Single-molecule approaches to study epiblast stem cell fate decision

项目摘要 由于损伤或老化引起的组织和器官衰竭正在成为世界范围内的主要健康问题， 估计费用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 可以通过利用在实验室环境中产生的功能体细胞来利用这些方法。多能外胚层 干细胞（EpiSC）可以作为一个很好的模型，以确定如何指导细胞命运，以创造功能性的干细胞。 体细胞然而，即使使用多能干细胞的最佳化学定义的分化方案， 细胞谱系特化的控制仍然很差。除了化学信号外，现在广泛接受的是， 来自细胞外基质（ECM）的物理信号在细胞命运决定中起关键作用。然而，尽管如此， 单独通过这种机械力对细胞谱系特化的控制不能得到改善，这可能是由于 在单分子水平上缺乏对力的精确控制， 和机械路径。为了弥补这一差距，拟议的研究旨在提供一个机制框架， 基于化学和单分子力的单个EpiSC命运决定（自我更新和分化） 接近。中心假设是，化学和单分子力线索的协同效应通过 细胞-ECM和细胞-细胞相互作用可以比以前更有效地控制命运决定。的 长期目标是开发新的方法来控制多能细胞定向分化为所有细胞， 三层生殖层为此，提出以下三个目标。具体而言，目标1将侧重于 了解单分子力介导的EpiSCs向中胚层分化的机制 脉通过单个αvβ3整联蛋白将力传递到单个细胞中将由张力计控制 栓绳这些基于DNA的可破裂系链可以精确地限制单分子水平的力的大小。 与化学信号一起，这种对机械通路的精确控制和特异性靶向可能导致 对细胞分化成中胚层的上级控制。在目标2中，单细胞自我更新的机制 将通过定义由自我更新促进配体组成的微环境来鉴定EpiSC，所述配体例如 E-钙粘蛋白。在目标3中，单个EpiSC的分化将通过Notch途径通过工程化的低分化的EpiSC来定义。 一种称为“纳米溜溜球”的耐张力计系绳，用于激活力依赖性Notch信号传导。拟议 工作将阐明详细的分子，化学和机械途径，有助于特定的谱系 嵄 彸诺丅最后，三名本科生和两名研究生将获得严格的研究经验， 以及在干细胞、细胞力学和生物物理学领域的深入研究。学生将进行 实验，分析和总结数据，并准备手稿，同时推进建议 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.