Human cytomegalovirus infection inhibits CXCL12- mediated migration and invasion of human extravillous cytotrophoblasts.

BACKGROUND: During the first trimester of pregnancy, a series of tightly regulated interactions govern the formation of a highly invasive population of fetal-derived extravillous cytotrophoblasts (EVT). Successful pregnancy is dependent on efficient invasion of the uterine wall and maternal spiral arteries by EVT. Dysregulated trophoblast invasion is associated with intrauterine growth restriction, birth defects, spontaneous abortion and preeclampsia. A number of soluble growth factors, cytokines, and chemokines modulate this process, fine-tuning the temporal and spatial aspects of cytotrophoblast invasion. In particular, the CXCL12/CXCR4 axis has been shown to specifically modulate cytotrophoblast differentiation, invasion, and survival throughout early pregnancy. Infection with human cytomegalovirus (HCMV) has been associated with impaired differentiation of cytotrophoblasts down the invasive pathway, specifically dysregulating the response to mitogens including epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In this study, the effect of HCMV infection on the CXCL12-mediated migration and invasion of the EVT cell line SGHPL-4 was investigated. RESULTS: Infection with HCMV significantly decreased secretion of CXCL12 by SGHPL-4 cells, and induced a striking perinuclear accumulation of the chemokine. HCMV infection significantly increased mRNA and total cell surface expression of the two known receptors for CXCL12: CXCR4 and CXCR7. Functionally, HCMV-infected SGHPL-4 cells were unable to migrate or invade in response to a gradient of soluble CXCL12 in transwell assays. CONCLUSIONS: Collectively, these studies demonstrate that HCMV impairs EVT migration and invasion induced by CXCL12. As HCMV has the ability to inhibit EVT migration and invasion through dysregulation of other relevant signaling pathways, it is likely that the virus affects multiple signaling pathways to impair placentation and contribute to some of the placental defects seen in HCMV-positive pregnancies.

Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu. We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.