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: 9238940
• 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/9238940
• 关键词: Activins; Address; Affect; Alpha Cell; Architecture; BMP4; Basic Science; Behavior; Biological Assay; CRISPR/Cas technology; Cell physiology; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Competence; Complex; Cultured Cells; Data; Data Set; Development; Distal; Ectoderm; Elements; Embryo; Embryonic Development; Endoderm; Experimental Designs; Genetic Transcription; Geometry; Germ Layers; Human; Human Development; Human Engineering; Income; 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; Preclinical Drug Evaluation; 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; computerized data processing; data modeling; embryo tissue; gastrulation; genome editing; human embryonic stem cell; human embryonic stem cell line; inhibitor/antagonist; innovative technologies; morphogens; 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.

项目概要 在早期胚胎发生过程中，多能细胞通过整合多个信号来指定它们的命运 在小鼠胚胎中，不同的时间、地点和不同的动态，三种信号之间的相互作用。 途径 - BMP4、Wnt 和 Nodal - 启动胚胎胚层的诱导和模式化。 此外，这些途径与人类发展的相关性尚不清楚。 整合信号通路来定义细胞命运尚不清楚，在此我们建议使用人类胚胎。 干细胞（hESC）了解单个细胞如何处理这些动态信号以产生离散的信号 为了解决这个问题，我们开发了两项创新技术，以精确控制。 配体应用的时机并遵循信号传感器 SMAD4 的行为，我们用它来 其次，控制 hESC 集落大小和的微图案 几何形状，表明响应 BMP4，在圆形集落中培养的 hESC 自组织成放射状 离散胚胎胚层的对称模式这概括了近端-远端轴。 在这次竞争性更新中，我们结合了两种技术的优势 通过微流体向 CRISPR 编辑的 hESC 细胞系提供独特的配体呈递动态 四个独立的信号报告基因 hESC 系，荧光标记 SMAD1， SMAD2、SMAD4 和 β-CATENIN 将用于可视化信号传导，以及一种三重标记的命运报告基因 CRISPR 编辑的荧光标记 SOX2、BRACHYURY 和 SOX17 的细胞系将用于监测命运 我们的 CRISPR 报告基因系将用于测量信号动态和命运获取。 当细胞呈现 BMP4、Wnt3A 和 Activin/Nodal 可以作为指定浓度的持续步骤，也可以作为指定持续时间的步骤之一。 在 goal1 中，三个配体将被呈现给我们的信号报告基因 CRISPR 系，以实现三个特定目标。 跟踪四个标记信号传感器的行为并确定每个传感器的动态行为 在 Target2 中，我们将使用相同的方法，通过测量 的活性来评估路径输出。 三个配体的转录生产者，并使用我们的三重命运报告基因 CRISPR 系列来追踪命运 这两种方法将在信号动力学、转录之间建立定量联系。 在目标 3 中，从目标 1 和目标 2 获得的大型数据集将用于对目标进行建模。 信号转导动力学，并提供解释 hESC 自组织的数学范式。 我们三个目标的解决将对我们对动态整合的理解产生重大影响 信号传导潜在途径人类细胞命运规范与基本理解直接相关， hESCs 的研究和临床应用。