Single-molecule approaches to study epiblast stem cell fate decision

项目总结 组织和器官衰竭，无论是由于受伤或衰老，正成为全球范围内的主要健康问题， 估计成本为每年医疗保健总费用的一半。为了解决这个问题，组织工程学 可以通过利用在实验室环境中创造的功能身体细胞来利用这些方法。多能上胚层 干细胞(EpiSCs)可以作为一个很好的模型来确定如何引导细胞命运来创造功能性 身体细胞。然而，即使采用了最好的化学定义的多能干细胞分化方案， 对细胞谱系特征的控制仍然很差。除了化学信号外，现在人们普遍认为 细胞外基质(ECM)的物理信号在细胞命运的决定中起着至关重要的作用。不过， 仅靠这种机械力无法改善对细胞谱系规格的控制，这可能是因为 在单分子水平上缺乏对作用力的精确控制，化学信号之间缺乏协同作用 和机械路径。为了解决这一差距，拟议的研究旨在提供一个机制框架， 基于化学和单分子作用力的单EpiSC命运决策(自我更新和分化) 接近了。中心假设是化学和单分子作用力的协同效应通过 细胞-细胞外基质和细胞-细胞相互作用可以比以前更有效地控制命运决定。这个 长期目标是开发新的方法来控制多能细胞定向分化为ALL 三层胚层。为此，提出了以下三个目标。具体地说，目标1将专注于 理解单分子力介导的EpiSCs向中胚层分化的机制 血统。通过单个αvβ3整合素向单细胞传递的力将由张力计控制 系绳。这些基于DNA的可断裂的系链可以精确地限制单分子水平上的作用力。 与化学信号一起，这种对机械通路的精确控制和特定靶向可能导致 更好地控制细胞分化为中胚层。在目标2中，个体的自我更新机制 EpiSCs将通过定义由自我更新促进配体组成的微环境来识别，例如 E-钙粘附素。在目标3中，单个EpiSCs的分化将通过Notch途径由工程化的Low- 公差张力计系绳称为“Nano-Yoyo”，以激活力依赖的Notch信号。建议数 工作将阐明促成特定血统的详细的分子、化学和机械途径。 承诺。最后，三名本科生和两名研究生将获得严谨的研究经验 以及在干细胞、细胞力学和生物物理学领域的深入研究。学生们将进行 实验，分析和总结数据，同时准备稿件，提出建议 科学议程。

PROJECT SUMMARY Tissue and organ failure, either due to injury or aging, are becoming a major health problem worldwide with an estimated cost of one-half of the total annual healthcare expenses. To address this issue, tissue engineering approaches can be leveraged by utilizing functional body cells created in a laboratory setting. Pluripotent Epiblast Stem Cells (EpiSCs) can serve as an excellent model to determine how to direct cell fate for creating functional body cells. However, even with the best chemically-defined differentiation protocol of pluripotent stem cells, the control of cell-lineage specification remains poor. Besides chemical signaling, it is now widely accepted that physical signals from the extracellular matrix (ECM) play a crucial role in cell fate determination. Nevertheless, control of cell-lineage specification by such mechanical forces alone could not be improved possibly due to the lack of precise control of forces at the single-molecule level and the lack of synergy between chemical signaling and mechanical pathways. To address this gap, the proposed study aims to provide a mechanistic framework of single EpiSC fate decisions (self-renewal and differentiation) based on chemical and single-molecule force based approaches. The central hypothesis is that the synergistic effect of chemical and single-molecule force cues via cell-ECM and cell-cell interactions can control fate decisions far more effectively than previously possible. The long-term goal is to develop novel approaches to control the directed differentiation of pluripotent cells into all three germ-layers. To this end, the following three aims are proposed. Specifically, Aim 1 will focus on understanding the mechanism of single-molecule force mediated differentiation of EpiSCs into the mesoderm lineage. The force transmission into single cells via single αvβ3 integrins will be controlled by tension gauge tethers. These DNA-based rupturable tethers can precisely limit the amount of force at the single-molecule level. Together with chemical signaling, such precise control and specific targeting of mechanical pathways may lead to superior control of cell differentiation into the mesoderm. In Aim 2, the mechanism of self-renewal of single EpiSCs will be identified by defining a microenvironment composed of self-renewal promoting ligands such as E-cadherin. In Aim 3, differentiation of single EpiSCs will be defined via the Notch pathway by engineered low- tolerance tension gauge tether called “nano-yoyo” to activate force-dependent Notch signaling. The proposed work will elucidate detailed molecular, chemical, and mechanical pathways that contribute to specific lineage commitments. Finally, three undergraduate and two graduate students will gain research experience in rigorous and intensive research in the areas of stem cells, cell mechanics, and biophysics. Students will conduct experiments, analyze and summarize data, and prepare manuscripts simultaneously advancing the proposed scientific agenda.