Embryonic Stem Cell Pluripotency and Early Differentiation

干细胞是发育和再生的基本单位，具有几乎无限的自我更新和分化能力，但最大的发育能力仅限于从植入前胚胎的内细胞团中分离出来的胚胎干细胞(Wobus&Boheler，2005)。在这个研究项目中，我们扩展了传统的基因组分析，以确定可能与ES细胞限制性基因启动子调控有关的顺式元件。我们采用的策略依赖于从基因表达序列分析(SAGE)图谱中生成问题特定列表(PSL)，以及随后的启动子分析，以确定PSL中基因之间在空间和方向上保守的多个顺式元件的框架。随后的实验数据表明，从这些模型中预测的两个转录因子B-Myb和Maz要么参与维持未分化的干细胞状态，要么参与分化的早期步骤。自那以后，我们一直专注于B-Myb的作用。 重要的是，转录因子B-Myb存在于所有增殖细胞中，在被设计移除该基因的小鼠中，胚胎在植入后不久就会由于内细胞团缺陷而在子宫中死亡。这种致命的表型通常被归因于G1期细胞周期中的增殖缺陷。我们最近的研究表明，缺乏B-Myb的小鼠胚胎干细胞(MES)的主要细胞周期缺陷发生在细胞周期的G2/M期。具体地说，短发夹状RNA敲除B-Myb会导致G2/M转运延迟，严重的有丝分裂纺锤体和中心体缺陷，以及多倍体。此外，许多暂时缺乏B-Myb的整倍体MES细胞变成非整倍体，不再被认为是活的。B-Myb在MES细胞中的敲除也降低了Oct4的RNA和蛋白质丰度，而B-Myb的过度表达略微上调了Pou5f1基因的表达。B-Myb和Oct4表达的协同变化至少部分归因于B-Myb在体外直接调节Pou5f1基因启动子活性的能力。最终，B-Myb的丢失和与Oct4相关的丢失导致在激活caspase介导的程序性细胞死亡之前早期分化标志物的增加。 这些发现使我们得出结论，适当的B-Myb表达对维持染色体稳定和多能性的ES细胞至关重要，而它的缺失会促进染色体的不稳定，从而导致非整倍体或与分化相关的细胞死亡。

Stem cells represent basic units of development and regeneration characterized by nearly unlimited self-renewal and differentiation capacities, but the greatest developmental capacity is restricted to embryonic stem (ES) cells isolated from the inner cell mass of pre-implantation embryos(Wobus & Boheler, 2005). In this research project, we have extended traditional genomic analyses to identify cis-elements that might be implicated in the control of ES cell-restricted gene promoters. The strategy that we employed relied on the generation of a Problem Specific List (PSL) from Serial Analysis of Gene Expression (SAGE) profiles, and subsequent promoter analyses to identify frameworks of multiple cis-elements conserved in space and orientation among genes from the PSL. Subsequent experimental data suggested that two transcription factors, B-Myb and Maz, predicted from these models are implicated either in the maintenance of the undifferentiated stem cell state or in early steps of differentiation. We have since focused on the role of B-Myb. Importantly, the transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Our research has recently shown that the major cell cycle defect in murine embryonic stem (mES) cells lacking B-Myb occurs in G2/M phase of the cell cycle. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. These findings lead us to conclude that appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, while its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.