Human Reproduction, Vol. 17, No. 5, 1351-1357,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Ultrastructure of the early human feto-maternal interface co-cultured in vitro
1 Institute of Reproductive and Developmental Biology, Wolfson & Weston Research Centre for Family Health, Hammersmith Hospital, Du Cane Road, London W12 0NN, 2 Electron Microscopy Unit, Department of Histopathology, 6th Floor, Laboratory Block, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
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Abstract |
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BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8–12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.
Key words: co-culture in vitro/EVT/feto-maternal interface/fusion of plasma membranes
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Introduction |
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Although interest in human development in utero dates back to antiquity, there is still a dearth of knowledge of the early events of human placentation. Hence the search continues for the better understanding of these events, using in-vitro co-culture models of early human placental villous and decidua parietalis tissues (Kliman et al., 1990
; Lewis et al., 1993; Vicovac et al., 1993, 1995; Babawale et al., 1996) or of cells from these tissues. In early human development, a local breakdown of the syncytial layer of the placenta occurs as it comes in contact with maternal decidua, whilst the underlying cytotrophoblast cells proliferate and form columns of invasive cytotrophoblast cells which form the anchoring villi (Boyd and Hamilton, 1970; Benirschke and Kaufmann, 1990). The anchoring villi (as the name implies) physically connect the placenta to the maternal uterine wall. Although very little is known about the chronology of these events in man, breakthrough of the cytotrophoblast may occur as early as day 12 of pregnancy in the rhesus monkey (Enders et al., 1983).
The invasive cytotrophoblast cells, commonly known as extravillous trophoblast (EVT) cells, follow two distinct pathways of differentiation (Pijnenborg et al., 1980). In the first pathway, EVT cells invade the decidualized endometrium, as well as the inner third of the myometrium. Some of these EVT cells further differentiate by fusing with one another to form the giant cells of the placental bed. It is this spatial confinement of trophoblast cells to the decidualized endometrium and the inner third of the myometrium that distinguish them from invasive cancer cells (Fisher and Damsky, 1993). In the second pathway, EVT cells invade the uterine spiral arteries and adopt a vascular phenotype (Zhou et al., 1997). This involves replacement of the vascular smooth muscle cells and endothelium by trophoblast, and the formation of distensible vessels for the provision of large volumes of blood to the feto-maternal interface. The initial remodelling of the uterine spiral arteries seems to involve cells migrating through the decidua to the target vessels (Pijnenborg et al., 1980), and occurs within the first trimester of pregnancy. This is limited to the decidual portion of these vessels. Remodelling of the inner myometrial portion occurs at 14–18 weeks gestation and is mediated by intravascular migration of EVT cells (Pijnenborg et al., 1980). In some abnormalities of pregnancy such as pre-eclampsia, trophoblast invasion of the uterine spiral arteries does not proceed beyond the decidual portions of the spiral arteries (Brosens et al., 1972; Moodley and Ramsaroop, 1989). Conversely, in other pathological conditions of pregnancy, trophoblast invasion of the decidua and the myometrium becomes excessive, resulting in placenta accreta (Hustin et al., 1990).
The fate of cytotrophoblast cells in the first trimester `floating villi' differs from those of the anchoring villi. In the floating villi, cytotrophoblast cells exist as polarized epithelial monolayers anchored to a basement membrane surrounding a mesenchymal stromal core containing fetal blood vessels (Boyd and Hamilton, 1970; Benirschke and Kaufmann, 1990; Aplin, 1991). These cytotrophoblast cells are highly proliferative and differentiate to form the outer syncytial covering of the villous tree (Boyd and Hamilton, 1966; Muhlhauser et al., 1993). Unlike the anchoring villi, the floating villi do not come in contact with the uterine wall but are bathed in maternal blood, thus forming the placental barrier where the exchange of oxygen, nutrients and waste products occurs between the mother and the fetus.
Models of the feto-maternal interface (Vicovac et al., 1995; Babawale et al., 1996) have shown that floating villi can readily form structures resembling anchoring villi when placed in contact with appropriate substrates, and these villi retain the potential to differentiate. This study has assessed the ultrastructural features of the interface formed in vitro, and compared these findings with previous studies on the feto-maternal interface of human pregnancy.
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Tissues
First trimester chorionic placental villi and uterine decidual tissues were obtained from six patients who had therapeutic termination of pregnancy at 8–12 weeks gestation. The study was approved by the ethics committee at Hammersmith Hospital, and informed consent was obtained from all the patients prior to operation. The products were collected from the operating theatre, washed in phosphate-buffered saline (PBS) solution, pH 7.2, to remove blood clots and were transported to the laboratory in 150 ml pots containing warm sterile culture medium, RPMI 1640 (Sigma-Adrich Company Ltd, Poole, UK). Both tissues were examined under a dissecting microscope to ensure that the pieces selected were free from contamination: the decidua parietalis, for evidence of villous attachment and villous tissue for evidence of damage or of blood clots. Tissues that appeared unsatisfactory were not used in the study.
Incubation method
All incubations were carried out in 24-well culture plates (Corning Ltd, High Wycombe, UK) using RPMI 1640 culture medium supplemented with 10% fetal calf serum, 2 mmol/l L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin (Sigma-Adrich). From each of six patients, four co-cultures were set up, corresponding to 24, 48, 72 and 96 h of culture. Cultures were incubated in an atmosphere of 5% CO2:95% air at 37°C and set up as follows: a piece of villous tissue (0.5 mm3) was placed on decidual explant (2 mm3) already plated on a few drops of culture medium left undisturbed for ~30 min. Both tissue samples were from the same patient. Thereafter, 2 ml culture medium was added carefully to each culture chamber. Tissue samples were removed for processing at 24 h intervals and the culture medium in the remaining culture chambers was changed daily. Four control cultures of decidual explants alone (without the placental villi) were set up from each of the six patients, corresponding to 24, 48, 72 and 96 h of culture as per the experiment.
Tissue processing for transmission electron microscopy (TEM)
Co-cultured samples (24 samples) and control samples (24 samples) were fixed in 3% glutaraldehyde in cacodylate buffer (pH 7.2) for 30 min, post-fixed in 1% osmium tetroxide in cacodylate buffer (pH 7.2), dehydrated through ascending grades of alcohol and embedded in Araldite. Semi-thin sections were cut at 1 µm and stained in 1% Toluidine Blue in order to identify areas of interest for viewing on the electron microscope. Ultra-thin sections (60–80 nm) were taken from the selected areas, stained with uranyl acetate and lead citrate. The stained sections were then examined and areas of interest photographed using a Hitachi electron microscope, model HU12A, at an accelerating voltage of 75 kV.
Immunocytochemistry/cytokeratin immunodetection
Immunolabelling of trophoblast cells was carried out with the anti-cytokeratin antibody CAM 5.2 (International Cancer Research Fund, London, UK) using the mouse avidin–biotin indirect immunoperoxidase detection method on Araldite-embedded co-cultured placental tissue blocks sectioned at 1 µm thickness. Slides were placed in sodium ethoxide for 30 min to remove the Araldite (etching procedure) after which endogenous peroxidase activity was blocked using 3% hydrogen peroxide in tap water for 30 min at room temperature. The slides were then placed in 0.1% trypsin (Sigma-Adrich) in 0.1% CaCl2, pH 7.8 at 37°C for 10 min and then rinsed in PBS.
Serum blocking was carried out with 5% normal goat serum for 15 min to eliminate non-specific binding. The tissue sections were then incubated in CAM 5.2 at 1/10 dilution overnight at 4°C. Non-immune mouse serum was substituted for CAM 5.2 in tissue sections that served as the negative control. The sections were thereafter rinsed in 3x5 min changes of PBS and incubated with biotinylated goat anti-mouse secondary antibody (Dako, High Wycombe, UK) at 1/500 dilution for 1 h. After 3x5 min further changes of PBS, the sections were incubated with peroxidase-labelled streptavidin 1/500 (Boehringer, Lewes, UK) for 1 h. A final rinse of 3x5 min changes of PBS was carried out. Peroxidase activity was demonstrated by developing the sections in 0.05% 3,3'diaminobenzidine solution (DAB; Dako), with 0.03% hydrogen peroxide as substrate.
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Semi-thin histology
After 24 h of co-culture, absence of the syncytium at the tip of the anchoring villus, cytotrophoblast cell proliferation and differentiation into EVT cell columns were observed (Figure 1
). The syncytial layer was missing at the interface between placental villi and decidua, and the syncytial margins were displaced laterally as the `leading' cytotrophoblast cells appeared to have commenced invasion into the adjacent decidual stroma (Figure 1). Cytotrophoblast cells at the invading front were cytokeratin-positive (micrograph not shown). No zone of matrix degradation was observed around the invading trophoblast cells and distant migration of the EVT cells into the decidual explant had not commenced at this stage.
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After 48 h of co-culture there was a clear column of cells connecting the placental villi to the decidua (Figure 2
), thus mimicking the anchoring villi seen in vivo in human placentation. EVT cells were identified in serial sections of Figure 2 by cytokeratin immunostaining (Figure 3), which revealed some EVT cells on the outer surface of the placental villi and within the decidua (arrowheads) and a syncytial knot (arrow). Fewer cytotrophoblast cells were seen in this location than are normally present in vivo. This might be because of the plane of cut of the section, or loss of some of the cytotrophoblast cells in the placental villi during experimentation, even though blunt forceps were used in placing the villi and decidual tissues together. The decidual stroma appeared less dense after 48–96 h of culture than it was after 24 h of co-culture.
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Sections of the decidual explant that had been in culture for 96 h served as controls and were stained for anti-cytokeratin CAM 5.2. Only cytokeratin-positive cuboidal uterine epithelial glandular cells were observed. There were no contaminant EVT cells within the decidual matrix (Figure 4
).
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Transmission electron microscopy
After 24 hours of co-culture, normal placental villous structures were still preserved. An outer syncytial layer containing a nucleus showing perinuclear chromatin condensation was observed overlying an inner layer of cytotrophoblast cells (Figure 5
). Some areas of the underlying cytotrophoblast cell plasma membrane were observed showing early signs of fusion to the overlying syncytium (5* and 5•). Higher magnification of the interfaces of the plasma membranes of cytotrophoblast cells and the overlying syncytium (Figures 6, 7 and 8) showed variable extents of contact and apparent fusion between the plasma membranes. Areas of apparent membrane degradation could also be seen (Figure 8).
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Cytotrophoblast cells rest on the basal lamina overlying the fetal mesenchymal core (Figure 9
). The fetal mesenchymal core consisted of a heterogeneous cell population, collagen fibrils, reticular, mesenchymal and Hofbauer cells and new blood vessels (micrograph not shown).
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EVT cells at the interface with the decidua generally showed a similar structure throughout the co-culture period of 96 h. Typical TEM images are shown to demonstrate these points. EVT cells at the leading edge of the cell column had invaded the decidual matrix (Figure 10
), and showed a pointed morphology. A remnant of the syncytium (S) appears to be in poor condition, and may have been be damaged during the remodelling processes of EVT invasion.
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There was also extensive cell–cell contact between the trophoblast cell column and decidual cells (Figure 11
), which seems to show desmosomes and the associated intermediate filaments connecting trophoblast and decidual cells. There were no signs of cellular degeneration in the decidual matrix (Figure 12), suggesting that the processes of trophoblast invasion are tightly regulated to prevent this and that the co-cultured tissues are viable in culture. Some trophoblast cells appeared to be very active, with large nucleoli (Figure 13) indicative of intense biosynthetic activity. The lamellopodic processes (locomotor apparatus) of some of the active trophoblast cells were observed to have invaded the adjacent decidual matrix (Figure 13).
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The ultrastructural integrity of the EVT cells remained intact for up to 96 h of co-culture (Figures 11–14
), with dilated Golgi, rough endoplasmic reticulum and healthy looking mitochondria (Figures 11, 12 and 14). Inclusions of lipid, auto or phagolysosomes and remnants of glycogen granules could also be seen (Figure 14), all of which suggest that these cells were metabolically very active.
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Discussion |
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There are many previous studies on human anchoring villi and the decidua, so the in-vivo structures and their likely functional correlates have been well described (Enders, 1968
; Boyd and Hamilton, 1970; Jones and Fox, 1991; Jones and Jauniaux, 1995). Our observations generally agree well with the earlier studies, in that similar features are seen. A critical point is that the interface in our study was generated by in-vitro co-culture of placental villi with decidual explants, whereas in earlier investigations the same interface formed in vivo. The similarities will be considered in more detail below, but the broad agreement indicates that this model is valid for investigating the feto-maternal interface of human pregnancy. The validity of these in-vitro models has already been suggested on the basis of light microscopy studies of co-cultures (Vicovac et al., 1995; Babawale et al., 1996) and by electron microscopy of the development of trophoblast cell columns on model matrices (Aplin et al., 1999).
The tissues comprising the feto-maternal interface showed the anticipated structures in this study. The decidua contained no low-molecular weight cytokeratin-positive cells in the stromal compartment (Figure 4), and the placental villi showed a multi-layered structure of mesenchymal core, basal lamina, cytotrophoblast and syncytium (Figures 5 and 9). Differentiation of cytotrophoblast into syncytium appears to involve fusion, dissolution, or both of the intervening plasma membranes between the cells (Figures 7 and 8). These findings are consistent with earlier work, and indicate that they occur in vitro as a dynamic process to maintain the syncytial structural integrity and function (Enders, 1965; Boyd and Hamilton, 1966; Morrish et al., 1997; Mayhew et al., 1999).
Previous studies have shown that desmosomal proteins are expressed in human placenta and trophoblast, and these may be involved in the functional differentiation of human and guinea pig trophoblast (Firth et al., 1980; Douglas and King, 1990; Winterhager et al., 1999, 2000; Cronier et al., 2001). The presence of desmosomes within the cell column (Figure 11) suggests that cell adhesion was well maintained between the cells in culture. There was extensive cell–cell contact between the trophoblast cell column and decidual cells (Figures 1–3, 11,13). This was particularly apparent in Figure 13, and seems to show desmosomes and the associated intermediate filaments connecting trophoblast and decidual cells. Further studies are needed to confirm this unexpected finding but, if correct, this is a clear indication of the lack of reaction between these genetically distinct cells. The expression of integrins changes within trophoblastic cell columns as they differentiate and invade either the decidua or the blood vessels (Damsky et al., 1994; Vicovac et al., 1995; Vicovac and Aplin, 1996) and it is possible that the desmosomes, in their role in maintaining adequate cell–cell adhesion, may be involved in this process.
Previous work has shown that all cytotrophoblast cells express low-molecular weight cytokeratins (Khong et al., 1986; Aplin et al., 1999). Not all cytotrophoblast expressed CAM 5.2 in the semi-thin sections used for immunohistochemistry (Figure 3), which was unexpected, as our earlier work on this model showed general positive staining (Babawale et al., 1996). We attribute the apparent incompatibilities to the more rigorous procedures needed for semi-thin methodology, which affects the detection limit so that only cells with high levels of cytokeratin can be visualized.
Differentiation of cytotrophoblast to form cell columns and invasive EVT was clearly documented (Figures 1–3 and 10–11). The process of trophoblastic cell invasion does not seem to damage the decidual structures (Figures 10–13), but rather there is continued healthy contact between invading trophoblast, decidual cells and decidual matrix. Some of the factors implicated in regulation have already been identified (matrix metalloproteinase-2 and -9, tissue inhibitors of metalloprotineases-1 and -2) (Librach et al., 1991; Pollete et al., 1994; Babawale et al., 1995; Bischof et al., 1998) and the co-culture model may be a useful technique to explore the roles of these factors further. Invasion is obviously an active process, and is consistent with the depletion of glycogen storage as well as the presence of an active nucleoli and lamellipodic processes in some of the trophoblast cells observed (Figures 11 and 14). The nature of the lamellae observed (Figure 12) requires further investigation, as such structures are not common in connective tissue.
In summary, our findings indicate that this model has features at the cellular and sub-cellular levels that resemble those observed in vivo in human pregnancy, and support our observations that this model is useful for the study of human placentation.
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Acknowledgements |
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This work was supported by medical research grants from The Sir Halley Stewart Trust (Cambridge, UK) to M.O.B. and The Robert McAlpine Foundation (Kent, UK) to M.H.F.S. The authors express their thanks to Ms Ann Kane for her assistance with photoimaging and Trevor Gray for help with some of the electron micrographs.
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Notes |
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3 Present address: Division of Pathology, School of Clinical Laboratory Sciences, University of Nottingham Medical School, Queens' Medical Centre, Nottingham NG7 2UH, UK
4 To whom correspondence should be addressed. E-mail: mark.sullivan{at}ic.ac.uk
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Submitted on July 12, 2001; resubmitted on November 19, 2001; accepted on January 7, 2002.
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