Self-Renewal and Differentiation: Molecular Events that Commit ES Cells to Exit t

项目2：自我更新和分化：使ES细胞退出细胞周期的分子事件 多能国家（James Thomson，PI） A.具体目标 在低浓度（4 ng/ml）或缺乏外源性bFGF的情况下，BMP 4诱导人ES细胞形成同质群体 滋养层，胎盘的外层。然而，我们最近发现，在高 (100 ng/ml）浓度的bFGF、BMP 4反而诱导人ES细胞形成细胞群， 不再表达滋养层标志物，而是瞬时表达短尾，中胚层或 中内胚层标志物，并随后表达内胚层和中胚层标志物的混合物。在这 项目，我们将研究BMP如何诱导人类ES细胞退出多能状态，并致力于 分化，并研究FGF如何介导这些不同的发育结果，以响应相同的 诱导剂。了解ES细胞如何退出多能状态以及为什么这种退出通常是不可逆的， 实现有效的重编程（项目3）和理解这些关键的早期谱系决定的核心 将允许更有效地区分特定的临床相关谱系。 我们将实现以下目标： 目标1。我们将建立一个详细的时间过程中的基因表达在人类胚胎干细胞的BMP 4- 诱导分化，无论是在存在（短尾阳性结果）和不存在（绒毛膜 促性腺激素阳性结果）的bFGF，并将这些变化与承诺退出 多能国家承诺将通过在连续更长的时间段内添加BMP 4来测量， 去除BMP 4，然后在几天后检查有多少细胞保留多能性标记。的 这一目标的假设是，数量承诺曲线将与 直接控制这些分化事件的基因的表达水平。我们将在接下来的时间里 与承诺最密切相关的转录因子，我们将下调基因， 在承诺期间下调的RNA干扰，以确定足以介导 向短尾畸形阳性群体分化。初步结果表明，GATA 2和GATA 3是 在缺乏bFGF的情况下，两者都足以介导滋养层分化，因此我们乐观地认为， 单个转录因子将足以介导人ES细胞分化为短尾畸形 在bFGF存在下的阳性群体。 目标2.我们将使用全基因组染色质免疫沉淀芯片（ChIP芯片）来定位 TGF β/激活素激活的Smad 2/3和BMP激活的Smad 1/5/8的基因组结合位点， 诱导分化这一目标的中心假设是，Smad 2/3直接激活表达 在人类胚胎干细胞中的关键多能性因子，直接抑制基因的表达， 否则促进分化，并在相同的启动子与Smad 1/5/8竞争，Smad 1/5/8具有 相反的效果。第二个假设是bFGF在细胞凋亡过程中会改变Smad 1/5/8的DNA结合位点。 BMP诱导的分化，导致观察到的不同发育结果。 目标3。我们将利用全基因组染色质免疫沉淀芯片（ChIP-chip）技术， BMP 4诱导分化过程中GATA 2和GATA 3的结合位点。这一目标的假设是 GATA 2和GATA 3直接负调控关键多能性基因的转录， 正调控自身的转录，因此一旦它们被Smads诱导，GAT A表达 成为自我维持和BMP-独立的，因此分化继续，即使在BMP去除。一 第二种假设是bFGF在BMP诱导的细胞凋亡过程中改变GATA 2/GATA 3的DNA结合位点。 分化，导致观察到的不同发育结果。 目标4。我们将鉴定出BMP 4诱导的磷酸化蛋白质与BMP-2诱导的磷酸化蛋白质之间的差异。 滋养层细胞分化（无bFGF）和BMP诱导的短尾阳性细胞分化（高 bFGF）来鉴定FGF信号传导的介体，所述介体引起这些趋化因子之间的转换。 发展成果。这一目标的假设是，有限数量的差异磷酸化 转录因子的表达与BMP后观察到的不同发育结果有偶然关系。 在存在或不存在bFGF的情况下诱导。差异化承诺曲线将再次成为 关注相关的磷酸化事件。

Project 2: Self-Renewal and Differentiation: Molecular events that commit ES cells to exit the pluripotent state. (James Thomson, PI) A. SPECIFIC AIMS In low (4 ng/ml) or absent exogenous bFGF, BMP4 induces human ES cells to form a homogenous population of trophoblast, the outer layer of the placenta. However, we have recently found that in the presence of high (100 ng/ml) concentrations of bFGF, BMP4 instead induces human ES cells to form a population of cells that no longer express trophoblast markers, but instead transiently express brachyury, a mesoderm or mesendoderm marker, and subsequently express a mixture of endoderm and mesoderm markers. In this project, we will study how BMPs induce human ES cells to exit the pluripotent state and commit to differentiation, and study how FGF mediates these divergent developmental outcomes in response to the same inducer. Understanding how ES cells exit the pluripotent state and why this exit is generally irreversible is central to achieving efficient reprogramming (Project 3), and understanding these key early lineage decisions will allow more efficient differentiation to specific clinically-relevant lineages. We will accomplish the following aims: Aim 1. We will establish a detailed time course of gene expression in human ES cells upon BMP4- induced differentiation, both in the presence (brachyury positive result) and absence (chorionic gonadotropin positive result) of bFGF, and correlate these changes with the commitment to exit the pluripotent state. Commitment will be measured by adding BMP4 for successively longer time periods, removing BMP4, and then examining how many cells retain markers of pluripotency several days later. The hypothesis of this aim is that the quantitative commitment curve will be most closely correlated with the expression levels of the genes that directly control these differentiation events. We will subsequently overexpress the transcription factors most closely associated with commitment, and we will downregulate genes by RNA interference that are downregulated during commitment to identify those which are sufficient to mediate differentiation to a brachyury-positive population. Preliminary results demonstrate that GATA2 and GATA3 are both individually sufficient to mediate trophoblast differentiation in the absence of bFGF, so we are optimistic that a single transcription factor will be sufficient to mediate human ES cell differentiation to a brachyury positive population in the presence of bFGF. Aim 2. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map the genomic binding sites of TGFp/Activin-activated Smad 2/3 and BMP-activated Smad 1/5/8 during BMP4- induced differentiation. The central hypothesis of this aim is that Smad 2/3 directly activates the expression of key pluripotency factors in human ES cells, directly suppresses the expression of genes that would otherwise promote differentiation, and competes at the same promoters with Smad 1/5/8 which has the opposing effects. A second hypothesis is that bFGF will change the DNA binding sites of Smad 1/5/8 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 3. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map genomic binding sites of GATA2 and GATA3 during BMP4-induced differentiation. The hypothesis of this aim is that GATA2 and GAT A3 directly negatively regulate the transcription of key pluripotency genes, and directly positively regulate their own transcription, so that once they are induced by Smads, GAT A expression becomes self-sustaining and BMP-independent, and thus differentiation continues even upon BMP removal. A second hypothesis is that bFGF will change the DNA binding sites of GATA2/GATA3 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 4. We will identify proteins that are differentially phosphorylated between BMP4-induced trophoblast differentiation (no bFGF) and BMP-induced brachyury positive cellular differentiation (high bFGF) to identify mediators of FGF signaling that cause the switch between these divergent developmental outcomes. The hypothesis of this aim is that differential phosphorylation of a limited number of transcription factors is casually related to the different developmental outcomes observed after BMP induction in the presence or absence of bFGF. Differentiation commitment curves will again become key for focusing attention to relevant phosphorylation events.