Surface Proteomic Signature Of Murine Embryonic Stem Cells

Embryonic stem (ES) cells are pluripotent stem cells derived from preimplantational embryos. It possesses two basic properties:Self-renewal and pluripotency. Because of these two properties, ES cells hold great potentials in basic studies in developmental biology and regenerative medicine. The mechanism of self-renewal is a basic question in ES cell biology. Complicated extracellular signals and intracellular molecular networks determine the self-renewal of ES cells. Cell surface proteins largely link the extracellular signals and intracellular molecular networks. They are critical for the self-renewal of ES cells and are also critical tools for quality control of ES cells. Therefore, it is critical to systematically explore the surface proteome for the dissection of self-renewal mechanisms and discovery of new surface markers of mES cells.We labeled the surface proteins of mES cells with the membrane impermeable EZLink-Sulfo-NHS-SS-Biotin and purified the surface proteins with streptavidin conjugated Latex beads. The proteins were then digested in gel and analyzed by LC-MS/MS.3468 proteins, of which 1699 were membrane proteins, were identified from the study. To our knowledge, it's the largest dataset concerning mES cell surface proteins till now. The surface proteome. of mES cells are complicated, composed of 513 types of signal molecules,295 types of transporters, and 122 types of adhesion and anchorage proteins, etc. Moreover some protein pairs that could form paracrine/autocrine pathways like Wnts/Wnt receptors, IGFs/IGF receptors and Semaphorin/Semaphorin receptors were identified. These results indicated that mES cells relied on complicated signal network and material transport system to maintain self-renewal.Using immunocytochemistry and flow cytometry analysis, we demonstrated that mES cells heterogeneously expressed five'selected surface proteins, Tie-1,GM-CSFRa,EGFR,BMPR2 and CD4. The expression frequency varied from 10% to 30%, and the expression level are highly varied too. We also demonstrated that the heterogeneous expression of the five proteins is consisted between different mES cell lines and single cell cloned sublines and the expression frequency is relatively stable. It indicated that mES cells are divided into subpopulations by surface proteins and different subpopulations may have different proliferation and differentiation potential. mES cells are highly corporative populations. The interactions between subpopulations may be critical for self-renewal maintenance of mES cells.We also found that a large variety of differentiation-associated proteins like CD4, CD34, IL1R11, NCAM-L1, PAI-3 and Tie-1 were expressed in undifferentiated mES cells and demonstrated that mES cells globally expressed differentiation-associated surface genes on protein level. It set up the basis for studying the relationship between global expression and self-renewal of ES cells.The global expression of differentiation associated surface proteins highly resembled the promiscuous expression of tissue specific genes in medullary thymic epithelia cells(mTEC). The promiscuous expression in mTEC cells is largely controlled by Aire gene. We demonstrated that Aire was expressed in mES cells and the expression decreased with differentiation. Moreover, we demonstrated that Aire regulated the expression of some differentiation associated surface proteins in mES cells and affected the clonogenicity and the expression of Oct4 and Nanog genes. It set up a new direction of studies of the mechanism that regulate global gene expression in ES cells and maintain the self-renewal of ES cells.In conclusion, we generated the largest dataset of surface proteins of mES cells and declared the complicated surface proteome of mES cells. We demonstrated that mES cells heterogeneously expressed various surface proteins and the expression frequency is a stable property of mES cells. We for the first time demonstrated that mES cells expressed a large variety of differentiation associated surface proteins. We preliminarily demonstrated that Aire is a mechanism that regulate the global gene expression in mES cells and are functional related to self-renewal. It indicated functional relationships between global expression and self-renewal of mES cells.