Embryonic stem cell differentiation: A key to unraveling the cellular mechanisms of patterning

在受精卵发育成胚胎的过程中，协调受精卵形态和功能变化的过程的复杂性、复杂性和有效性是生物学中最令人着迷和最基本的问题之一。在过去的几十年里，对早期胚胎发育至关重要的遗传网络和信号通路已经被确定并详细描述。然而，单个信号通路的复杂性，特别是它们的相互作用和对时间的依赖，迄今为止阻碍了将时间信号线索明确地归因于细胞的特定行为的尝试。本文提出了一种缩小这一差距的策略，通过提出一套专门的实验来适应人类胚胎干细胞（hESC）细胞培养中信号通路活性和细胞命运的定量模型。该项目建立在教授所在实验室的两项最新发现的基础上。美国纽约洛克菲勒大学的Siggia和Brivanlou。在最近的实验中，他们可以证明，将生长因子均匀地应用于圆形限制的干细胞菌落，足以在人类胚胎的胚层中重建空间模式，突出了通过细胞间通信和菌落几何形状进行自组织的重要性。在理论方面，Siggia教授的团队最近开发了一个框架，用于将一个简约的定量模型与秀丽隐杆线虫的实验数据相匹配，以描述细胞命运决策作为两种信号通路的功能。这种方法绕过了途径的分子细节，用最少数量的聚集变量描述了它们的有效作用。我们提出了一组实验，可以在上述框架内进行量化。人类胚胎干细胞将在不同的限制几何形状中生长，途径的活性，特别是TGF-ß和Wnt，以及细胞命运将在细胞分辨率上通过免疫荧光染色和细胞命运和途径活性的活荧光报告来确定。这些数据将以空间和时间解决的方式提供信号通路活动与细胞命运的映射，并将提供有关导致特定命运模式的细胞间通信的前所未有的信息。然后，一个适用于胚胎干细胞的细胞命运的定量数学模型将与这些数据相匹配。拟议的研究不仅将为早期哺乳动物胚胎发生，特别是人类胚胎发生提供独特的见解。由于这些通路的特征在物种间高度保守，并且与成人的广泛细胞过程相关，因此拟议研究的影响范围从胚胎发生延伸到器官发生、再生医学和癌症等多种主题。

The complexity, intricacy and efficacy of the process that orchestrates the changes in form and function of the fertilized egg as it develops to the embryo presents one of the most fascinating and fundamental problems of biology. Over the past decades, the genetic networks and signaling pathways that are key to the development of the early embryo have been identified and described in detail. However, the unmitigated complexity of the individual pathways, in particular their interaction and dependence on time have so far prevented attempts to unambiguously attribute temporal signaling cues to a specific behavior of a cell. This proposal presents a strategy for closing this gap by suggesting a dedicated set of experiments to which a quantitative model of signaling pathway activity and cell fate in cell cultures of human embryonic stem cells (hESC) is to be fitted. The project builds on two recent findings of the host laboratories of Profs. Siggia and Brivanlou at Rockefeller University, New York, USA. In recent experiments, they could show that uniform application of growth factors to circularly confined stem cell colonies is sufficient to recreate the spatial patterning into the germ layers of the human embryo, highlighting the importance of self-organization through cell-to-cell communication and the geometrical shape of the colonies. On the theoretical side, Prof. Siggia's group has recently developed a framework for fitting a parsimonious, yet quantitative model to experimental data from Caenorhabditis elegans in order to describe cell fate decision making as a function of two signaling pathways. This approach circumvents the molecular details of the pathways and describes their effective action with a minimal number of aggregate variables. We propose a set of experiments that will be amenable to quantification within the framework described above. Human embryonic stem cells will be grown in different confining geometries and the activity of pathways, in particular TGF-ß and Wnt, as well as the cell fates will be determined at cellular resolution using immunofluorescent staining and live fluorescent reporters of cell fate and pathway activity. This data will provide a mapping of the signaling pathway activity to cell fates in a space and time resolved manner and will also provide unprecedented information about the cell-to-cell communication that leads to a particular fate pattern. A quantitative mathematical model of cell fate adapted to the case of embryonic stem cells will then be fitted to the data. The proposed research not only will provide unique insight into early mammalian embryogenesis, in particular in the human. Because of the characterization of pathways highly conserved across species and relevant for a wide range of cellular processes in the adult, the impact of the proposed research extends from embryogenesis to topics as diverse as organogenesis, regenerative medicine, and cancer.