Trophoblast

Human term placentas were collected from normal vaginal or cesarean deliveries performed by the faculty of the Department of Obstetrics, Gynecology and Women's Health at the Missouri Women’s and Children’s Hospital, University of Missouri School of Medicine. All samples were processed immediately following delivery. Several cubic millimeters of the basal plate surface were removed sharply. Villous tissue was gently scraped free from vessels and connective tissue using the blunt edge of a scalpel. After washing thoroughly with DPBS, the tissue was cut into small pieces. Every wet gram of minced tissue was digested three times in 5 ml of a digestion enzyme medium containing 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I in DMEM at 37 °C for 20 min each cycle in a shaking water bath. Enzymatic degradation was stopped by adding an equal volume of complete DMEM containing 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The mix was filtered through a 100 μm strainer (Thermo Fisher Scientific, Waltham, MA, USA) and then a 40 μm strainer (Thermo Fisher Scientific) to remove contaminating tissue debris. Cell suspensions were collected and centrifuged at 350 g for 10 min at 4 °C. Cell pellets were resuspended in 5 ml of complete DMEM, layered on the top of a preformed Percoll gradient (65 %, 55 %, 50 %, 45 %, 35 %, 30 % and 25 %) and centrifuged at 730 g at 4 °C without braking for 30 min. The layer between the 45 % and 35 % Percoll aliquots containing trophoblast cells (density 1.050-1.060 g/ml) was collected, suspended in complete DMEM and centrifuged at 350 g at 4 °C for 10 min. The resulting cell pellet was resuspended in PBS, cells were counted and the suspension was re-centrifuged at 350 g at 4 °C for 10 min.

EV pellets from 3 samples derived from each study group (HP and GVC) were labeled with pkh67 green fluorescent cell linker (MINI67-1 KT SIGMA) as described (Hazan-Halevy et al., 2015 (link)). Labeled EV samples were washed and concentrated with Amicon^® Ultra-15 Centrifugal Filter Units—10,000 NMWL (MERK) and then by Zeba Spin Desalting Columns0.5 ml (Thermo Scientific). Pkh67 green EVs (0.3 × 10E10). HVT cells were seeded on a 24-well plate in a recommended trophoblast medium. When cells reached 100% coverage, 8 × 10E4, they were washed with PBS. Pkh67 green EVs were added to the cells in serum free media for 6 h. At the end of incubation, cells were washed twice and detached with trypsin. Internalization of EVs into trophoblast cells was quantified by flow cytometry and documented by ZOE™ Fluorescent Cell Imager (Bio-Rad).

To distinguish between maternal and placental involvement in miRNA expression, we exposed primary cell cultures from early stage human placenta villus trophoblast (HVT) to EV pellets that were obtained from the HP and GVC groups. The primary ECs representing the maternal side were also exposed to EVs of NP, as well as EVs of HP and women with GVC.