Background: Self-renewal and pluripotency maintenance ofembryonic stem cell (ESC) could be controlled by the impact ofculture condition in cell molecular mechanism. Understandingthis mechanism is important for translating stem cell technologiesto clinical applications. In this study, we aim to evaluatethe proteome of mouse ESC-cultured underground state conditionscontain ۲i and
R۲i (Royan ۲ inhibitors of MEK and TGFβsignaling pathways) in comparison with serum.Materials and Methods: By the shotgun proteomics approach,we investigated the proteome of cells grown under ۲i, R۲i, andserum culture conditions. According to differentially expressedproteins, we choice loss of function approach to evaluating therelated signaling pathway. The findings were validated by cellcycle analysis and gene expressions of the cells with flow cytometryand qRT-PCR, respectively.Results: Out of ۱۷۴۹ proteins identified, ANOVA analysis revealed۱۷۱ differentially expressed proteins (P<۰.۰۵) in ۲i, R۲iand serum. Among them, ۱۲۰ proteins were significantly upregulatedand ۵۱ proteins were significantly down-regulated between۲i and
R۲i versus serum. Gene ontology (GO) analysis ofdifferentially abundant proteins showed that highest enrichmentpathways in ۲i- and R۲i-cultured cells were associated with themetabolic process, glycolysis, gluconeogenesis and amino acidbiosynthesis. Glycolysis was highlighted for energy productionand used to maintain high levels of glycolytic intermediates tosupport cell proliferation which correlated with rapid cell cyclingin ۲i and R۲i-grown cells. Focal adhesion, integrin signaling,and RNA transportation signaling pathways significantlydown-regulated under ground state conditions. In terms of focaladhesion signaling, we confirmed the shotgun proteomics datafor the integrins family by qRT-PCR, which showed reducedexpression of the integrin subunits in ۲i and
R۲i conditions.Integrins activation by Mn۲+ in ۲i and
R۲i cultures resulted inreduced Nanog level and increased the expression of lineagemarker genes. The serum culture had more prominent phosphorylationof focal adhesion kinase (FAK) compared to ۲i andR۲i cultures which were related to increasing of focal adhesionsignaling pathway. Therefore, reduced focal adhesion enabledmESCs to be maintained in an undifferentiated state.Conclusion: Our results provided an insight into the key proteinpathways, which are involved in self-renewal, and pluripotencymaintenance employed by ESCs in ground state conditionsdifferent from that of serum culture.