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

自我更新和分化：使 ES 细胞退出的分子事件

• 批准号: 8381275
• 负责人: James Alexander Thomson
• 金额: $ 36.96万
• 依托单位: MORGRIDGE INSTITUTE FOR RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8381275
• 关键词: Activins; Attention; BMP4; Binding Sites; Brachyury protein; Cell Differentiation process; Cells; Chorionic Gonadotropin; Commit; DNA Binding; Development; Endoderm; Event; Excision; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Maps; Measures; Mediating; Mediator of activation protein; Mesoderm; Molecular; Outcome; Phosphorylation; Placenta; Population; Proteins; RNA Interference; Signal Transduction; Time; chromatin immunoprecipitation; clinically relevant; embryonic stem cell; human embryonic stem cell; overexpression; pluripotency; poly(L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10)); promoter; response; self-renewal; transcription factor; trophoblast

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.

项目2：自我更新和分化：承诺ES细胞退出的分子事件 多能状态。(詹姆斯·汤姆森，少年派) A.具体目标 在低浓度(4 ng/ml)或不含外源性bFGF的条件下，BMP4可诱导人ES细胞形成同源群体 滋养层细胞，胎盘的外层。然而，我们最近发现，在高水平的存在下 (100 ng/ml)浓度的bFGF、BMP4诱导人ES细胞形成一组细胞， 不再表达滋养层标志，而是瞬时表达短小胚层、中胚层或 中胚层标记，随后表达内胚层和中胚层标记的混合物。在这 项目中，我们将研究BMP如何诱导人ES细胞退出多能性状态并致力于 分化，并研究成纤维细胞生长因子如何调节这些不同的发育结果来应对相同的 诱导者。理解ES细胞如何退出多能性状态以及为什么这种退出通常是不可逆的是 实现有效的重新编程的核心(项目3)，并理解这些关键的早期世系决策 将允许更有效地分化为特定的临床相关谱系。 我们将实现以下目标： 目的1.我们将建立人ES细胞在BMP4-BMP4作用下基因表达的详细时间进程。 诱导分化，在存在(短促阳性结果)和不存在(绒毛膜)两种情况下 促性腺激素阳性结果)，并将这些变化与退出 多能状态。承诺将通过在连续更长的时间段内添加BMP4来衡量， 去除BMP4，几天后检查有多少细胞保留了多能性标记。这个 这一目标的假设是，量化承诺曲线将与 直接控制这些分化事件的基因的表达水平。我们随后将过度表达 与承诺最密切相关的转录因子，我们将通过以下方式下调基因 RNA干扰在承诺过程中被下调，以确定那些足以介导的RNA干扰 分化为短视阳性的人群。初步结果表明，GATA2和GATA3是 两者单独足以在缺乏bff的情况下调节滋养层细胞的分化，因此我们持乐观态度。 一个单一的转录因子就足以将人类胚胎干细胞的分化调节到短小的水平 碱性成纤维细胞生长因子存在的阳性群体。 目的2.我们将使用全基因组染色质免疫沉淀芯片(ChlP-ChIP)来定位 TGFp/激活素激活的Smad2/3和BMP激活的Smad1/5/8在BMP4- 诱导分化。这一目的的中心假设是Smad2/3直接激活表达 在人类ES细胞中的关键多能性因子中，直接抑制将 否则促进差异化，并与Smad 1/5/8在相同的启动子上竞争 相反的效果。第二种假说是bFGF会改变Smad1/5/8的DNA结合部位。 BMP诱导分化，导致观察到的不同发育结果。 目的3.我们将使用全基因组染色质免疫沉淀芯片(ChlP-ChIP)来定位基因组 BMP4诱导分化过程中GATA2和GATA3的结合位点这个目标的假设是 GATA2和GATA3直接负调控关键多能性基因的转录，并直接 正向调节自身转录，使其一旦被Smads诱导，GAT A表达 变得自我维持和不依赖BMP，因此即使去除BMP也会继续分化。一个 第二种假设认为，在BMP诱导过程中，bFGF会改变GATA2/GATA3的DNA结合部位 差异，导致观察到的不同发育结果。 目的4.我们将鉴定BMP4诱导的差异磷酸化的蛋白质 滋养层细胞分化及BMP诱导的短缩细胞分化(高分化 碱性成纤维细胞生长因子)来确定导致这些分歧之间切换的成纤维细胞生长因子信号的介体 发展成果。这个目的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随意相关 在碱性成纤维细胞生长因子存在或不存在的情况下进行诱导。差异化承诺曲线将再次成为 关注相关的磷酸化事件。