King Charles C
Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2010;584:151-77. doi: 10.1007/978-1-60761-369-5_9.
New challenges will arise as research into human embryonic stem (hES) cell differentiation moves from optimization and overcoming technical hurdles to mechanistic considerations. An immediate need will be to culture hES cells in the absence of contaminating feeder layers and allow for the preparation of purified DNA, RNA, and proteins to analyze changes in microRNA levels, gene expression, protein expression, and signal transduction. Purified, uniform populations of hES cells will allow researchers to better explore the biochemical mechanisms by which differentiation occurs.Much recent work has focused upon genetic analysis of different stem cell populations. Expected variabilities between pluripotent hES cells, mesoderm, ectoderm, and definitive endoderm have been observed in microarray profiles (1-7). Interestingly, there also appears to be significant heterogeneity in mRNA expressed in different hES cell lines (8, 9). One approach to better understand how changes in mRNA levels in differentiating stem cells and individual hES cell lines relate to cell function is to study changes in signal transduction and global changes in protein expression. This chapter describes the methods routinely employed to prepare cells for analysis by traditional biochemistry (fractionation and western blotting) and proteomic analysis (2D electrophoresis/mass spectrometry and free-flow isoelectric focusing).
随着人类胚胎干细胞（hES）分化研究从优化和克服技术障碍转向机制性考量，新的挑战将会出现。当务之急是在不存在污染性饲养层的条件下培养hES细胞，并能够制备纯化的DNA、RNA和蛋白质，以分析微小RNA水平、基因表达、蛋白质表达及信号转导的变化。纯化、均一的hES细胞群体将使研究人员能够更好地探索分化发生的生化机制。近期的许多工作都集中在对不同干细胞群体的基因分析上。在微阵列图谱中已观察到多能hES细胞、中胚层、外胚层和定形内胚层之间预期的变异性（1 - 7）。有趣的是，不同hES细胞系中表达的mRNA似乎也存在显著的异质性（8, 9）。一种更好地理解分化干细胞和单个hES细胞系中mRNA水平变化如何与细胞功能相关的方法是研究信号转导的变化以及蛋白质表达的整体变化。本章描述了用于制备细胞以通过传统生物化学方法（分级分离和蛋白质印迹法）和蛋白质组学分析方法（二维电泳/质谱分析法和自由流等电聚焦法）进行分析的常规方法。
