Murphy Christopher L, Polak Julia M
Tissue Engineering Center, Imperial College School of Medicine, Chelsea and Westminster Campus, London, United Kingdom.
Tissue Eng. 2002 Aug;8(4):551-9. doi: 10.1089/107632702760240472.
Embryonic stem (ES) cells are pluripotent cell lines that possess virtually unlimited self-renewal and differentiation capacity. Such characteristics make them potentially an invaluable cell source for diverse tissue-engineering applications. In vitro ES cell differentiation occurs spontaneously in three-dimensional structures termed "embryoid bodies" that mimic postimplantation embryonic tissue. HPRT, beta-tubulin, and GAPDH are commonly used as internal RNA standards in ES cell-derived gene transcription studies so that corrected sample mRNA levels can be obtained for (semi) quantitative gene expression data. However, if reliable data is to be obtained, it is essential that such housekeeping gene expression remains constant, and this has not been demonstrated for differentiating ES cell cultures, which represent a mixed and changing population of cells with time in culture. Therefore, in the present study, we tested the suitability of these housekeeping genes to act as true internal standards for differentiating murine ES cells cultured as embryoid bodies. PCR-amplified gene-specific products were quantified from digital images of ethidium bromide-stained gels using a computer software package. Both HPRT and beta-tubulin mRNA levels varied markedly in spontaneously differentiating and growth factor-supplemented (TGF-beta) ES cell cultures (p < 0.001, ANOVA), while GAPDH expression remained relatively constant (p > 0.2). Our results demonstrate the importance of fully validating housekeeping gene expression in in vitro ES cell gene transcription studies and suggest that GAPDH may be a suitable candidate to act as an internal RNA standard, while both HPRT and beta-tubulin appear to be inappropriate. Finally, we demonstrate enhanced mesodermal differentiation of ES cell-derived cultures by treatment with TGF-beta through significant upregulation of Brachyury T expression, with a concomitant decrease in expression of the undifferentiated ES cell marker Oct-4.
胚胎干细胞（ES细胞）是具有几乎无限自我更新和分化能力的多能细胞系。这些特性使它们有可能成为各种组织工程应用中非常宝贵的细胞来源。体外ES细胞分化在称为“胚状体”的三维结构中自发发生，这些结构模拟植入后胚胎组织。次黄嘌呤磷酸核糖转移酶（HPRT）、β-微管蛋白和甘油醛-3-磷酸脱氢酶（GAPDH）通常用作ES细胞衍生基因转录研究中的内部RNA标准，以便能够获得校正后的样本mRNA水平用于（半）定量基因表达数据。然而，如果要获得可靠的数据，至关重要的是这种管家基因的表达保持恒定，而对于分化的ES细胞培养物尚未证明这一点，分化的ES细胞培养物在培养过程中代表着一个随时间混合且不断变化的细胞群体。因此，在本研究中，我们测试了这些管家基因作为真正内部标准用于分化为胚状体培养的小鼠ES细胞的适用性。使用计算机软件包从溴化乙锭染色凝胶的数字图像中对PCR扩增的基因特异性产物进行定量。在自发分化和添加生长因子（转化生长因子-β，TGF-β）的ES细胞培养物中，HPRT和β-微管蛋白的mRNA水平均有显著变化（p<0.001，方差分析），而GAPDH的表达保持相对恒定（p>0.2）。我们的结果证明了在体外ES细胞基因转录研究中充分验证管家基因表达的重要性，并表明GAPDH可能是用作内部RNA标准的合适候选者，而HPRT和β-微管蛋白似乎都不合适。最后，我们证明通过用TGF-β处理，ES细胞衍生培养物的中胚层分化增强，这是通过短尾相关转录因子（Brachyury T）表达的显著上调以及未分化ES细胞标志物八聚体结合转录因子4（Oct-4）表达的相应降低实现的。
