Abstract
Although human term placenta-derived primary cytotrophoblasts (pCTBs) represent a good human syncytiotrophoblast (STB) model, in vitro culture of pCTBs is not always easily accomplished. Y-27632, a specific inhibitor of Rho-associated coiled-coil containing kinases (ROCK), reportedly prevented apoptosis and improved cell-to-substrate adhesion and culture stability of dissociated cultured human embryonic stem cells and human corneal endothelial cells. The Rho kinase pathway regulates various kinds of cell behavior, some of which are involved in pCTB adhesion and differentiation. In this study, we examined Y-27632’s potential for enhancing pCTB adhesion, viability and differentiation. pCTBs were isolated from term, uncomplicated placentas by trypsin–DNase I–Dispase II treatment and purified by HLA class I-positive cell depletion. Purified pCTBs were cultured on uncoated plates in the presence of epidermal growth factor (10 ng/ml) and various concentrations of Y-27632. pCTB adhesion to the plates was evaluated by phase-contrast imaging, viability was measured by WST-8 assay, and differentiation was evaluated by immunofluorescence staining, expression of fusogenic genes and hCG-β production. Ras-related C3 botulinum toxin substrate 1 (Rac1; one of the effector proteins of the Rho family) and protein kinase A (PKA) involvement was evaluated by using their specific inhibitors, NSC-23766 and H-89. We found that Y-27632 treatment significantly enhanced pCTB adhesion to plates, viability, cell-to-cell fusion and hCG-β production, but showed no effects on pCTB proliferation or apoptosis. Furthermore, NSC-23766 and H-89 each blocked the effects of Y-27632, suggesting that Y-27632 significantly enhanced pCTB differentiation via Rac1 and PKA activation. Our findings suggest that Rac1 and PKA may be interactively involved in CTB differentiation, and addition of Y-27632 to cultures may be an effective method for creating a stable culture model for studying CTB and STB biology in vitro.
Highlights
The syncytiotrophoblast (STB), a large multinucleated syncytium, covers the surface of placental villi, is in direct contact with the maternal blood and contributes to unique placental functions such as gas exchange, nutrition and ion supply to the fetus, waste transfer, pregnancyspecific hormone production and the immunological barrier [1]
It is impossible to culture placenta-derived STB, previous studies demonstrated that term human placenta-derived primary cytotrophoblasts, which are the progenitor of STB, can be obtained [7,8,9]
We examined the effects of a Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, on primary cytotrophoblasts (pCTBs) adhesion, viability and differentiation in vitro
Summary
The syncytiotrophoblast (STB), a large multinucleated syncytium, covers the surface of placental villi, is in direct contact with the maternal blood and contributes to unique placental functions such as gas exchange, nutrition and ion supply to the fetus, waste transfer, pregnancyspecific hormone production and the immunological barrier [1]. A trophoblast cell line, BeWo, is the most popular STB model for placental research [2]. Experiments using trophoblast cell lines have some limitations, since they merely fuse spontaneously [3], and their gene expression profile correlates weakly with that of CTBs [4,5,6]. A primary culture STB model has been used to overcome these limitations. In contrast to trophoblast cell lines, pCTBs reportedly differentiate into STB spontaneously (i.e., fuse to form syncytia and produce STB-specific proteins and hormones such as human chorionic gonadotropin β subunit (hCG-β) and placental lactogen), and they have been suggested to be a good model for STB [7, 10, 11]. There has been an urgent need to improve pCTB adhesion and differentiation in vitro
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