Culture and preparation of human embryonic stem cells for proteomics-based applications.

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).