Human Placental Trophoblasts Are Resistant to Trypanosoma cruzi Infection in a 3D-Culture Model of the Maternal-Fetal Interface

Figures

• 

  FIGURE 1. JEG-3 cells grown in the RWV bioreactor fuse to form syncytia. (A) Micrographs of JEG-3/HBMEC spheroids cultured for 20 days. Cell membranes were stained with Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, red). Left: bright-field. Center: Texas Red® DHPE. Right: 4’, 6-diamindino-2-phenylindole (DAPI). Arrows point to cell coated beads (spheroids). (B) Staining of actin filaments in monolayers of 2D JEG-3 cells (left) or 3D-cultured cells, either detached from beads (center) or on 3D-grown spheroids (right). Cells were fixed/permeabilized and stained with Alexa Fluor® 680 phalloidin (red). Arrows point to syncytia. Slides were mounted with Vectashield mounting medium containing DAPI (blue) to visualize nuclei. Fluorescent micrographs are representative of three independent experiments and were acquired on a Keyene BZ-9000 fluorescence microscope. Scale bars: 50 μm in (A) and 30 μm in (B).

• 

  FIGURE 2. JEG-3 cells but not HBMECs express cytokeratin-19. (A) Immunostaining of 2D JEG-3 cells, HBMECs, 3D-cultured cells detached from beads, and spheroids, as indicated. Cell monolayers and spheroids were fixed/permeabilized and stained with rabbit anti-CYT-19 followed by Alexa Fluor 488 anti-rabbit IgG (H + L) antibodies (green). Slides were mounted with Vectashield mounting medium containing DAPI to visualize nuclei (blue). Fluorescent micrographs were acquired on a Keyene BZ-9000 fluorescence microscope. Scale bars: 10 μm (2D JEG-3 and HBMEC panels); 30 μm (3D JEG-3 cells detached from beads and spheroids). (B) Flow cytometry analysis for CYT-19 expression in 3D-cultured cells after detachment from beads, 2D JEG-3 and HBMEC cells, as indicated. Gating scheme to identify singles and exclude dead cells based on LIVE/DEADTM violet dye staining is shown. Data are representative of two independent experiments.

• 

  FIGURE 3. 3D-cultured JEG-3 cells express markers associated with syncytiotrophoblasts’ differentiation. (A) ELISA assay for β human chorionic gonadotrophin (β hCG) from culture supernatants of 2D JEG-3 cells, 3D HBMECs and 3D-grown spheroid cultures, as indicated. Data represent the mean ± SD of two independent vessels or 2D cultures with samples tested in duplicates. (B) qRT-PCR analysis of human placental lactogen (hPL) mRNA from two independent vessels of spheroid cultures (3D) or conventional flasks of JEG-3 cells (2D). Fold change in gene expression was determined by the 2 –ΔΔCt method with samples normalized to β actin. Boxes represent first and third quartiles with a line at the median. Whiskers indicate maximum and minimum data values. (C) qRT-PCR analysis of endothelial nitric oxide synthase (eNOS) mRNA from two independent vessels of spheroid cultures (3D) or conventional flasks of JEG-3 cells (2D). Fold change in gene expression calculated and plotted as in (B). In all panels, asterisks indicate statistically significant differences calculated by the unpaired t-test. **P ≤ 0.01; ****P ≤ 0.0001.

• 

  FIGURE 4. 3D-grown JEG-3 cells resist T. cruzi infection. (A) Monolayers of JEG-3 cells (2D JEG-3) grown in 96-well plates and spheroids (3D JEG-3) transferred from the RWV bioreactor to 96-well plates, were infected with T. cruzi-luminescent parasites at a ratio of 5 parasites per live cell. The infection was conducted under static conditions and monitored over time by measuring intracellular nanoluciferase activity at 0 (T = 0), 1 (T = 1), and 3 (T = 3) dpi. Luminescence was quantitated in relative light units (RLU) and normalized to the number of live cells. Data represent the mean ± SD of three technical replicates and is representative of 3 independent experiments. Asterisks indicate statistically significant differences calculated by an unpaired t-test. **P ≤ 0.01; ns: non-significant. (B) 3D JEG-3 cells were infected with CellTraceTM Far Red- labeled trypomastigotes at a ratio of 5 parasites per live cell under slow rotation in the RWV bioreactor. Sixteen hours post-infection, cells were detached from the beads, stained for CYT-19 and analyzed by flow cytometry. The gating strategy used to identify singles and exclude dead cells (based on LIVE/DEADTM violet dye staining) is shown.…

• 

  FIGURE 5. 3D cultures of JEG-3 cells release paracrine factors that prevent T. cruzi infection of non-trophoblastic cells. (A) LLC-MK2 or (B) HBMEC cells were preincubated with supernatants from 2D JEG-3 (2D CM), 3D JEG-3 (3D CM) or non-conditioned medium (NCM) (LLC-MK2 or HBMECs culture medium) for 24 h followed by infection with T. cruzi-luminescent parasites (5 parasites per live cell). Nanoluciferase activity was measured as relative luminescence units (RLU) at 0 (T = 0), 1 (T = 1), and 3 (T = 3) dpi. Data were normalized to the number of live cells and represent the mean ± SD of three technical replicates. The fold change RLU values per live cell over T = 0 were plotted. The experiment was repeated two times with similar results. In all panels, asterisks indicate statistically significant differences calculated using an unpaired t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.