Differentiation of mouse embryonic stem cells into gonadotrope-like cells in vitro
Differentiation of mouse embryonic stem cells into gonadotrope-like cells in vitro
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DOI：
10.1016/j.jsgi.2005.01.004
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发表时间：
2005-05-01
期刊：
影响因子：
--
通讯作者：
Sasaki, K
中科院分区：
文献类型：
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作者：
Zhao, X;Teng, RF;Sasaki, K
目的：本研究旨在探讨小鼠胚胎干细胞（ES细胞）在体外向促性腺激素细胞（gonadotropics.METHODS）分化的潜能。通过5天的悬滴培养，ES细胞被诱导形成称为胚状体（EBs）的多分化结构。采用逆转录聚合酶链反应（RT-PCR）、Western blotting和免疫细胞化学等方法检测不同培养时间点EB细胞促性腺激素标志物的基因表达。ES细胞中表达的同源盒基因（Hesx 1）、LIM同源盒蛋白3（Lhx 3）、配对样同源域因子1（Prop 1）、加塔结合蛋白2（GATA 2）、促卵泡激素β在第6天EB检测FSH β和LH β mRNA，并在整个培养过程中维持至第56天。FSH β和LH β蛋白从第6天开始在EB中表达。FSH β和LH β的免疫荧光标记显示，特异性染色仅限于某些分化的EB细胞的细胞质。随着EB培养时间的延长，阳性细胞数明显增多。单激素细胞和双激素细胞都存在，主要是在EBs内的集群和周围的EBs.CONCLUSION：这些结果表明，小鼠ES细胞可以产生成熟的促性腺激素样细胞在EBs的最外层的细胞之间稀疏分布。这也表明，EBs可能作为一种新的模型系统，研究促性腺激素的发育和功能。
OBJECTIVE: This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes.METHODS: Undifferentiated ES cells were maintained on mitomycin C-inactivated fibroblasts in the presence of leukemia inhibitory factor (LIF). By a 5-day hanging drop culture devoid of them, ES cells were induced to form multidifferentiated structures called embryoid bodies (EBs). Reverse transcriptase-polymerase chain reaction, (RT-PCR), Western blotting, and immunocytochemistry were used to analyze gene expression of gonadotrope markers in EBs at different time points during the culture.RESULTS: Homeo box gene expressed in ES cells (Hesx1), LIM homeobox protein 3 (Lhx3), paired like homeodomain factor 1 (Prop1), GATA binding protein 2 (GATA2),follicle-stimulating hormone beta (FSH beta), and luteinizing hormone beta (LH beta) mRNAs were detected at day 6 EBs and maintained throughout the culture to day 56. FSH beta and LH beta proteins were expressed in EBs from day 6 onward. Immunofluorescent labeling of FSH beta and LH beta showed that specific staining was restricted to the cytoplasm of some differentiated EB cells. With the prolongation of EB culture, the number of positive cells increased significantly. Both monohormonal and bihormonal cells were present, mainly in clusters within EBs and sparsely distributed among the outermost cells surrounding the EBs.CONCLUSION: These results indicate that mouse ES cells can give rise to mature gonadotrope-like cells in EBs. It also shows that EBs may serve as a novel model system to study the development and function, of gonadotropes.