Mechanical regulation of transcription in dental epithelial stem cells through cell packing and tissue forces

项目摘要 有效利用躯体干细胞修复受损组织或生物工程器官是一个重要目标。 在再生医学方面。然而，经临床证实的干细胞在治疗中的应用仍然有限。 今天的医学。翻译障碍在很大程度上是因为我们缺乏精确控制干细胞的能力。 增殖和分化，这是安全和有效的临床使用的关键。为了克服这一挑战， 我们首先必须加深对器官中正常干细胞调节的了解。除了生化信号外， 理论上，细胞拉力和推力所施加的组织机械力可以作为一种信号机制 调节成体干细胞的基因表达和各种细胞过程。然而，调制和 这些力信号在3D组织中的影响被极大地忽视了，留下了悬而未决的问题 干细胞如何感知和解释力量。我们和其他人已经证明了老鼠门牙是一种强大的 研究成体上皮干细胞的模型系统和我们先前已经证明的转录辅助因子 YAP和染色质抑制在门牙上皮干细胞的调控中起重要作用。 我们的初步研究表明，细胞的机械变形和细胞的几何形状都与致密 填塞可以影响YAP的表达和门牙干细胞壁龛中的抑制性染色质标记。这个 因此，小鼠切牙为研究细胞组织如何协调机械力提供了一个有价值的在体平台 通过YAP和染色质控制干细胞功能的信号。在此应用程序中，我们建议检验假设 这种致密的细胞堆积调节组织力对核变形的影响，而核变形又调节YAP 核进入和H3K27me3介导的牙上皮干细胞转录抑制。要测试这一点，请执行以下操作： 目标1将描述野生型门牙的力模式、大小和核硬度，特别是在 密集排列的牙齿上皮干细胞和较松散排列的转运扩增细胞。 目标2将研究细胞几何形状和填充的变化如何影响组织力模式，核变形， YAP定位和染色质状态。我们将进行机械救援实验，以检验力量的作用。 目标3将阐述层蛋白A在调节核僵硬和异染色质形成中的功能作用。 牙齿上皮干细胞，以及它对细胞堆积的伸缩反应。 总之，这些研究将提供对组织力量如何控制牙齿干细胞的机制的理解。 并产生对牙科研究人员以及干细胞和再生细胞都有普遍兴趣的研究结果 医学界。

Project Summary Effective utilization of somatic stem cells to repair injured tissues or to bioengineer organs is an important goal in regenerative medicine. However, clinically-proven application of stem cells in therapies remains limited in medicine today. The translational hurdles are in large part due to our lack of ability to precisely control stem cell proliferation and differentiation, which is critical for safe and effective clinical use. To overcome this challenge, we must first deepen our knowledge of normal stem cell regulation in organs. In addition to biochemical signals, tissue mechanical forces exerted by cell pulling and pushing can in theory serve as a signaling mechanism to regulate gene expression and various cellular processes in adult stem cells. However, the modulation and influence of these force signals within a 3D tissue are dramatically understudied, leaving open questions around how stem cells sense and interpret forces. We and others have demonstrated the mouse incisor as a powerful model system to study adult epithelial stem cells and we have previously shown that the transcription co-factor Yes-associated protein (YAP) and chromatin repression are important for regulating incisor epithelial stem cells. Our initial studies indicate that both mechanical deformation of cells and the cell geometry associated with dense packing can influence the expression of YAP and repressive chromatin marks in the incisor stem cell niche. The mouse incisor thus provides a valuable in vivo platform to study how cellular organizations coordinate mechanical signals to control stem cell functions via YAP and chromatin. In this application, we propose to test the hypothesis that dense cell packing modulates the effect of tissue forces on nuclear deformations, which in turn regulate YAP nuclear entry and H3K27me3-mediated transcriptional repression in the dental epithelial stem cells. To test this: Aim 1 will characterize the force patterns, magnitude, and nuclear stiffness in wild type incisors, specifically in the densely packed dental epithelial stem cells and the more loosely packed transit amplifying cells. Aim 2 will study how changes in the cell geometry and packing affect tissue force patterns, nuclear deformations, YAP localization, and chromatin states. We will perform mechanical rescue experiments to test the role of forces. Aim 3 will address the functional role of lamin A in regulating nuclear stiffness and heterochromatin formation in the dental epithelial stem cells, as well as its scaling response to cell packing. Together, these studies will deliver a mechanistic understanding of how tissue forces control dental stem cells and yield findings that will be of general interest to both dental researchers and to the stem cell and regenerative medicine communities.