The Human Endogenous Retrovirus Ervw-1 Regulates Trophoblast Stem Cell Proliferation.

The human endogenous retrovirus ERVW-1 drives differentiation of cytotrophoblast into syncytiotrophoblast, the fetal portion of the placenta responsible for secretion of hormones and nutrient exchange. ERVW-1 also presents in cytotrophoblast cells; however, its role in maintaining this undifferentiated population of cells is poorly understood. The objective of this study was to examine the role of ERVW-1 in the maintenance of proliferation of human trophoblast stem cells (TSC). We hypothesized that ERVW-1 regulates undifferentiated trophoblast cell proliferation during placental development. Prospective research study. To assess the role of ERVW-1, we used the CT27 line of TSC first described by Okae et al. in 2018. TSC were treated with lentivirus containing either shRNA designed to knockdown ERVW-1 (KD) or a scramble control (SC). To select successfully transduced cells, we treated wild type (WT), SC, and KD cells with 2 μg/mL puromycin. After 48 hours, all wild type cells treated with puromycin were dead. We continued puromycin selection for 5 additional days to ensure successful selection. After puromycin selection, RNA was isolated from WT, SC, and KD cells. First strand complementary DNA was synthesized using the BioRad iScript Reverse Transcriptase kit and real-time quantitative PCR (RT-qPCR) was performed to confirm knockdown of ERVW-1. After confirmation of knockdown, WT, SC, and KD cells were plated at a concentration of 5,000 cells/well of a 12-well tissue-culture treated plate. Cells were collected after 24, 36, 48, and 72 hours and counted to assess cell doubling time (n= 4 replicates). Finally, to assess trophoblast cell proliferation and health, we used RNA to determine gene expression of placental growth factor (PGF) in all 3 cell types using RT-qPCR (n= 3 replicates). Knockdown cells had significantly reduced expression of ERVW-1 mRNA compared to SC (73.5% reduction, p<0.05) and WT cells (80.9% reduction, p<0.05). ERVW-1 knockdown cells had an average doubling time of 26±1.74 hours compared to the 15.43±0.46 and 16.18±0.6 hour doubling time of the SC and WT cells, respectively. These data show that ERVW-1 KD cells have a significantly (p<0.005) longer cell doubling time compared to WT and SC cells. There was no difference in cell proliferation between WT and SC cells. Additionally, mRNA levels of PGF were significantly decreased in the KD cells compared to SC control cells (p=0.05). As PGF is known to increase trophoblast cell proliferation and reduce apoptosis, these data further suggest that decreased expression of ERVW-1 causes altered cell proliferation in TSC. ERVW-1 is important in maintaining cell proliferation in undifferentiated TSC. The decreased expression of PGF is also interesting as PGF is known to regulate cell proliferation as well as trophoblast vascularization. As reduced ERVW-1 and PGF has been implicated in placental pathologies including preeclampsia, these data provide context for the role of ERVW-1 in placental cell function outside of its classically understood role in regulating placental cell fusion.