Stable isotope labeling by SILAC-based quantitative proteomics analysis provides an unprecedented tool for the study of mechanisms underlying the self-renewal and differentiation of human embryonic stem cells (hESCs). While we recently reported a chemically defined SILAC culture system specific for a rare cell proteomic reactor (R. Tian et al., Mol. Cell. Proteomics 2011, 10, M110.000679), total hESC yield, prolonged self-renewal capacity (i.e.<12 days), and laborious procedure remain substantial hurdles for its conventional application in hESC studies. Here, we devised an enhanced SILAC culture system consisting of a new chemically defined SILAC-medium and a novel culture protocol. As a result, with much less culture maneuvers, approximately 40-fold greater hESCs were produced than the system reported previously. Moreover, the enhanced SILAC culture system was sufficient to support the self-renewal of hESCs for >60 days and was also highly reproducible. As such, it provides a new platform that can be readily adapted by general laboratory for further comprehensive SILAC-based proteomics analysis of hESCs and induced pluripotent stem cells.
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