Embryonic Stem Cell Pluripotency and Early Differentiation

干细胞代表发育和再生的基本单位，其特征在于几乎无限的自我更新和分化能力，但最大的发育能力限于从植入前胚胎的内细胞团分离的胚胎干（ES）细胞（Wobus & Boheler，2005）。在这个研究项目中，我们已经扩展了传统的基因组分析，以确定顺式元件，可能涉及控制ES细胞限制性基因启动子。我们采用的策略依赖于从基因表达系列分析（SAGE）谱生成问题特异性列表（PSL），以及随后的启动子分析，以鉴定来自PSL的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，从这些模型预测的两个转录因子，B-Myb和Maz，涉及未分化干细胞状态的维持或分化的早期步骤。从那以后，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，并且在经工程改造以去除该基因的小鼠中，胚胎在植入后由于内细胞质量缺陷而在子宫内死亡。这种致死表型通常归因于G1期细胞周期阶段的增殖缺陷。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。具体而言，通过短发夹RNA敲低B-Myb导致通过G2/M的延迟转运、严重的有丝分裂纺锤体和中心体缺陷以及多倍性。此外，许多瞬时缺乏B-Myb的整倍体mES细胞变成非整倍体，并且不再被认为是存活的。在mES细胞中敲低B-Myb也降低了Oct 4 RNA和蛋白质丰度，而B-MYB的过度表达适度上调pou 5 f1基因表达。B-Myb和Oct 4表达的协调变化至少部分是由于B-Myb在体外直接调节pou 5 f1基因启动子活性的能力。最终，B-Myb的丢失和相关的Oct 4的丢失导致在激活半胱天冬酶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表达是至关重要的染色体稳定和多能性ES细胞的维护，而它的缺乏促进染色体不稳定，导致非整倍体或分化相关的细胞死亡。基于最近尚未发表的ChIP芯片分析，我们现在预测B-MYB涉及胚胎干细胞所有三个独特性状的调节：多能性，短细胞周期和以平衡基因为特征的表观遗传状态。 由于NIH iPS计划的NIH资助，我们现在能够扩大该项目以检查人类iPS细胞的细胞表面标记物。作为对照，我们最初提出并开始检查人ES细胞作为对照，但由于NIH指令，在排除hES细胞的情况下，已经包括了额外的iPS细胞系。该计划的基本原理和目的如下：体外产生的人诱导多能干细胞（iPSC）系是异质的，并表现出各种各样的分化和治疗潜力。这些性状类似于造血干细胞（HSC）亚群，其在再增殖和分化潜力方面不同。然而，功能性造血重建可以通过使用谱系标记来预测。如果与HSC一样，人iPSC（hiPSC）治疗潜力是确定性的而不是随机的，那么合乎逻辑的是假设新的细胞表面蛋白质组将充当在效力和分化潜力方面不同的重编程细胞的独特群体的替代物。为了开始鉴定这些谱系标志物和标志物组的过程，我们将使用最先进的生物化学方法和质谱法标记、捕获和鉴定细胞表面糖蛋白。细胞表面蛋白质组的分析将包括至少3至5个人iPSC（皮肤成纤维细胞和脂肪衍生）系。将数据与专有的细胞表面蛋白质图谱进行比较，以鉴定其表达仅限于多能细胞的蛋白质。通过流式细胞术，我们将测试单个抗体，然后测试一组抗体，以确定它们从诱导分化的ES和iPS细胞中可靠地鉴定和分离多能细胞亚群的能力。然后测试所选抗体组分离用附加型构建体在体外产生的真正重编程体细胞的能力。我们的实验室已经使用小鼠ESC（R1和D3）和iPSC（TTF 1和2D 4）建立了主要研究证据，其中我们通过流式细胞术成功鉴定了新的细胞表面差异（定量和定性）。由于维持多能性、特化和分化的核心分子程序在小鼠和人之间很好地保守，我们提出的关于人细胞（ES和iPS）的工作的结果与我们完成的小鼠数据集结合将促进患者特异性iPSC和亚群的分离和表征，无论年龄或性别如何，具有更广泛的分化和增强的治疗潜力。

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. Based on recent ChIP-chip analyses which are not yet published, we now predict that B-MYB is implicated in the regulation of all three unique traits of embryonic stem cells: pluripotency, a short cell cycle, and an epigenetic state characterized by poised genes. Because of NIH funding from the NIH iPS initiative, we have now been able to expand this project to examine cell surface markers of human iPS cells. As a control, we originally proposed and began examining human ES cells as a control, but due to NIH directives, additional iPS cell lines have been included at the exclusion of hES cells. The rationale and aims of this program are as follows: Human induced pluripotent stem cell (iPSC) lines generated in vitro are heterogeneous and exhibit a wide variety of differentiation and therapeutic potentials. These traits are analogous to hematopoietic stem cell (HSC) sub-populations that differ in their repopulating and differentiation potentials. Functional hematopoietic reconstitution, however, can be predicted through the use of lineage markers. If, as with HSCs, human iPSC (hiPSC) therapeutic potential is deterministic and not stochastic, then it is logical to assume that novel cell surface protein panels will serve as surrogates for unique populations of reprogrammed cells that differ in potency and differentiation potential. To begin the process of identifying these lineage markers and marker panels, we will label, capture and identify cell surface glycoproteins using state of the art biochemical methods and mass spectrometry. Analyses of the cell surface proteome will include at least 3 to 5 human iPSC (skin fibroblast- and fat-derived) lines. Data will be compared to a proprietary Cell Surface Protein Atlas to identify proteins whose expression is restricted to pluripotent cells. Using flow cytometry through a reiterative process, we will test individual antibodies and then panels of antibodies for their ability to reliably identify and isolate subpopulations of pluripotent cells from ES and iPS cells induced to differentiate. Selected antibody panels will then be tested for their ability to isolate bona fide reprogrammed somatic cells generated in vitro with episomal constructs. Proof-of-principal studies have already been established in our lab using mouse ESCs (R1 and D3) and iPSCs (TTF1 and 2D4), where we successfully identified novel cell surface differences (quantitative and qualitative) by flow cytometry. Since the core molecular program underlying maintenance of pluripotency, specification and differentiation is well conserved between mouse and human, the results from our proposed work on human cells (ES and iPS) coupled with our completed mouse dataset will promote the isolation and characterization of patient specific iPSCs and subpopulations, regardless of age or gender, with a broader differentiation and enhanced therapeutic potential.