Combining dynamics of ligand presentation with dynamics of hESC response in colonies with defined architecture

将配体呈递动力学与具有明确结构的集落中 hESC 响应动力学相结合

• 批准号: 9755445
• 负责人: ALI H BRIVANLOU
• 金额: $ 33.9万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-19 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755445
• 关键词: Activins; Address; Affect; Architecture; BMP4; Basic Science; Behavior; Biological Assay; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Competence; Complex; Cultured Cells; Data; Data Set; Development; Distal; Drug Screening; Ectoderm; Elements; Embryo; Embryonic Development; Endoderm; Experimental Designs; Genetic Transcription; Geometry; Germ Layers; Human; Human Development; Human Engineering; Individual; Kinetics; Laboratories; Ligands; Link; MADH2 gene; MADH4 gene; Mathematics; Measures; Mesoderm; Microfluidics; Modeling; Monitor; Movement; Mus; Nodal; Nuclear Translocation; Output; Paracrine Communication; Pathway Analysis; Pathway interactions; Pattern; Process; Radial; Recording of previous events; Regenerative Medicine; Reporter; Reporting; Resolution; SOX17 gene; Signal Pathway; Signal Transduction; Specific qualifier value; Stem cells; System; Technology; Time; Tissues; Transducers; Transforming Growth Factor beta; Video Microscopy; beta catenin; cell fate specification; clinical application; clinical implementation; computerized data processing; data modeling; embryo tissue; gastrulation; genome editing; human embryonic stem cell; human embryonic stem cell line; inhibitor/antagonist; innovative technologies; morphogens; real time monitoring; response; self organization; time use

Project Summary During early embryogenesis, pluripotent cells specify their fates by integrating multiple signals delivered at different times, places, and with different dynamics. In the mouse embryo, interplay between 3 signaling pathways - BMP4, Wnt, and Nodal - initiate the induction and patterning of embryonic germ layers. However, the relevance of these pathways to human development is not understood. Furthermore, how multiple dynamic signaling pathways are integrated to define cellular fate is unclear. Here we propose to use human embryonic stem cells (hESCs) to understand how individual cells process these dynamic signals to generate discrete fates. To address this, we developed 2 innovative technologies. First, microfluidics to precisely control the timing of ligand application and follow the behavior of the signal transducer SMAD4, with which we demonstrated that TGFβ signaling was adaptive. Second, micropatterns to control hESC colony size and geometry, to show that in response to BMP4, hESCs cultured in circular colonies self-organize into radially symmetric patterns of discrete embryonic germ layers. This remarkably recapitulates the proximal-distal axis of the gastrulating mouse embryo. In this competitive renewal, we combine the strengths of both technologies to deliver distinct dynamics of ligand presentation by microfluidics to CRISPR-edited hESC lines cultured in micropatterned colonies. Four independent signaling-reporter hESC lines that fluorescently tag SMAD1, SMAD2, SMAD4, and β-CATENIN will be used to visualize signaling, and one triple-tagged, fate-reporter CRISPR-edited line that fluorescently tags SOX2, BRACHYURY, and SOX17, will be used to monitor fate acquisition. Our CRISPR-reporter lines will be used to measure signaling dynamics and fate acquisition, with single cell resolution and in real-time by video-microscopy, when cells are presented with BMP4, Wnt3A, and Activin/Nodal either as a persistent step of defined concentration, or as one of defined duration. We propose three specific aims. In aim1, the three ligands will be presented to our signaling-reporter CRISPR lines, to follow the behavior of the four tagged signal transducers and to determine the dynamic behavior of each pathway. In aim2, using the same approach, we will evaluate pathway output by measuring the activity of transcriptional reporters for the three ligands, and use our triple fate-reporter CRISPR line to follow fate acquisition. These two approaches will establish a quantitative link between signaling dynamics, transcriptional output, and fate determination. In aim3, large datasets obtained from aims1 and 2, will be used to model the kinetics of signal transduction, and provide a mathematical paradigm to explain hESC self-organization. The resolution of our three aims will have a strong impact on our understanding of the dynamic integration of signaling pathways underlying human cell fate specification with direct relevance to both basic understanding, and clinical applications of hESCs.

项目摘要 在早期胚胎发育过程中，多能细胞通过整合多种信号来决定它们的命运。 不同的时间，不同的地点，不同的动力。在小鼠胚胎中，3个信号之间相互作用 途径-BMP4、Wnt和Nodal-启动胚胎胚层的诱导和图案化。然而， 这些途径与人类发展的相关性尚不为人所知。此外，如何多重动态 信号通路被整合到一起来定义细胞的命运尚不清楚。在这里，我们建议使用人类胚胎 干细胞(HESCs)了解单个细胞如何处理这些动态信号以产生离散的 命运。为了解决这个问题，我们开发了两项创新技术。第一，用微流控技术精确控制 配基应用的时间和遵循信号换能器Smad4的行为，我们使用它 证明转化生长因子β信号转导途径具有适应性。第二，控制hESC集落大小和 几何学，以表明响应BMP4，培养在圆形菌落中的hESCs自组织成放射状 离散胚胎胚层的对称图案。这极大地概括了近端-远端轴。 原肠小鼠胚胎。在这场竞争性更新中，我们结合了这两种技术的优势，以 通过微流控技术向CRISPR编辑的hESC细胞提供不同的配体呈递动力学 微图案化的菌落。四个独立的信号报告hESC系，其荧光标记SMAD1， Smad2、Smad4和β-catenin将用于可视化信令，以及一个三重标记的命运报告程序 CRISPR编辑的荧光标记SOX2、Brachyury和SOX17的行将用于监测命运 收购。我们的CRISPR记者线路将用于测量信号动态和命运获取， 单细胞分辨率和实时视频显微镜，当细胞被BMP4，WNT3a，和 激活素/结节作为规定浓度的持久步骤，或作为规定持续时间的步骤。我们建议 三个具体目标。在AIM1中，这三种配体将呈现给我们的信号报告CRISPR线路，以 跟踪四个标记的信号换能器的行为，并确定每个 路径。在AIM2中，使用相同的方法，我们将通过测量路径输出的活性来评估路径输出 三个配体的转录记者，并用我们的三重命运-记者CRISPR线跟踪命运 收购。这两种方法将在信号动力学、转录 产出，和命运的决定。在目标3中，从目标1和目标2获得的大数据集将被用来对 信号转导动力学，并为解释hESC自组织提供了一个数学范式。这个 我们三个目标的解决将对我们理解动态整合产生强烈的影响 与人类细胞命运规范的信号通路直接相关的既有基本理解， 以及人类胚胎干细胞的临床应用。