An optimized embryonic stem cell model for consistent gene expression and developmental studies - A fundamental study
An optimized embryonic stem cell model for consistent gene expression and developmental studies - A fundamental study
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DOI：
10.1160/th05-05-0338
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发表时间：
2005-10-01
影响因子：
6.7
通讯作者：
Sachinidis, A
中科院分区：
文献类型：
--
作者：
Gissel, C;Voolstra, C;Sachinidis, A
胚胎干细胞（ES）的体外分化导致组织特异性体细胞的产生，并且可能代表对体内细胞分化和发育的一般理解的有力工具。大多数ES细胞系的培养需要小鼠胚胎成纤维细胞（MEF），这可能会影响ES细胞的遗传分化程序。我们比较了在MEF非依赖性CGR 8 ES细胞系和MEF依赖性D3 ES细胞系中关键发育基因的表达谱。使用新霉素抗性MEFs，我们证明了即使在去除MEFs后，MEFs也能够污染D3 ES细胞。随后，建立了CGR 8 ES细胞向各种胚层细胞分化的最佳分化条件。通过RT-PCR分析和微阵列分析进行了分化中的CGR 8细胞中的详细基因表达研究，表明与未分化的ES细胞相比，所评估的基因在3天或10天的胚状体（EB）中表达的一般趋势。定义了各种功能基因类别内的亚组，其特异性地一致上调或下调。有趣的是，目前的结果表明，向胚层形成的发育过程是不可逆的，大多数独立的培养条件。值得注意的是，凋亡和线粒体核糖体基因分别下调和上调10日龄的EB，而与3日龄的EB相比，而细胞外信号调节激酶（ERK）1/2的活性随着进行性发展而下降。本文为ES细胞分化和基因表达研究提供了一个平台。
In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and development in vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEF-dependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RT-PCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were down- and up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK) 1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.