The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells
The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells
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DOI：
10.1007/s10616-016-9994-x
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发表时间：
2016-07
期刊：
影响因子：
2.2
通讯作者：
Xiawei Li;Zhiying Li;Dongxia Hou;Yuhang Zhao;Chen Wang;Xueling Li
中科院分区：
文献类型：
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作者：
Xiawei Li;Zhiying Li;Dongxia Hou;Yuhang Zhao;Chen Wang;Xueling Li
体外培养子宫内膜上皮细胞是研究胚胎着床和滋养层分化的重要工具。本研究建立了牛内皮细胞与滋养层干细胞共培养体系，并利用该体系研究了有蹄类动物双核细胞的形成。采用Ⅰ型胶原酶和Ⅰ型脱氧核糖核酸酶从黄体同侧的子宫角分离培养的EECs，具有典型的上皮细胞形态，表达牛子宫上皮细胞标志物IFNAR 1、IFNAR 2、Erα、PGR、ESR 1和KRT 18。KRT 18阳性细胞达99%。EECs在体外培养20代后，其形态学和子宫上皮标志基因表达无明显变化。bTS细胞在双重抑制剂系统中建立，并表现出典型的滋养层干细胞特征。当bTS细胞与内皮细胞一起培养时，bTS细胞粘附于内皮细胞，如同粘附于饲养细胞一样。双核细胞在共培养的第4天开始出现，并达到约18.47%的分化细胞。在第6天和第12天对共培养的bTS细胞进行定量实时PCR或免疫荧光分析。结果显示，在bTS细胞中，KRT 18的表达水平下调，而滋养细胞分化标志物MASH 2、HAND 1、GCM 1和CDX 2的表达水平上调。结论：采用胶原酶I和脱氧核糖核酸酶I联合诱导，可从黄体同侧子宫角获得牛内皮细胞，建立牛内皮细胞-bTS细胞共培养体系是研究bTS细胞向滋养层双核细胞分化的理想工具。
Endometrial epithelial cells (EECs) cultured in vitro are valuable tools for investigating embryo implantation and trophoblast differentiation. In this study, we have established the bovine EECs and trophoblast stem-like (TS) coculture system, and used it to investigate the binucleate cell formation of ungulates. The EECs was derived from the uterine horn ipsilateral to the corpus luteum by using collagenase I and deoxyribonuclease I, which exhibited typical epithelial morphology and were expressing bovine uterine epithelial marker such asIFNAR1,IFNAR2,Erα,PGR,ESR1andKRT18.The cells immunostained positively by epithelial and trophectoderm marker cytokeratin 18 (KRT18) and stromal marker vimentin antibodies, and the KRT18 positive cells reached 99 %. The EECs can be cultured for up to 20 passages in vitro with no significant morphology changes and uterine epithelial marker gene expression alteration. The bTS cells were established in a dual inhibitor system and exhibited typical trophoblast stem cell characteristics. When bTS cells were cultured with EECs, the bTS cells adhered to the EECs as adhering to feeder cells. Binucleate cells began appearing on day 4 of coculture and reached approximately 18.47 % of the differentiated cells. Quantitative real-time PCR or immunofluorescence analyses were performed on bTS cells cocultured at day 6 and day 12. The results showed that the expression level ofKRT18was down-regulated while the expression level of trophoblast differentiation markerMASH2,HAND1,GCM1andCDX2was up-regulated in bTS cells. In conclusion, bovine EECs can be obtained from the uterine horn ipsilateral to the corpus luteum via treatment with collagenase I and deoxyribonuclease I, and the EECs-bTS cells coculture system presents an ideal tool for studying the differentiation of bTS cells to trophoblast binucleate cells.