Placental sample collection
The placental tissues of first-trimester pregnancy (6-12 weeks) and second-trimester pregnancy (13-34 weeks) were obtained from the legal abortion and those of full-term pregnancy (36-40 weeks) were obtained by the cesarean section. All placental specimens were collected from the Department of Obstetrics and Gynecology of Shengjing Hospital of China Medical University (Shenyang, China). This study was approved by the Ethics Committee of China Medical University. All participants were healthy women between the ages of 25-40 years and had no history of infection, underlying disease, or obstetric complications.
The blood-free villi of early pregnancy and the avascular villi between the maternal side and fetal side in the second-trimester and full-term pregnancy were selected. Each placental sample was collected immediately after miscarriage or delivery and then placed in cold phosphate-buffered saline (PBS).
Cell lines
The HTR-8/SVneo cell line was transfected with simian virus 40 large T antigen (SV40) using normal human first-trimester extravillous cytotrophoblasts (evCTBs) [26, 27]. HUVECs derived from the umbilical vein endothelium were used as a laboratory model for the study of endothelial cell functions, such as angiogenesis. The HTR-8/SVneo cell line (Chinese Academy of Sciences, China) and HUVECs cell line (Chi Scientific, China) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 2 mM L-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Gibco, USA) in a 5% CO2 incubator at 37℃ (standard culture conditions). The culture medium was replaced every 3-5 days. After the cells reached 80% confluence, the cells were harvested with 0.25% trypsin (Gibco, USA) and stored in a -80℃ freezer for subsequent experiments.
Primary hPDMSCs and primary CTBs isolation, culture, and identification
Primary hPDMSCs isolation and culture
hPDMSCs were obtained according to our previous study protocol [23,24]. The placental villi tissue from the different pregnancy periods was dissected into small pieces and extensively rinsed in PBS with 5% penicillin/streptomycin to remove the blood. After being mechanically minced again, the minced placental tissue was laid uniformly in 10 cm2 Petri dishes until semidry and cultured in DMEM supplemented with 10% FBS, 2% penicillin/streptomycin, and 2 mM L-glutamine under standard culture conditions. The next day, the supernatant was discarded, the cells were washed with PBS three times, and new medium was added. After 3-7 days, the cells crawled out of the tissue mass. The culture medium was replaced every 5 days. After 15-21 days, the placental tissue fragments were removed, and hPDMSCs were digested with 0.25% trypsin and passaged. While the cells reached 80% confluence, they were passaged at a ratio of 1:2. hPDMSCs at passages 3-5 were stored a -80℃ freezer and used subsequent experiments.
Primary hPDMSCs identification
Primary hPDMSCs at passage 3 (P3) were used to measure cell surface marker expression and differentiation capacity.
Immunophenotype analysis of primary hPDMSCs
hPDMSCs were harvested with trypsin, washed twice with PBS, centrifuged at 1000 rpm for 10 minutes, and resuspended in 1 ml of PBS at a final concentration of 5×105/ml. Then, the harvested cells were incubated with the following phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, or PerCP-conjugated antibodies for 30 minutes at 4℃: anti-CD34-PE, anti-CD45-FITC, anti-CD73-PE, anti-CD90-PE, and anti-CD105-PerCP (all from B&D, USA). After being incubated, the cells were washed, centrifuged, suspended in PBS, and analyzed by flow cytometry.
Differentiation of primary hPDMSCs
Endothelial cell differentiation
For endothelial cell differentiation, primary hPDMSCs were cultured in endothelial differentiation medium. The medium was prepared with DMEM with 0.1 mM β-mercaptoethanol (Sigma, USA), 50 ng/ml recombinant human vascular endothelial growth factor (rh-VEGF, Sigma, USA), 10 ng/ml basic fibroblast growth factor (rh-bFGF, Sigma, USA), and 5% FBS. The differentiation medium was replaced every 3-5 days. On the 8th-10th day, the differentiated cells were fixed with 4% paraformaldehyde, and immunocytochemical analysis was performed with mouse anti-human von Willebrand factor (vWF, Maxim, China) according to the manufacturer’s instructions [23].
Osteogenic differentiation
To detect the osteogenic differentiation ability of hPDMSCs, hPDMSCs were cultured in osteogenic differentiation medium and identified by Alizarin red according to the manufacturer’s instructions for the human umbilical cord mesenchymal stem cell osteogenic differentiation kits (Chem-bio, China). Ascorbic acid, β-glycerophosphate, dexamethasone, penicillin/ streptomycin, and FBS were added to the basal medium to form the complete osteogenic induction medium. When the hPDMSCs were more than 60% confluent, they were cultured in complete osteogenic induction medium. The induction medium was replaced every 3 days for 2-4 weeks. After induction, the cells were fixed with 4% paraformaldehyde for 20 minutes, stained with Alizarin red solution for 15 minutes, and imaged.
Adipogenic differentiation
Similar to osteogenic differentiation, adipogenic differentiation and oil-red O detection were performed using an adipogenic differentiation induction kit according to the manufacturer's instructions. First, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine, insulin, penicillin/streptomycin, and FBS were added into the basal medium to form complete adipogenic differentiation induction medium A. Adipogenic differentiation maintenance medium B was formed by adding insulin, penicillin/streptomycin, and FBS to the basal medium. When hPDMSCs were 80% confluent, the cells were cultured with complete adipogenic induction medium A for 72 hours, and then the medium as replace with maintenance adipogenic induction medium B and further incubated for 24 hours, which was one cycle. Three to five cycles were repeated. When obvious lipid droplets appeared in the cell, the cells were cultured only in adipogenic differentiation maintenance medium B, and new medium B was replaced every 2 days. When the lipid droplets were large enough, the culture ended. Then, the induced cells were fixed with paraformaldehyde, stained with oil-red O, and imaged.
Primary first-trimester CTBs isolation, culture, and identification
Primary CTBs isolation and culture
Primary single CTBs were isolated from human first-trimester villi as described previously with some modifications [28, 29]. First, following the same steps as those of primary hPDMSCs isolation, the placental villi were minced into small pieces and rinsed extensively with PBS. Then, the digestion mixture containing 0.25% trypsin and 0.1 mg/ml DNAse I (Biodee, China) was added and incubated in a 37℃ shaking water bath for 30 minutes. The digested suspension was filtered through a nylon mesh (200 µm) and terminated with FBS. The remaining tissue was digested with the digestion mixture containing 1 mg/ml collagen I (Invitrogen, USA) and 0.1 mg/ml DNAse I in a 37℃ shaking water bath for 20 minutes. The digested suspension was filtered, and digestion was terminated again. The whole digestion procedure was repeated at least 3 times. Then, the whole-cell suspension was filtered through a nylon mesh (100 µm) and centrifuged at 1000 rpm for 10 minutes. The cell pellet was resuspended in 3 ml DMEM with 10% FBS, gently layered on the top of a preformed noncontinuous Percoll gradient (Phamaiva, USA) (75%-25%), and centrifuged at 3000 rpm for 30 minutes. Single cells were collected between the 45% and 35% Percoll aliquots, resuspended, and counted. The freshly isolated cell suspension was seeded at a concentration of 1×108 cells/dish into 10 cm2 Petri dishes to collect CM or analyze by flow cytometry (or at a concentration of 1×105 cells/dish into 3 cm2 Petri dishes for identification). After being cultured for 1 hour, the cell suspension was transferred to new Petri dishes and incubated overnight. The next day, the nonadherent cells were removed, and new medium was added. Once the cells in the 10 cm2 Petri dishes were more than 90% confluent, the medium was replaced with serum-free DMEM, and the primary CTBs-CM was collected at different time points. The isolated cells in the 3 cm2 Petri dishes were passaged for identification.
Primary first-trimester CTBs identification
Immunofluorescence staining was performed as previously described [16, 30]. Briefly, human first-trimester placental tissue sections, primary CTBs, or HTR-8 cells were fixed, blocked, and incubated at 4℃ overnight with rabbit anti-cytokeratin 7 (CK7, Abcam, USA) and mouse anti-vimentin (Vim, Abcam, USA) to identify trophoblasts. The nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, USA). The placental sections, primary CTBs, or HTR-8 cells were observed under the fluorescence microscope (Leica, Germany), and pictures were taken. At the same time, the harvested primary CTBs were incubated with anti-CK7 and anti-Vimentin, and analyzed by flow cytometry.
Conditioned medium preparation and experiments
Placental cells-derived conditioned medium
hPDMSCs at passages 3 to 5 and isolated primary CTBs cultured for 1-2 days were used to collect conditioned medium. To harvest the conditioned medium, the cells were cultured with 10% FBS in DMEM until the cells were greater than 90% confluent and washed with PBS to remove detached cells. Then, the medium was replaced with serum-free DMEM. At different time points (6, 12, 24, 48, and 72 hours), the cell culture supernatant was collected and centrifuged at 3500 rpm for 20 minutes to remove detached cells and cellular debris. Then, the placental cell CM was filtered (0.22 μm), adjusted with serum-free medium to 10 ml/5×107 cells, and frozen at -80℃ for future experiments.
Sub-cultured placental tissue-derived conditioned medium
The nonvascular placental stromal tissue between the maternal surface and fetal surface was selected for the collection of sub-cultured placental tissue conditioned medium. These placental samples were mechanically minced into small pieces and washed with PBS supplemented with 5% penicillin/streptomycin. Then, the wet placental villi tissue (1 cm3/dish) was laid uniformly in 10 cm2 Petri dish until the tissue was semidry, and serum-free DMEM was added. The next day, the supernatant was discarded, the tissue was washed, and the medium was replaced with 10 ml of serum-free DMEM. At different time points (1, 3, 5, 7, 10, and 14 days), the supernatant of the cultured placental tissue was collected, centrifuged, filtered, and then adjusted with serum-free medium to 10 ml/dish. Finally, the sub-cultured placental tissue-derived CM was stored at -80℃ for further experiments.
Experimental groups
All in vitro HUVECs experiments were performed with conditioned medium from placental cells or sub-cultured full-term placental tissue that was collected at different time points.
Placental cell types included primary cytotrophoblasts from early pregnancy placenta (early-CTBs), human mesenchymal stem cells from early pregnancy placenta (early-hPDMSCs), human mesenchymal stem cells from middle pregnancy placenta (middle-hPDMSCs), and human mesenchymal stem cells from full-term placenta (term-hPDMSCs).
The different time points included 6, 12, 24, 48, and 72 hours for placental cells, and 1, 3, 5, 7, 10, and 14 days for sub-cultured placental tissue.
HUVECs proliferation assay
First, HUVECs were seeded into 24-well plates at a concentration of 1.0×104 cells/well. The cells were cultured overnight in standard culture medium supplemented with 10% FBS, the medium was discarded, and 1 ml of conditioned medium from the different experimental groups supplemented with 5% FBS (only DMEM was used as the control) was added. At different time points (placental cells-derived conditioned medium groups: 1, 2, 3, to 6 days; sub-cultured placental tissue-derived conditioned medium groups: 12, 24, 36, and 48 hours), the cells were harvested and counted. The proliferation ability of HUVECs was measured by counting the number of cells using a Countstar automated cell counter (ALIT Life Science, USA).
HUVECs adhesion assay
To assess the adhesion ability of HUVECs, a cell adhesion assay was performed with slight modifications according to WOOD [31]. Briefly, the 96-well plates were coated with conditioned medium (50 µl/well) overnight in a 5% CO2 incubator at 37℃. The next day, the CM was removed, and the 96-well plates were air-dried in the biosafety cabinet. Then, HUVECs (0.5×103 cells/well) were suspended in 100 µl of conditioned medium and seeded into the corresponding CM-coated 96-well plates. After being incubated for 2 hours, the cells were washed to remove nonadherent cells. Adherent cells were fixed with 4% paraformaldehyde and stained with 0.1% gentian violet. Three to five visual fields per well were randomly selected to take pictures and count cells.
HUVECs wound-healing assay
The horizontal migration ability of HUVECs was evaluated by using a wound healing assay. The cells were seeded into 24-well plates and grown to confluence. The confluent cells were scratched with a pipette tip, washed three times with PBS to remove the loose cells, and photographed. After being incubated for 18 hours in different CM with 5% FBS, the cells were fixed, and pictures were taken in 3 random fields. The cell-free area between the confluent cells was measured by ImageJ (NIH, USA) and calculated by the following formula:
The cell-free clear distance (vertical scratch in the picture)=the clear area/height of the picture; the cell-free clear distance (horizontal scratch in the picture)=the clear area/width of the picture.
The migration distance=the cell-free clear distance at 0 hours–the cell-free clear distance at 18 hours.
The ratio of the migration distance between the experimental group and the control group was calculated as the relative ratio of the migration distance, which represents cell migration ability [32, 33].
HUVECs transwell migration assay
HUVECs vertical migration was assessed by using a transwell migration assay, which was performed with a 24-well transwell insert (8 μm pore size, Corning Costar, USA). Next, 250 µl of different conditioned medium containing 20% FBS was added to the lower chamber, and 100 µl of HUVECs suspension containing 3% FBS was seeded into the upper chamber at a density of 2×104 cells/well. After being incubated for 18 hours, the nonmigratory cells on the upper surface of the membrane were gently removed with cotton swabs. The migrated cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% gentian violet. Three to five areas were randomly selected to take pictures and count [34, 35].
HUVECs transwell invasion assay
The transwell invasion assay was carried out with Matrigel invasion chambers with 8.0 μm PET membranes (Corning Biocoat, USA). HUVECs suspension (100 µl, 8×104 cells/well) supplemented with 3% FBS were seeded into the upper Matrigel-coated chamber, and 250 µl of different conditioned medium supplemented with 20% FBS was added into the lower chamber. Then, the following steps were the same as those in the transwell migration assay. After being incubated for 18 hours, the noninvasive cells were removed, and the invasive cells were fixed, stained, and photographed.
In- vitro Matrigel tube formation assay
The angiogenic ability of HUVECs in vitro was detected by using the Matrigel tube formation assay. Briefly, Matrigel (50 µl/well, BD, USA) was added to precooled 96-well plates and polymerized at 37℃ for 2 hours to form a thin gel layer. DMEM supplemented with VEGF-A was used as a positive control, and DMEM without any additive was used as a negative control. HUVECs (3×104 cells/well) were suspended in conditioned medium supplemented with 2% FBS and seeded onto 96-well plates containing Matrigel. After being incubated for 15 hours, the capillary-like structures were observed under a light microscope, and pictures of 3-5 visual fields/well were taken. The total length of the tubular structure was analyzed by using the Angiogenesis Analyzer plug-in for ImageJ software (NIH, USA) [36, 37].
Angiogenesis Antibody Array analysis of placental cells- or sub-cultured placental tissue-derived conditioned medium
Angiogenic cytokines/proteins in the conditioned medium were analyzed with a human angiogenesis antibody array C series 1000 kit (RayBiotech, USA). This array consists of two membranes containing 43 factors related to angiogenesis, each of which is repeated twice. The cytokine array protocol was carried out according to the manufacturer’s instructions. Briefly, the array membrane was blocked with 5% BSA for 30 minutes and incubated in 1 ml of conditioned medium at 4℃ overnight. After being washed thoroughly to remove unbound substances, the membrane was incubated with a cocktail of biotin-labeled cytokine antibodies at room temperature for 2 hours. Then, the membrane was incubated with HRP-streptavidin at room temperature for 1 hour. Finally, the chemiluminescent signal of each factor on the array was acquired by ChemiDoc XRS (Bio-Rad, USA), and the value of integrated optical density (IOD) was measured with ImageJ software. The IOD value of each spot was corrected by subtracting the background value and was standardized with the IOD value of the positive control spots on the same membrane to obtain the relative level of each factor [12, 38, 39].
Statistical Analysis
All experiments were repeated three times. All results are expressed as the mean ± SD. Statistical analysis was performed with Prism 7 software (GraphPad, USA). One-way ANOVA was performed to compare the differences within groups, and two-way ANOVA was used to assess the difference between groups. Statistical significance was considered as p<0.05.