Skip to Content
Merck
  • The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.

The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.

PloS one (2015-11-12)
Dongxia Hou, Min Su, Xiawei Li, Zhiying Li, Ting Yun, Yuhang Zhao, Manling Zhang, Lihua Zhao, Rongfeng Li, Haiquan Yu, Xueling Li
ABSTRACT

Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetic acid, glacial, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.8-100.5%
Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Water, Deionized
Sigma-Aldrich
Methanol, BioReagent, ≥99.93%
Sigma-Aldrich
Acetic acid solution, suitable for HPLC
Sigma-Aldrich
Water, ACS reagent
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Acetic acid, glacial, ≥99.99% trace metals basis
Sigma-Aldrich
Water, BioPerformance Certified
Sigma-Aldrich
Sodium chloride, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Sodium chloride solution, 0.85%
Supelco
D-(+)-Glucose, analytical standard
Sigma-Aldrich
D-Glucose-12C6, 16O6, 99.9 atom % 16O, 99.9 atom % 12C
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Streptomycin sulfate salt, powder
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
E-Toxate Water, endotoxin, free
Sigma-Aldrich
Water, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Sodium pyruvate, ReagentPlus®, ≥99%
Sigma-Aldrich
Water, PCR Reagent
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Sodium pyruvate, BioXtra, ≥99%
Sigma-Aldrich
Sodium pyruvate, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99%
Sigma-Aldrich
D-(+)-Glucose, suitable for mouse embryo cell culture, ≥99.5% (GC)
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC), BioXtra
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)