Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells: Proteomics of Embryonic Stem Cell Differentiation

This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/stem.2067 ; During mammalian pre-implantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes. ; This work was supported by the European Union 7th Framework Program (PRIME-XS project grant number 262067 to K.S.L., L.G and C.M.M), the Biotechnology and Biological Sciences Research Council (BBSRC grant number BB/L002817/1 to K.S.L and L.G.), as well as a HFSP grant (RGP0029/2010) and a European Research Council (ERC) Advanced Investigator grant to A.M.A. C.S was supported by an EMBO long term fellowship and a Marie Curie IEF. L.T.Y.C. and K.K.N. were supported by the Medical Research Council (MRC, UK, MC_UP_1202/9) and the March of Dimes Foundation (FY11-436). We also thank Professor Steve Oliver and Dr. A.K.Hadjantonakis for helpful discussions and advice