Sohnis and Forman Families Center for Stem Cell and Tissue Regeneration Research, Ruth & Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Stem Cell Rev Rep. 2012 Mar;8(1):137-49. doi: 10.1007/s12015-011-9286-y.
The regulatory pathways responsible for maintaining human embryonic stem cells (hESCs) in an undifferentiated state have yet to be elucidated. Since these pathways are thought to be governed by complex protein cues, deciphering the changes that occur in the proteomes of the ESCs during differentiation is important for understanding the expansion and differentiation processes involved. In this study, we present the first quantitative comparison of the hESC protein profile in the undifferentiated and early differentiated states. We used iTRAQ (isobaric tags for relative and absolute quantification) labeling combined with two dimensional capillary chromatography coupled with tandem mass spectrometry (μLC-MS/MS) to achieve comparative proteomics of hESCs at the undifferentiated stage, and at 6, 48, and 72 h after initiation of differentiation. In addition, two dimensional electrophoresis (2-DE) was performed on differentiating hESCs at eleven points of time during the first 72 h of differentiation. The results indicate that during the first 48 h of hESC differentiation, many processes are initiated and are later reversed, including chromatin remodeling, heterochromatin spreading, a decrease in transcription and translation, a decrease in glycolytic proteins and cytoskeleton remodeling, and a decrease in focal and cell adhesion. Only 72 h after differentiation induction did the expression of the homeobox prox1 protein increase, indicating the beginning of developmental processes.
维持人类胚胎干细胞（hESCs）未分化状态的调控途径尚未阐明。由于这些途径被认为是由复杂的蛋白质线索控制的，因此解析在分化过程中 ESCs 蛋白质组中发生的变化对于理解涉及的扩增和分化过程非常重要。在这项研究中，我们首次对未分化和早期分化状态下 hESC 蛋白质谱进行了定量比较。我们使用 iTRAQ（相对和绝对定量的同重同位素标记）标记结合二维毛细管色谱与串联质谱（μLC-MS/MS），在未分化阶段和分化开始后 6、48 和 72 h 时对 hESCs 进行比较蛋白质组学分析。此外，在分化的 hESCs 中，在分化的前 72 小时内的 11 个时间点进行二维电泳（2-DE）。结果表明，在 hESC 分化的前 48 h 内，许多过程被启动，随后被逆转，包括染色质重塑、异染色质扩散、转录和翻译减少、糖酵解蛋白和细胞骨架重塑减少以及焦点和细胞粘附减少。只有在分化诱导后 72 h，同源盒蛋白 prox1 的表达才增加，表明发育过程的开始。
