A Two-Colour Fluorescence Assay for the Measurement of Syncytial Fusion between Trophoblast-Derived Cell Lines
Introduction
Syncytialization, the fusion of cells into a multinuclear syncytium is a normal morphogenic feature in myotube formation (van den Eijnde et al., 2001), osteoclast formation (Vignery, 2000) and in the formation of placental syncytiotrophoblast (Benirschke and Kaufmann, 2000; for review see Pötgens et al., 2002). The regulation of syncytial fusion of trophoblast is only poorly understood. A protein called syncytin (the envelope protein of an endogenous retrovirus, HERV-W) appears to play an important role (Blond et al., 2000; Mi et al., 2000). Syncytin was shown to induce syncytial fusion upon transfection of its cDNA into various cell lines. Syncytin was found to be continuously expressed in the basal syncytiotrophoblast membrane (Blond et al., 2000; Lee et al., 2001; Mi et al., 2000). However, syncytiotrophoblast fuses with cytotrophoblast cells only under special conditions, (a) in late stages of differentiation of cytotrophoblast and (b) when the syncytiotrophoblast shows degenerative signs (Kaufmann, Gentzen and Davidoff, 1977; Benirschke and Kaufmann, 2000). The respective regulation of the fusion process may be a matter of local differences in syncytin surface density in the syncytiotrophoblast, but may also be a question of the availability of its putative receptor, RDR (RD114 receptor), also known as neutral amino acid transporter system B (ATB(0)) (Rasko et al., 1999; Tailor et al., 1999; Blond et al., 2000). Also additional factors, such as the externalization of phosphatidylserine (Lyden, Ng and Rote, 1993; Adler, Ng and Rote, 1995; Katsuragawa et al., 1997; Huppertz et al., 1998; Guilbert et al., 2002), or hitherto unknown factors, may play a role in regulating this process. Additional in vitro studies will be necessary to fully understand the regulation of this fusion process.
Several in vitro systems for trophoblast fusion have been developed. Primary trophoblast cells isolated from placenta fused spontaneously, and at a higher rate in the presence of EGF (Morrish et al., 1997; Crocker et al., 2001). Trophoblast-derived choriocarcinoma cell lines BeWo (Lyden, Ng and Rote, 1993) and JAR (Adler, Ng and Rote, 1995) have been shown to form multinuclear structures as well, while this process was enhanced by treatment with forskolin. In previous studies syncytial trophoblast fusion was assessed by morphological examination, often aided by the use of anti-desmoplakin antibodies to show the absence of intercellular junctions. Using this methodology, however, syncytialization is difficult to distinguish from endomitosis, a process in which multinuclear plasmodia are produced after nuclear division without subsequent cell division.
To overcome these problems, in this study we present a trophoblast fusion assay based on differential staining of two separate batches of cells with green-fluorescent and red-fluorescent cytoplasmic dyes. After fusion between green- and red-labelled cells, the resulting syncytia were easily discriminated by fluorescence microscopy by proof of mixed colors in cytoplasm and nuclei of the multinucleated structures. Using this assay we found differences in fusion behaviour between various well-known choriocarcinoma cell lines. The presence or absence of intercellular fusion in these cell lines could not be explained solely by differential expression of the genes encoding syncytin and/or RDR.
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Cell culture
Human choriocarcinoma cell lines BeWo, JAR, and JEG3 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cell lines K562 (human erythroleukemia), A375 (human melanoma) and HT1080 (human fibrosarcoma) were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). All lines but K562 were cultured at 37°C in 5 per cent CO2in HAM's F12 medium (Sigma, Taufkirchen, Germany), supplemented with 10 per cent fetal
Results
Choriocarcinoma cell line BeWo subjected to the fusion assay occasionally showed cell–cell fusion. Single cells stained with cytoplasmic dyes CT green or CT orange in fluorescence microscopy showed green or red-fluorescent cytoplasm, respectively, but nuclei were stained even more intensely. After fusion, syncytia were visible with a double-fluorescent (yellow to orange in composed pictures) cytoplasm surrounding multiple (between 2–20) nuclei (Figure 1B, C). Nuclear sizes resulting after
Discussion
In this paper we present a cell–cell fusion assay. The use of two fluorescent dyes that diffusely mix after cell–cell fusion, allows easy, reproducible and observer-independent detection of syncytial fusion by routine fluorescence microscopy. This assay differs from most conventional assays based on morphological criteria, on desmoplakin immunohistochemistry, or on diffusion of dye microinjected into cells. These latter assays can indicate the presence of multinuclear cells, but not whether
Acknowledgments
We thank Uta Zahn and Anke Gärtner for excellent technical support. This work was supported by grants of the German Research Council (DFG: FR1245/3-3 to HGF and PK), and of the Rockefeller Foundation: RF96020#76 and RF99021#114 to PK and, HGF).
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2014, PlacentaCitation Excerpt :After 48 h, the samples were observed on a Motic BA310 epifluorescence microscope, and images were obtained with a digital Moticam 5 camera and analyzed with Fiji/Image J. For quantitation of fusion events, at least twenty pictures were taken randomly from each sample. The figures from the different pictures were summed up to calculate the fusion index, which was defined as the average percentage of nuclei in double-fluorescent cell [22]. All experiments were performed in triplicate using three different placentas.