Research ArticleLive cell image analysis of cell–cell interactions reveals the specific targeting of vascular smooth muscle cells by fetal trophoblasts
Introduction
During human pregnancy normal fetal growth and development is dependent on the placenta, the highly differentiated organ which serves as the interface between the mother and the growing fetus and allows exchange of oxygen, nutrients and waste molecules. The function of the placenta is precisely regulated and changes in placental development can have a dramatic effect on fetal growth and well-being. Maternal blood is carried to the placenta via the uterine spiral arteries. As demand for blood supply by the growing fetus increases, more blood needs to be delivered. To meet this increased demand, the spiral arteries undergo a physiological change in which disappearance of normal muscular and elastic structure results in remodelled and dilated arteries and increased maternal blood flow to the fetus [1], [2], [3].
In the first trimester fetal trophoblast cells differentiate into an invasive cell population that, as part of the normal course of pregnancy, leave the placenta, infiltrate through the decidua and part of the myometrium to reach the spiral arteries (but not veins) and play an active role in the remodelling process. The mechanisms controlling trophoblast migration and spiral artery invasion and remodelling are crucial for understanding normal and abnormal pregnancies. Failure of artery remodelling has been described in several complications of pregnancy such as pre-eclampsia and fetal growth restriction [4], [5]. Pre-eclampsia is the most common defect affecting 7–10% women during their first pregnancy and accounts for significant fetal and maternal morbidity and mortality.
Despite the importance of trophoblast invasion and vascular remodelling, these processes are still not well understood. Clinical and basic research studies have focused on identifying various cells and mechanisms involved in these processes revealing that the remodelling process relies on a complex co-ordination of micro-environmental factors and cell–cell interactions. It has been shown that co-culture of trophoblasts with vascular smooth muscle cells (VSMC) or endothelial cells leads to induction of cell death by apoptosis in the vascular cells which may be important in the remodelling of spiral arteries [6], [7], [8]. However, the influence of interaction between trophoblast and vascular cells on their behaviour has not been studied. The aim of this study was to investigate the interaction between primary first trimester human cytotrophoblasts (TC) and human VSMC and, in particular, to investigate whether TC are specifically targeting VSMC.
A number of methods have been used to measure cell migration including transwell chambers [9], under-agarose migration [10], agarose drop [11] and wound healing assays [12]. Since the development of computer assessed video microscopy [13], [14], [15], [16], [17], [18] it has been possible to record sequences of several hundred images in a single experiment, generating a large data set and a requirement for fast data analysis methods. Computer assessed video microscopy automatically records sequences of images by means of a microscope equipped with an attached image acquisition system, typically a charged-coupled device (CCD) camera and a computer. This method has advantages over other in vitro assay methods because it can yield additional parameters reflecting the dynamic migration behaviour of individual cells and whole cell populations. Often sequences are analysed manually as tracking cells automatically is more challenging due to changes in shape and appearance of cells as they migrate.
In this study we developed a new analysis technique to quantify relative cell–cell migration for cells in co-culture; in this instance addressing the importance of the interaction between TC and VSMC. We used time-lapse phase-contrast microscopy to monitor cells and incorporated a semi-automatic cell tracking technique to obtain cell positions over time. From the positions of cells in co-culture, the movement behaviour of each of the cell types was analysed. Furthermore, we analysed whether TC were specifically targeting VSMC which would be important in the understanding of spiral artery remodelling in early pregnancy. This method will be useful to study simultaneous cell motility and cell–cell attraction of different cell types in co-culture in a broad range of experiments.
Section snippets
Reagents
Reagents were purchased from the following sources: Tissue culture medium and fetal bovine serum, Sigma-Aldrich Inc. (Saint Louis, MO, USA); Matrigel™, BD Discovery Labware (Bedford, MA, USA); and CellTracker™ Orange, Invitrogen Corp. (Carlsbad, CA, USA).
Cell culture and co-culture experiments
Informed consent was obtained for all placental tissue used in this study and all procedures were performed in accordance with the Wandsworth Local Research Ethical Committee.
First trimester primary cytotrophoblast (CTB) were cultured in
TC are more motile in co-culture with VSMC
The tracking software calculated individual cell trajectories for each cell type. A graphical representation of the cell movement was created in order to observe the difference in cell directionality and distance travelled by the migrating cells (Figs. 4A–C). In these representations, the starting positions of all tracks were moved to the same origin and tracks were coloured according to cell type. Fig. 4B shows tracks for TC migration in mono-culture and Fig. 4C shows tracks for VSMC in
Discussion
The newly developed tracking software has shown that it is possible to simultaneously track two different cell types independent of their appearance or movement behaviour. The tracking algorithm is based on determining cell positions in subsequent images by performing an intensity search in the neighbourhood of the previous position. A new cell position was set to the position of the lowest intensity within this search space, which corresponds to the densest area such as the nucleus. A
Acknowledgment
RJK was supported by the Wellcome Trust (grant number 069939).
References (28)
- et al.
Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy
Placenta
(1983) - et al.
Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy
Placenta
(1980) - et al.
The uterine spiral arteries in human pregnancy: facts and controversies
Placenta
(2006) - et al.
Invasive trophoblasts stimulate vascular smooth muscle cell apoptosis by a fas ligand-dependent mechanism
Am. J. Pathol.
(2006) - et al.
Osteoblast-derived oxysterol is a migration-inducing factor for human breast cancer cells
J. Biol. Chem.
(2003) - et al.
Are growth factors chemotactic agents?
Exp. Cell Res.
(1995) - et al.
A fast and robust quantitative time-lapse assay for cell migration
Exp. Cell Res.
(2005) - et al.
Trophoblast invasion of spiral arteries: a novel in vitro model
Placenta
(2002) - et al.
Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta
Lab. Invest.
(2001) - et al.
Pre-eclampsia and maternal anaemia display reduced apoptosis and opposite invasive phenotypes of extravillous trophoblast
Placenta
(2003)