Fibroblast growth factor 4

After obtaining the ethical approval from forensic medicine and clinical toxicology department, Cairo University, the informed consents were obtained from healthy mother donors.
HP-MSCs were isolated and characterized as described in Nasser et al. [20 (link)]. After placenta tissue’ collection, the internal chorion, and decidua membranes were detached. The separated tissue was enzymatically digested by shaking with a mixture of trypsin, dispase, and collagenase IV at 37 °C for 60 min. HP-MSCs were collected by strainer and were cultured in T75 flasks in mesenchymal stem cells basal medium (MSCBM, Lonza). It was supplemented with 10 % fetal bovine serum and 25 ng/mL fibroblast growth factor 4 (R&D System, Minneapolis, MN) at 37 °C in an atmosphere of 5% CO₂ and 3% O₂.
The propagation of cells was performed after detaching the adherent monolayer by adding trypsin 5% at 37 °C for 15 min. The first patch was propagated after three days, and the subsequent patches were propagated every two days for six passages.
Cellular phenotype identification was made by fluorescence-activated cell sorting analysis. The identity of HP-MSC was evaluated by the presence of CD29 and CD90 with the absence of CD45 and CD34 expressions.

Blastocyst-derived rat trophoblast stem (TS) cells were used to evaluate the effects of OPN on trophoblast cell invasion. Rat TS cells were generously provided by Michael Soares (University of Kansas Medical Center, Kansas City, KS), and were cultured as described previously (Asanoma et al., 2011 (link)). Briefly, TS cells were maintained in RPMI-1640 medium (ThermoFisher Scientific) supplemented with 20% (v/v) FBS (ThermoFisher Scientific), 100 μM 2-mercaptoethanol (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 50 units/mL penicillin, 50 μg/mL streptomycin, fibroblast growth factor 4 (25 ng/mL; R&D Systems), activin A (10 ng/mL, R&D systems) and heparin (1 μg/mL; Sigma-Aldrich). 70% of the media was preconditioned by mitomycin C–treated mouse embryonic fibroblasts prior to being added to rat TS cells. Cells were maintained at 37°C with 5% CO₂, and were subcultured by light trypsinization prior to reaching confluency. To induce differentiation, TS cells were cultured for 6 days as above but without mouse embryonic fibroblast-conditioned media, fibroblast growth factor 4, activin A, or heparin. Media were replenished daily.