Embryonic Stem Cell Pluripotency and Early Differentiation

干细胞代表了再生医学的明显选择，对人类发育和毒理学研究非常宝贵。特别是多能干细胞（PSC）的蛋白质组学景观尚未明确定义;因此，这一研究领域将大大受益于旨在更好地表征这些细胞群的协同努力。最近，我们已经开始开发干细胞潜能的概述，突出PSC异质性的类型和实际意义，并提供了一个独特的和快速发展的资源中的多能蛋白质组的当前观点的详细分析。我们的目标一直是提供具体的见解，目前的状态，已知的蛋白质组的小鼠和人类PSC。这是通过将已发表的数据整合到统一的PSC蛋白质组中来实现的，以便于识别可能为干细胞状态提供信息的蛋白质，并揭示我们目前观点有限的领域。这些分析提供了对PSC蛋白质组学分析所面临的挑战的深入了解，并揭示了一个领域，即细胞表面亚蛋白质组，这将特别受益于加强研究工作。 最近，我们已经开始通过细胞表面捕获技术对质膜N-糖蛋白胞外结构域进行靶向鉴定，发现小鼠胚胎和诱导多能干细胞上有500种细胞表面可及蛋白，其中包括187种以前未在小鼠多能干细胞中报道的蛋白。这个新的资源包括91个CD分子，代表了多能状态的功能性表面分子“条形码”。从这种“条形码”中出现的是多能状态的信息标记物，其可用于分选PSC的活亚群，包括从重编程细胞的异质混合物中分离iPSC。这种靶向策略的结果代表了一种重要的干细胞资源，将加速用于免疫分型和分离多能干细胞的表面蛋白标记物和亲和试剂的开发。 最后，我们扩展了我们以前对B-MYB及其在未分化干细胞中的作用的研究。我们已经发现，B-MYB，一种细胞周期调节的磷蛋白和转录因子，对内细胞团的形成至关重要，是维持ESCs自我更新的转录和共调节网络的核心。从表型上看，B-MYB在胚胎干细胞和诱导多能干细胞中稳健表达，并且主要以低磷酸化状态存在。B-MYB的敲低导致涉及S、G2和M期的细胞周期异常，以及关键细胞周期调节因子如ccnb 1和plk 1的表达减少。通过对对照和B-MYB缺陷细胞进行基因表达谱分析，ChIP芯片实验和综合计算分析，我们已经揭示了一个高度复杂的B-MYB介导的转录网络，指导ESC自我更新。该网络包括所有细胞周期阶段的关键调节因子以及多能性转录因子、表观遗传调节因子和分化决定因子。B-MYB沿着与E2 F1和c-MYC优先共调节细胞周期靶基因。同时，B-MYB与OCT 4、SOX 2和NANOG共靶基因一起与干细胞分化、胚胎发育和表观遗传控制显著相关。此外，B-MYB的缺失导致ESC中存在的转录层次结构的破坏。这些结果加上功能研究表明，B-MYB积极上调自我更新的关键因素，包括对细胞周期进展和命运决定重要的关键调控蛋白。

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. 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.