Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells

Tatyana A. Prokhorova, Kristoffer T. G. Rigbolt, Pia T. Johansen, Jeanette Henningsen, Irina Kratchmarova, Moustapha Kassem, Blagoy Blagoev
2009 Molecular & Cellular Proteomics  
Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome
more » ... us that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations. Molecular & Cellular Proteomics 8:959 -970, 2009. Human embryonic stem cells (hESCs) 1 are stem cells derived from the blastocyst inner cell mass. They are pluripotent; thus they are able to differentiate into any human cell type. The self-renewal capacity and pluripotency make hESCs an ideal system to study the processes of cell development and differentiation. Moreover hESC research is highly relevant for regenerative medicine, which aims at replacing or restoring tissue damaged by disease or injury through transplantation of functional hESCs (1, 2). However, factors responsible for maintaining the undifferentiated and pluripotent nature of hESCs are still largely unknown. Before hESCs can be used for transplantation into the human body, reliable and reproducible protocols for differentiating them into specific cell types are needed. To create such protocols we need to develop a thorough understanding of the mechanisms maintaining the undifferentiated pluripotent nature of hESCs and those guiding their differentiation into specific lineages. A number of factors involved in the maintenance of pluripotency have been described over the last few years (3). It has also been demonstrated that overexpression of some of these factors in somatic cells is sufficient to turn them into pluripotent stem cells very similar to hESCs (4 -8). However, it is apparent that the processes occurring during such transformation are extremely complex. A large number of factors and pathways are involved in maintaining the pluripotent state and regulating self-renewal and differentiation. The process of specific hESC differentiation into distinct cell types is even less understood. Most current attempts to directionally differentiate hESCs are based on sequential application of empirically selected growth factors and consequent selection for markers expressed in the target cell types (9). A more sys-
doi:10.1074/mcp.m800287-mcp200 pmid:19151416 pmcid:PMC2689770 fatcat:2qj7pkrgibbphjxlmzdqgca2uu