A novel three-dimensional in vitro system to study trophoblast-endothelium cell interactions

Abstract

Introduction: Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial-trophoblast interaction and signaling in a three-dimensional system.

Method of study: Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in Matrigel(TM) OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex.

Results: Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4-8 hr and achieves complete replacement of the endothelium by 72-96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium-trophoblast interaction.

Conclusion: We report a novel three-dimensional in vitro and in vivo system of trophoblast-endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation.

Figure 1.. Endothelial tube formation.

Light microscopy images after overnight incubation of immortalized human endometrial endothelial cells (1.0 × 10⁵) grown as a monolayer, (a) or in Matrigel-coated wells (b). 10× magnification.

Figure 2.. Trophoblast migration in the three-dimensional Matrigel system toward differentiated endothelial cells.

Human endometrial endothelial cells were plated in Matrigel overnight (1.0 × 10⁵) and then co-cultured with PKH26 labeled first trimester trophoblast (8.0 × 10⁴) (red). (a) Low magnification (25×) of the tubes; (b) trophoblast cells added to the culture migrate toward the endothelial cells (25×). (c-h) Sequential migration of trophoblast cells (100×) over a period of 2 hours (c), 4 hours (d and e) and 8 hours (f), 24 hours (g) and 48 hours (h).

Figure 3.. Trophoblast-endothelium interaction.

Sequential distribution of trophoblast and human endometrial endothelial cells in the three-dimensional Matrigel system can be observed by labeling each cell type with a different fluorescent dye. (a) Trophoblast cells reached the walls of the tubes (red) and distribute between and on top of the endothelial cells (green). (b) Appearance of trophoblast cells (red) together with endothelial cells (green). (c and d) Composite picture of red fluorescently labeled trophoblast cells and green endothelium reveals areas of yellow color product of the combination of red and green. 10× magnification.

Figure 4.. Co-culture of human endometrial endothelial cells (HEECs) and trophoblast maintain endothelial cell structure.

HEECs were plated in Matrigel overnight (1.0 × 10⁵) and then co-cultured with first trimester trophoblast (8.0 × 10⁴) for a period of 21 days. Tubes integrity remained intact in the wells containing transformed trophoblast-endothelial cells (a); HEECs cultured alone began to breakdown after 48 hours (b). Primary trophoblast migration in the three-dimensional Matrigel system. HEECs were grown in Matrigel overnight (1.0 × 10⁵) and then co-cultured with PKH26 labeled primary culture of first trimester trophoblast (8.0 × 10⁴) over a period of 24 hours (c) and 48 hours (d). 10× magnification.

Figure 5.. Specificity of the endothelium-induced migration.

Human endometrial endothelial cells were grown in Matrigel overnight (1.0 × 10⁵) and then co-cultured with THP-1 cells and migration monitored by light microscopy. (a) THP-1 monocytes remained at the top of the well and did not show any migratory effect. 10× magnification (b) THP-1 cells incubated with transformed trophoblast/endothelium tubes migrate and distribute on the lumen of the tubes. 25× magnification.

Figure 6.. Effect of lipopolysaccharide (LPS) on differentiated and undifferentiated endothelial cells.

Light microscopy of human endometrial endothelial cells (HEECs) plated with Matrigel (top panel) or without Matrigel (bottom panel) and treated with LPS at 5 or 50 μg/mL for 24 hours. HEECs cultured in Matrigel treated with LPS lose their structure in a dose-dependent manner (top panel), while cells in monolayer do not show any morphological change on the presence of LPS (bottom panel).

Figure 7.. Human endometrial endothelial cells (HEECs)’ cytokine profile in response to lipopolysaccharide (LPS) treatment.

Differentiated (Matrigel) and undifferentiated (monolayer) HEECs were treated with 50 μg/mL LPS for 24 hours after which cell-free supernatant was collected and cytokine/chemokine secretion analyzed using a multiplex bead assay. Stimulation with LPS significantly increased levels of MCP-1, IL-8 (a), GRO-α, and IL-6 (b) in both groups of cells as compared with the no treatment (*P < 0.05). However, a differential response was seen in the levels of migration inhibitory factor (c) in cells cultured in Matrigel as compared with the no treatment (*P < 0.05).

Figure 8.. Long-term response of transformed trophoblast/endothelial tubes to lipopolysaccharide (LPS).

Both short-term and long-term cultured trophoblast cells stimulated with 50 μg LPS do not show any change on cytokine production (a and c); however, stimulation of short-term co-cultured tubes (24 hours) with 50 μg LPS showed a significant increase in GRO-α, MCP-1, and IL-8 secretion (b). The long-term co-cultured tubes (5 days) produced high levels of cytokines but only IL-8 secretion increased following LPS treatment (d). *P < 0.05 **P < 0.01 as compared with the no treatment control group.

Figure 9.

Lipopolysaccharide (LPS) disrupts trophoblast migration toward differentiated human endometrial endothelial cells (HEECs). HEECs were plated (1.0 × 10⁵) with Matrigel and treated with 50 μg/mL LPS for 24 hours and migration monitored by fluorescent microscopy. (a) Trophoblast co-cultured with non-treated endothelial cells migrated and aligned on top of the tubes. (b) Trophoblast co-cultured with LPS treated HEECs began to clump within the lumen of the tubes.