Comparative transcriptional control of establishment, maintenance and collapse of naive pluripotency in rodents and primates in vivo

无论是从为研究建立动物模型的角度来看，还是从为药物筛选或细胞替代疗法生产患者特有的人体组织的角度来看，能够在培养中繁殖并保持成为体内任何组织类型的能力的干细胞系的价值都是巨大的。从小鼠早期胚胎的创始组织中提取的干细胞，被称为多能胚胎干细胞，通过观察它们对将ES细胞整合到宿主胚胎后产生的动物的贡献，已经被证明可以生成任何类型的成人组织，包括生殖细胞。然而，人类ES细胞具有不同于小鼠ES细胞的特征；这在其分子属性、形态和培养要求上是显而易见的。事实上，人类胚胎干细胞与更成熟的小鼠胚胎中发现的细胞更相似，这些胚胎已经植入子宫，被认为已经为分化做好了准备。与小鼠ES细胞相比，这些细胞更难生长，在分化能力上也受到更多限制。为了治疗的相关性，非常希望了解如何从人类胚胎中捕获更类似于小鼠ES细胞的细胞。尽管已经有人试图制造出这样的细胞，无论是通过控制它们生长的培养条件，还是通过可以将成年细胞类型恢复到更接近胚胎条件的“重新编程”策略，但还不可能捕获在这种状态下生长的人类细胞。我们认为，克服这一障碍的唯一方法是了解这些创始人胚胎细胞是如何在小鼠胚胎中形成的，从中可以很容易地获得真正的ES细胞系。最终目标是利用这一知识作为蓝图，探索早期人类胚胎(或作为人类模型的非人类灵长类胚胎)的发展，并开发替代途径，从灵长类动物中捕获类似的多能细胞。除了发现如何从人类胚胎中提取理想类型的干细胞外，这一知识还将用于从成人组织和患者样本中产生有用的细胞。在这个项目中，我们计划扩展我们以前的工作，在动物发育阶段自然产生多能细胞之前、期间和之后产生详细的小鼠胚胎的分子画像。我们将利用遗传修饰策略来探索主控基因在这一过程中的重要性。此外，我们可以在培养基中加入高度特异的化学物质，激活或抑制不同的细胞行为，以揭示这些控制基因是如何打开或关闭的。我们已经开始识别啮齿动物(小鼠)和灵长类(绒猴)胚胎之间的分子差异。我们将扩展这一概念，然后测试绒猴胚胎对我们预测可能有利于发育的外部信号的反应。我们将利用这些信息来针对特定的途径，这些途径可以被增强或抑制，从而使多能细胞能够被捕获，从而为药物筛选和最终细胞替代疗法创造有用的组织提供必要的起点。

The value of stem cell lines that can multiply in culture and retain the capacity to become any tissue type in the body is immense, both from the point of view of generating animal models for research, and to produce patient-specific human tissue for drug screening or cell replacement therapy. Stem cells derived from the founder tissue of the foetus in the early mouse embryo, known as pluripotent embryonic stem (ES) cells, have been proven to generate any type of adult tissue, including the germ cells, by observing their contribution to animals produced after integrating ES cells into a host embryo. Human ES cells, however, have characteristics distinct from mouse ES cells; this is evident in their molecular attributes, morphology and culture requirements. In fact, human ES cells resemble much more closely the cells found in more mature mouse embryos that have already implanted in the uterus and are considered to be 'primed' for differentiation. These cells are harder to grow and are more restricted in their differentiation repertoire compared with mouse ES cells. In the interests of therapeutic relevance, it would be highly desirable to understand how to capture cells from human embryos that are more akin to mouse ES cells. Although attempts have been made to produce such cells, either by manipulating the culture conditions in which they are grown, or by means of 'reprogramming' strategies that can revert adult cell types to a more embryonic condition, it has not been possible to capture human cells that can thrive in such a state. We believe that the only way to overcome this obstacle is to understand how these founder embryonic cells are formed in mouse embryos, from which true ES cell lines can be readily obtained. The ultimate goal is to use this knowledge as a blueprint to probe the development of early human embryos (or non-human primate embryos as a model for humans) and exploit alternative pathways to capture similar pluripotent cells from primates. In addition to discovering how to derive the ideal type of stem cells from human embryos, this knowledge will be used to generate useful cells from adult tissues and patient samples. In this project we plan to expand on our previous work to produce a detailed molecular portrait of mouse embryos before, during and after the stage in animal development when pluripotent cells are naturally produced. We will make use of genetic modification strategies to explore the importance of master control genes in this process. In addition, we can add highly specific chemicals to the culture medium that activate or suppress different cellular behaviours to reveal how these control genes are turned on or off. We have already begun to identify molecular differences between rodent (mouse) and primate (marmoset) embryos. We will extend this concept and then test how marmoset embryos respond to external signals that we predict may be beneficial to development. We will use this information to target specific pathways that could be enhanced or inhibited to allow pluripotent cells to be captured, thereby providing the essential starting point to create useful tissues for drug screening and, ultimately, cell replacement therapy.