A Two-Colour Fluorescence Assay for the Measurement of Syncytial Fusion between Trophoblast-Derived Cell Lines

Borges, Marcus Kiiti; Bose, Patrick; Frank, Hans-Georg; Kaufmann, Peter; Pötgens, Andy J.G.

doi:10.1016/s0143-4004(03)00173-5

Cited by 58 publications

(40 citation statements)

References 20 publications

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“…17 Forskolin-treated, non-silenced BeWo cells showed enhanced cell-cell fusion compared with nonsilenced controls (13±0.08% versus 1.5±0.24% fusion events per number of nuclei, Po0.01) (Figure 1b and g and Supplementary Figure 1b). By contrast, GCM1 silencing in forskolin-treated BeWo cells resulted in reduced levels of cell-cell fusion compared with that in non-silenced forskolin-treated controls (Figure 1d and g and Supplementary Figure 1d).…”

Section: Gcm1mentioning

confidence: 91%

“…Second, BeWo cells were color-labeled with Cell Tracker fluorescent dyes (Invitrogen) for analysis and quantification as described earlier. 17 Forskolin (final concentration 25 mM) (Sigma, Oakville, ON, Canada) was added to accelerate syncytial fusion. 16 BeWo cells were assessed after 24 and 48 h for syncytial fusion using phase-contrast and fluorescent microscopy and quantified as described by Borges et al 17 After 48 h, RNA was extracted from the cells using the RNeasy kit (Qiagen, Mississauga, ON, Canada), and protein was extracted using RIPA lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

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Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast

et al. 2009

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Mammalian placentation is a highly regulated process and is dependent on the proper development of specific trophoblast cell lineages. The two major types of trophoblast, villous and extravillous, show mitotic arrest during differentiation. In mice, the transcription factor, glial cell missing-1 (Gcm1), blocks mitosis and is required for syncytiotrophoblast formation and morphogenesis of the labyrinth, the murine equivalent of the villous placenta. The human homolog GCM1 has an analogous expression pattern, but its function is presently unknown. We studied GCM1 function in the human-derived BeWo choriocarcinoma cell line and in first trimester human placental villous and extravillous explants. The GCM1 expression was either inhibited by siRNA and antisense oligonucleotides methods or upregulated by forskolin treatment. Inhibition of GCM1 resulted in an increased rate of proliferation, but prevented de novo syncytiotrophoblast formation in syncytially denuded floating villous explants. GCM1 inhibition prevented extravillous differentiation along the invasive pathway in extravillous explants on matrigel. By contrast, forskolin-induced expression of GCM1 reduced the rate of proliferation and increased the rate of syncytialization in the floating villous explant model. Our studies show that GCM1 has a distinct role in the maintenance, development and turnover of the human trophoblast. Alterations in GCM1 expression or regulation may explain several aspects of two divergent severe placental insufficiency syndromes, namely preeclampsia and intrauterine growth restriction, which cause extreme preterm birth. Successful mammalian pregnancy demands two key steps in placental development, both of which are regulated by differentiation of the epithelial trophoblast lineage. 1 First, maternal blood flow to the implantation site must increase exponentially to serve the demands of the rapidly growing fetus. This is achieved through the invasion of extravillous cytotrophoblasts (EV-CTs) from the base of the placenta into the maternal uterine stroma, where they surround and dilate the distal portions of the utero-placental arteries. 2 Second, the increased delivery of maternal blood to the placenta must be matched by an exchanger organ to transfer nutrients, remove waste products and respiratory gases between the mother and the developing fetus. This requirement is achieved through the expansion and differentiation of the chorionic villous trees of the placenta. These villi are covered by a villous trophoblast compartment comprising of villous cytotrophoblasts (V-CTs) beneath a continuous multinucleated layer of syncytiotrophoblast (SCT), which is in direct contact with maternal blood. 3 In the human placenta, EV-CTs of the proximal portion of the anchoring villus cell column are proliferative, but more distal cells differentiate, as they invade and disperse into the uterine stroma. Here these cells surround and replace the smooth muscle cells of maternal uterine arteries, converting them into high-flow dilated sinusoids. The ar...

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Section: Gcm1mentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast

et al. 2009

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“…Trophoblast stem cells (TSCs) have yet to be derived from human embryos (Roberts and Fisher, 2011), and, although 'trophoblast progenitors' have been cultured from first trimester chorionic mesenchyme (Genbacev et al, 2011), the true nature of these cells remains to be independently confirmed. Established human trophoblast cell lines represent immortalized or transformed versions of previously committed villous or extravillous lineages, and therefore are not good models to study lineage-specific differentiation (Janneau et al, 2002;Borges et al, 2003;Khoo et al, 1998). For these reasons, many researchers focus on mouse models, for which trophectodermderived TSCs have been established (Tanaka et al, 1998), and the placental contribution to embryonic phenotype can be easily studied (Rossant, 2007;Cockburn and Rossant, 2010;Rossant and Cross, 2001).…”

Section: Research Articlementioning

confidence: 99%

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state

et al. 2013

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SUMMARYThe placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63 + /KRT7 + CTB stem cell state, followed by formation of functional KLF4 + STB and HLA-G + EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63 + CTB stem cells to terminally differentiated trophoblast subtypes.

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“…Their differentiation into syncytiotrophoblasts does not occur spontaneously, but the syncytialization process can be induced by forskolin or 8-bromoadenosine 38,58-cyclic monophosphate treatment (Borges et al, 2003). For this reason, BeWo cells are used for studies on the maturation of trophoblasts: they can model either cytotrophoblasts of early gestation or syncytiotrophoblasts of late gestation.…”

Section: Human Choriocarcinoma Cell Linesmentioning

confidence: 99%

The Role of the Placenta in Fetal Exposure to Xenobiotics: Importance of Membrane Transporters and Human Models for Transfer Studies

Prouillac

Lecoeur²

2010

Drug Metab Dispos

176

127

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ABSTRACT:The placenta is a key organ in fetal growth and development because it controls maternal-to-fetal exchanges of nutrients and hormones. It also interferes with drug delivery to the fetus by expressing active membrane transporters and xenobiotic metabolism enzymes. Developing strategies to understand the role of the placenta in drug delivery is a challenge in toxicology. Despite common physiological functions, the placentas of different species are heterogeneous in their morphology and in their expression of membrane transporters and metabolizing proteins. These characteristics raise the difficulty of obtaining a good representative model of human placental transfer. To date, different in vitro, in vivo, and ex vivo tools have been used to elucidate transport and metabolism processes in the human placenta. This study recapitulates the typical features of human placenta and then presents the placental enzymes of xenobiotic metabolism, ATP-binding cassette transporters, solute carrier transporters, and their role in fetal exposure to xenobiotics. The study also compares the characteristics of different models of human placenta, in terms of membrane localization of transporters, and the expression of xenobiotic metabolism enzymes. The use of these models for toxicological studies, in particular xenobiotic transfer, is described, and the advantages and limits of each model are summarized.

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A Two-Colour Fluorescence Assay for the Measurement of Syncytial Fusion between Trophoblast-Derived Cell Lines

Cited by 58 publications

References 20 publications

Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast

Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state

The Role of the Placenta in Fetal Exposure to Xenobiotics: Importance of Membrane Transporters and Human Models for Transfer Studies

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