min by using RIPA buffer (Sigma, St. Louis, MO, USA). After centrifugation at 12,000 × g, the cell lysates were treated with LDS Sample Buffer. Then, SDS-PAGE was used to separate the protein mixtures, which were then shifted from the gel to a membrane of polyvinylidene fluoride (PVDF) (Bio-Rad, Hercules, CA). Next, 5% skim milk was used to block the membrane.
Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were used to incubate the blocked membrane. Then, secondary antibodies were used to incubate the membrane (1:1,000; CST, Danvers, MA). We detected and quantified the protein-antibody complexes using a chemiluminescence detection system (Bio-Rad, Hercules, CA).
Placental explant culture
Within a 10-min operation, all placentas were collected, which were then treated within 30 min and closely examined for any seeable abnormalities. After being thoroughly rinsed in PBS for 3 times to get rid of maternal blood, placental villous tissue was dissected into 8-mm3 pieces of tissue (2 mm × 2 mm × 2 mm). DMEM/F12 (4 ml per well) with 1% penicillin/streptomycin and amphotericin B (Gibco, Carlsbad, CA) was used to culture the placental explants in six-well dishes (Corning) in a hypoxic incubator for 48 h at 37 °C; the oxygen content was 2%. After CV-MSC CM treatment for 24 h, PBS was used to rinse the explants; after that, they were frozen in liquid nitrogen.
4% paraformaldehyde was used to fix the human term placental explants (n = 5) for 60 min. We embedded the tissues in paraffin, sliced them into 4-μm sections, and deparaffinized them. Then, the slides were boiling in 6.0-pH sodium citrate buffer (10 mM) for 7 min at a temperature of one hundred and twenty degrees Celsius for antigen retrieval. Hydrogen peroxide was used to block endogenous peroxidase for 10 min. We washed the slides three times for 5 min each with TBS, which contained 0.05% Tween 20 (TBS/T; Merck; Darmstadt, Germany); later, these slides were incubated with monoclonal anti-STAT3 antibodies (1:200) and anti-P62 antibodies (1:1,000) for 12 h at a temperature of four degrees Celsius. Diluted biotinylated secondary antibodies were used to incubate the sections for twenty min at a temperature of thirty-seven degrees Celsius. We visualized the target proteins via fresh DAB solution; then, we utilized haematoxylin as a tissue counterstain. Through an optical microscope (Olympus FV500, Tokyo, Japan), the expression of the target proteins was assessed by two observers independently. Via image-Pro Plus 5.1, the area, as well as the intensity of staining in five random regions (200× magnification), were analyzed; thus, the expression level of proteins was assessed.
One-way analysis of variance (ANOVA) or two-tailed Student’s t-tests was used to carry out statistical analyses; data were reported in the form of the mean ± standard deviation from more than three experiments, which were independently performed. If the P value was less than 0.05, it demonstrated a significant difference.