Fig. 5
Impaired motility of PE hCV-MSCs and reduced stimulatory capacity for placental organoids and trophoblastic cells. A HIPEC cells and hCV-MSCs from 1st trimester, term control, and term PE placentas were seeded into each Ibidi chamber. After 8 h, the chambers were removed and the hCV-MSCs started to migrate toward HIPEC cells. After 4 and 8 h, bright-field images were taken for analysis. For fluorescence visualization, the cells were stained with phalloidin (actin filaments, red), p-FAK (focal adhesion marker, green), and DNA (DAPI, blue). Representatives of the hCV-MSCs at the migrating front after 8 h are shown (left). Scale: 50 μm. The length of the cellular protrusions of hCV-MSCs toward HTR cells was quantified and are presented as scatter plot (right) showing mean ± SEM (n = 180 protrusions, pooled from three independent experiments. B, C Single HTR cells were tracked after the treatment with indicated medium (control medium or medium containing 50% supernatant from MSCs of 1st trimester, term or term PE placentas) using time-lapse microscopy to analyze their cell motility. Representative trajectories are depicted for individual cells (B) (n = 30 cells in each group). The velocity (C, left graph) and accumulated distance (C, middle graph) are evaluated for each individual treatment and the results from three experiments are depicted as scatter plots showing mean ± SEM (n = 90 cells). Unpaired Mann-Whitney U test was used for (A and C). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. D, E HIPEC cells were incubated with indicated medium (control medium or medium containing 50% supernatant from MSCs of 1st trimester, term, or term PE placentas) for 7 days. Afterwards, total RNAs were extracted from treated HIPEC cells for analyzing gene levels of MMP2 (E) and MMP9 (D). The data are based on three independent experiments and presented as RQ with minimum and maximum range and statistically analyzed. RQ: relative quantification of the gene expression. F–H Placental organoids were generated for 72 h by using hTSCCT cells and treated then for up to 7 days with 30% supernatants from 1st trimester, term or PE hCV-MSCs. The organoids were stained against β-hCG (red), pHH3 (green), and DNA (DAPI, blue) and microscopically evaluated. The results of the organoid area are presented as scatter graphs showing the mean ± SEM (n = 7-10 organoids, from three different hCV-MSC supernatants for each group) (F). Representative images of stained hTSCCT organoids treated with different supernatants for 7 days are shown (white dotted lines indicate measured areas, arrowheads depict pHH3 positive cells). Scale: 200 μm. Supernatants of treated hTSCCT organoids were also collected for the evaluation of β-hCG via ELISA (G). The results are from three experiments and presented as mean ± SEM in a scatter graph. Student’s t-test was used. ** p < 0.01, *** p < 0.001