Mitochondrial Dynamics in the Control of the Pluripotent States

在胚胎发育的初期，细胞开始具有产生形成新生有机体的所有细胞类型和组织的潜力。这种潜力被称为多能性，而那些多能性的细胞在再生医学上有着巨大的希望。这一前景不仅是因为它们未来在细胞治疗中的临床应用，还因为现在可以产生针对患者的多能细胞，用于“培养皿中的疾病”方法，以了解人类疾病。干细胞疗法正被用于治疗从糖尿病到神经退行性疾病的各种疾病，而基于干细胞的机制被认为是不同形式癌症的基础。在过去的几年里，人们已经投入了大量的努力来识别调节多能状态和控制分化的第一步的信号和基因调控网络，当细胞开始获得谱系特异性身份时。这些研究已经确定了提供多能性细胞身份的关键分子特征。与此相反，我们对多能状态下的细胞生物学变化知之甚少，例如细胞的不同细胞器如何适应分化初期遇到的不同环境。特别令人感兴趣的是线粒体，它不仅是细胞的动力源，而且还调节多种细胞过程，包括细胞对细胞死亡信号的反应能力，它们的分化潜力，以及它们如何对调节细胞身份和功能的不同信号通路作出反应。我们已经发现，在分化的第一阶段，细胞的线粒体形态发生了巨大的变化，这表明线粒体动力学在多能性退出期间发生了变化。此外，我们发现在分化的第一阶段，细胞改变了它们的代谢和对细胞死亡刺激的反应，对死亡信号变得异常敏感。在本提案中，我们将研究线粒体动力学如何影响这些变化。我们将通过在多能小鼠干细胞系和小鼠胚胎中进行的实验组合来做到这一点，这使我们能够很容易地确定我们的数据在体内的相关性，以及人类胚胎干细胞模型，这使我们能够深入了解我们的发现在人类中的保护作用。具体来说，我们将做三件事。首先，我们将在超微结构水平上详细研究细胞退出多能状态时线粒体形态发生的精确变化。我们将通过分析线粒体蛋白在多能性和分化细胞中的分布，以及在多能性退出时成像线粒体动力学来做到这一点。然后，我们将操纵线粒体动力学并研究这如何影响细胞身份。为此，我们将研究可能受线粒体变化影响的三个细胞过程，细胞代谢，细胞对细胞死亡刺激的反应能力，以及分化成形成胚胎的不同谱系的潜力。最后，我们将利用维持多能性和驱动分化的信号通路的重要知识，识别调节干细胞线粒体动力学和功能的新信号输入。总之，这些实验将为早期胚胎发育的一个重要但尚未得到充分研究的方面提供独特的见解，线粒体的动力学，以及解决它们如何影响分化过程。

At the beginning of development of an embryo, cells start off with the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer. Over the last few years considerable efforts have been invested into identifying the signalling and gene regulatory networks that regulate the pluripotent state and control the first steps of differentiation, when cells start to acquire a lineage specific identity. These studies have identified the key molecular features that provide pluripotent cell identity. In contrast to this, we know relatively little about the cell biology changes that accompany exit from the pluripotent state, for example how the different organelles of the cell adapt to the different environments they encounter during the first stages of differentiation. Of particular interest are mitochondria, that are not only the powerhouse of the cell, but also regulate a diverse range of cellular processes, including the ability of the cell to respond to cell death signals, their potential to differentiate, as well as how they respond to the different signalling pathways that regulate cell identity and function.We have identified that during the first stages of differentiation, cells undergo a dramatic change in the morphology of their mitochondria, suggesting that mitochondrial dynamics change during exit of pluripotency. Additionally we have found that the first stages of differentiation, cells alter their metabolism and their response to cell death stimuli, becoming hypersensitive to death signals. In this proposal we will investigate how mitochondrial dynamics impact of these changes. We will do this by using a combination of experiments performed in pluripotent mouse stem lines and mouse embryos, that allow us to readily determine the in vivo relevance of our data, as well as human embryonic stem cell models, that provide us with insight of the conservation in human of our findings. Specifically we will do three things.In the first place we will study in detail, at the ultrastructural level, the precise changes that occur in mitochondrial morphology as cells exit the pluripotent state. We will do this by analysing the distribution of mitochondrial proteins in pluripotent and differentiating cells, as well as by imaging mitochondrial dynamics upon exit of pluripotency. We will then manipulate mitochondrial dynamics and study how this affects cell identity. For this we will study three cellular processes that are likely to be affected by mitochondrial changes, the cells metabolism, it's ability to respond to cell death stimuli, and its potential to differentiate into the different lineages that will form the embryo. Finally, we will exploit the significant knowledge of the signalling pathways that maintain pluripotency and that drive differentiation, to identify novel signalling inputs that regulate mitochondrial dynamics and function in stem cells. Together, these experiments will provide a unique insight into an essential, but understudied aspect of early embryonic development, the dynamics of the mitochondria, as well as addressing how they impact on the process of differentiation.