Embryonic Stem Cell Pluripotency and Early Differentiation

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Stem cells represent obvious choices for regenerative medicine and are invaluable for studies of human development and toxicology. The proteomic landscape of pluripotent stem cells (PSCs), in particular, is not yet clearly defined; consequently, this field of research would greatly benefit from concerted efforts designed to better characterize these cell populations. Recently, we have begun to develop an overview of stem cell potency, highlight the types and practical implications of heterogeneity in PSCs and provide a detailed analysis of the current view of the pluripotent proteome in a unique and rapidly evolving resource. Our goal has been to provide specific insights into the current status of the known proteome of both mouse and human PSCs. This has been accomplished by integrating published data into a unified PSC proteome to facilitate the identification of proteins which may be informative for the stem cell state as well as to reveal areas where our current view is limited. These analyses provide insight into the challenges faced in the proteomic analysis of PSCs and reveal one area the cell surface subproteome that would especially benefit from enhanced research efforts. More recently, we have begun a targeted identification of plasma membrane N-glycoprotein extracellular domains by Cell Surface Capturing Technology revealed 500 cell surface accessible proteins on mouse embryonic and induced pluripotent stem cells, including 187 not previously reported in mouse pluripotent stem cells. This new resource includes 91 CD molecules and represents a functional surface molecule "barcode" for the pluripotent state. Emerging from this "barcode" are informative markers for the pluripotent state that can be used for sorting live subpopulations of PSCs, including isolating iPSCs from heterogeneous mixture of reprogramming cells. The results from this targeted strategy represent an important stem cell resource that will accelerate the development of surface protein markers and affinity reagents for immunophenotyping and isolating pluripotent stem cells. These data from pluripotent mouse and human ESCs have been recently published. Finally, we have extended our previous work on B-MYB and its role in undifferentiated stem cells. We have found that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain self-renewal of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells, and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we have unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases as well as pluripotency transcription factors, epigenetic regulators, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. Meanwhile B-MYB together with OCT4, SOX2, and NANOG co-target genes significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB actively up-regulates factors critical to self-renewal, including key regulatory proteins important for cell cycle progression and fate decisions. These data were also recently published.