title: The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.
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.
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Dongxia
authors.initials: D
authors.lastname: Hou
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Xiawei
authors.initials: X
authors.lastname: Li
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Li
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Ting
authors.initials: T
authors.lastname: Yun
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Yuhang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.|Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China.
authors.forename: Manling
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.|Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhao
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.|Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China.
authors.forename: Rongfeng
authors.initials: R
authors.lastname: Li
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Haiquan
authors.initials: H
authors.lastname: Yu
authors.affiliation: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, P. R. China.
authors.forename: Xueling
authors.initials: X
authors.lastname: Li
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression / Q000187:drug effects; D010312:Parthenogenesis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D034421:Sus scrofa; D014327:Trophoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000577:Amides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases