Associate Professor University of Western Ontario, Children's Health Research Institute London, Ontario, Canada
Abstract Body : Introduction: Extravillous trophoblast (EVT) migration, invasion, and subsequent remodeling of maternal spiral arteries is an essential component of placentation, and necessary for sufficient delivery of oxygen and nutrients to a growing fetus. Aberrant EVT invasion is associated with many complications of pregnancy, including preeclampsia, fetal growth restriction, preterm labor, and miscarriage. Although EVT invasion plays a fundamental role in placentation and fetal development, much is yet to be understood regarding the regulatory mechanisms behind this process. Previous studies suggest that chemokines, which encompass a large family of small cytokines that promote chemotaxis by binding to specific G-protein coupled receptors, may be important regulators of EVT invasion towards maternal spiral arteries. As such, the goal of this project was to evaluate the expression of chemokine receptors in recently-derived human trophoblast stem (TS) cells differentiated toward EVTs, and determine the effect of chemokine receptor activation on EVT invasiveness.
Hypothesis: We hypothesized that TS cells acquire chemokine receptors as they differentiate to EVTs, and that activation of these chemokine receptors by their ligands stimulates EVT migration and invasion.
Methods: We used a publicly-available RNA-sequencing dataset to evaluate expression of chemokine receptors in human EVTs and TS cells (Okae et al. Cell Stem Cell 2018). TS cells were cultured in the cytotrophoblast (CT) stem state or placed in EVT differentiation media for up to 6 days to induce EVT differentiation. Quantitative RT-PCR was used to measure gene expression of CCR1, CXCR4, and CXCR6. Matrigel-based invasion assays were performed using terminally differentiated (day 6) TS cell-derived EVTs in the presence or absence of 100-1000 ng/mL recombinant CCL5 or CXCL16. Data was analyzed using ANOVA followed by a multiple comparisons test, with P< 0.05 considered significant.
Results: Using publicly available RNA-sequencing data, we identified high expression of genes encoding three chemokine receptors – CCR1, CXCR4, and CXCR6 – in EVTs. Quantitative RT-PCR results showed that relative expression of both CCR1 and CXCR6 was increased as EVT differentiation progressed, but there was no change in CXCR4 expression. Specifically, CCR1 expression was increased by 4-fold on day 5 and 6 of EVT differentiation compared to CTs (N=4; P< 0.001). Likewise, expression of CXCR6 was increased by 4-fold after 4, 5, and 6 days of EVT differentiation compared to CTs (N=4, P < 0.0001). Preliminary results using Matrigel-based invasion assays show increased EVT invasion in wells with media containing CCL5 (ligand for CCR1) and CXCL16 (ligand for CXCR6) compared to controls.
Discussion: CCR1 and CXCR6 are induced during EVT differentiation, suggesting that activation of these chemokine receptors may promote directed EVT migration upon interacting with their ligands. Preliminary Matrigel-based invasion assays are consistent with this notion, as the addition of CCL5 or CXCL16 appears to promote EVT invasion.
Significance: Future work will further characterize the functional role of these chemokines and their receptors on EVT invasion. Overall, this research will provide key insights into the fundamental regulation of EVT invasion, thereby leading to a better understanding of the underlying causes of many pregnancy complications.
