A Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, enhances adhesion, viability and differentiation of human term placenta-derived trophoblasts in vitro

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.

RNA-Seq identifies genes whose proteins are transformative in the differentiation of cytotrophoblast to syncytiotrophoblast, in human primary villous and BeWo trophoblasts

“…TWIST1 39 was not expressed in BeWo cells at any timepoint, regardless of forskolin treatment, yet was consistently expressed in human placental cells, but with no marked up- or down-regulation during syncytialization. ROCK1 and RAC1 40 were expressed at similar levels across all time points and treatments. Both LAT1 and 4F2hc 41 were upregulated 3–4 fold in forskolin-treated BeWo cells at 72 hours.…”

Epitranscriptomic profiling in human placenta: N6‐methyladenosine modification at the 5′‐untranslated region is related to fetal growth and preeclampsia

“…[51][52][53][54] It has been recently reported that one cellular response to hypoxia-ischemia is mRNA stabilization and promotion of translation through m 6 A modification of the transcription factors JUN and MYC, demonstrating that mRNA degradation is not the only effect of m 6 A. In the future, using techniques such as first-stage cell culture of patient-derived villi, 56 we intend to investigate details of the relationships between various stresses and m 6 A levels especially at the 5′-UTR to further clarify the significance of m 6 A in placental mRNAs. Additionally, JUN is among the list of 5′-UTR DMGs among these categories (Supplemental Data).…”

The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1

“…ROCK inhibition is known to facilitate the growth and survival of human embryonic stem cells by inhibiting anoikis. ROCK inhibitors are now part of standard stem cell culture protocols [ 51 , 52 ]. Our results show that Y27632 can also increase the in vitro viability of MDA-MB-231 spheroids ( Supplementary Figure 8 ).…”