4- Protein identification Comparative Proteomics Analysis of Mouse Embryonic Stem Cell under Different Culture Conditions Sara Taleahmad 1, Azam Samadian1, Sepideh Mollamohammadi 1, Seyedeh-Nafiseh Hassani 1, Hossein Baharvand 1, Ghasem Hosseini Salekdeh 1,2 1- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR,. 2- Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran Objectives Methods Self-renewal and pluripotency maintenance of embryonic stem cell (ESC) could be controlled by impact of culture condition in cell molecular mechanism. Understanding this mechanism is important for translating stem cell technologies to clinical applications. In this study, we aim to evaluate the proteome of mouse ESC-cultured under ground state conditions contain 2i and R2i (Royan 2 inhibitors of MEK and TGFβ signaling pathways) in comparison with serum. 1- Protein extraction from samples 2- Trypsin In-gel digestion 3- Nanoflow LC-tandem mass bRPLC GO analysis Molecular Function Biological process Cellular component KEGG Pathway S 2i 6- Western blot confirmation 5- Quantitative proteomics analysis 4- Protein identification MS-MS Data Results KEGG pathway analysis of up/down regulated proteins By the shotgun proteomics approach a total of 1749 proteins were identified in cells. 177 differentially expressed proteins (p<0.05). KEGG pathway analysis of these proteins showed that proteins enriched in ground state conditions mainly involved in metabolic process, glycolysis and amino acid biosynthesis. Spliceosome and integrin signaling pathway significantly enriched in R2i (1) vs. 2i., whereas, TGF-beta signaling pathway enriched in 2i and serum compared to R2i condition. Higher in 2i, R2i Higher in serum 4 8 12 KEGG pathway analysis 2i- and R2i-grown cells used the glycolysis pathway for the generation of energy and intermediate products (A), and underwent rapid cell cycling by the overexpression of cell division and cell proliferation associated proteins (B). Low focal adhesion signaling promotes pluripotency of mESCs We investigated the role of ECM-integrin interaction to inhibit the self-renewal phenotype in mESCs. qRT-PCR analysis showed that over expression of integrins by Mn2+ reduced the expression level of Nanog and increased the expression levels of early lineage marker genes (A). Furthermore, the proportion of ALP-positive colonies decreasedd ICC indicated that induction of integrin activity decreased the abundance of Nanog in both the 2i- and R2i-grown cells (B). A B Serum 2i R2i A B mRNA fold change of MnCl2 treated cells vs. control Nanog FAK Nanog / FAK 2i-Ctrl R2i-Ctrl DAPI R2i-MnCl2 2i-MnCl2 ALP mRNA fold change of MnCl2 treated cells vs. control Conclusion This study revealed that mESCs cultured under 2i and R2i conditions used the glycolysis pathway for the generation of energy and intermediate products (2). 2i- and R2i-grown cells underwent rapid cell cycling as a feature of pluripotency by the overexpression of cell division and cell proliferation associated proteins. The activation of integrins by Mn2+, induced cell spreading and reduced the expressions of pluripotency marker gene. The self-renewal of mESCs was affected by cell-substratum adhesion. Reference:  1- Hassani et al., 2014. 2014 Stem Cell Rev.10(1):16-30. 2-Taleahmad et al., 2018. Cell J., 20 (3), 388-395.