Proteomics profiling of human embryonic stem cells in the early differentiation stage.

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.