E-cadherin Mechanotransduction, Pluripotency and the Warburg Effect

背景资料。胚胎干细胞(ESCs)是多能的，这意味着它们有能力分化为体内的任何细胞类型。我们观察到，当mESC聚集体在生物反应器中搅拌时，小鼠(M)ESCs的多能性得到促进。即使去除了维护因子LIF，也能保持多能性。此外，细胞重编程，即细胞去分化为多能性的过程，在生物反应器中发生的效率要高出1000倍。我们的初步数据表明，多能转录因子β-连环蛋白在搅拌引起的流体剪切作用下被移位到细胞核中。切应力可以通过机械转导来调节基因的表达。在一种途径中，流体剪切导致β-连环蛋白的核移位。E-钙粘蛋白是一种负责细胞间相互作用和聚集形成的蛋白质，当剪切力被感知时，移位被认为发生了。作为对剪切力的响应，β-连环蛋白可能被纽蛋白取代，纽蛋白的作用是通过将E-钙粘附素拴在细胞骨架上来加强细胞结构。*在细胞重新编程期间，细胞代谢从氧化磷酸化(即线粒体)转变为有氧糖酵解(非线粒体)。最近，β-连环蛋白也被证明通过上调糖酵解基因来促进癌细胞中的这种所谓的华宝效应。*目标。利用以下目标，我们将测试流体剪切是否通过机械转导促进mESC的多能性和细胞在生物反应器中的重编程：*1.β-连环蛋白机械转导。利用流动室和分子分析，结合药理学和遗传学方法，我们将确定β-连环蛋白是否从E-钙粘附素释放并移位到细胞核中作为剪切反应。*2.E-钙粘素机械接收。利用磁性扭曲细胞术和分子分析，结合药理学和遗传学方法，我们将确定剪切作用于E-钙粘素是否会导致β-连环蛋白核易位。*3.多能性与华宝效应。利用药理学和遗传学方法，我们将确定β-连环蛋白机械转导在生物反应器中的重新编程和扩张过程中是否促进多能性和有氧糖酵解。*影响。政府对多能干细胞(PSC)生物库的投资表明，生物反应器将成为未来的应用标准。作为PSCs生物反应器扩展的领导者，我们有能力产生重大影响。了解“机械多能性”背后的机制将有助于开发更有效和更负担得起的生物过程来生产PSCs及其细胞/组织衍生品。我们将继续在研究过程中培训学生和博士后，同时通过体验式学习和知识翻译帮助他们发展领导力和专业技能。

BACKGROUND. Embryonic stem cells (ESCs) are pluripotent meaning they have the ability to differentiate into any cell type in the body. We have observed that the pluripotency of murine (m)ESCs is promoted when mESC aggregates are stirred in bioreactors. Pluripotency is maintained even when the maintenance factor LIF is removed. Moreover, cellular reprogramming, the process of dedifferentiating cells to pluripotency, occurs 1000-fold more efficiently in the bioreactor. Our preliminary data indicates that the pluripotency transcription factor β-catenin is translocated into the nucleus in response to fluid shear caused by stirring.******Shear stress can modulate gene expression through mechano-transduction. In one pathway, fluid shear leads to the nuclear translocation of β-catenin. Translocation is thought to occur when shear is sensed by E-cadherin, a protein responsible for cell-cell interaction and aggregate formation. In response to shear, β-catenin may be displaced by vinculin, whose role is to strengthen cell architecture by tethering E-cadherin to the cytoskeleton. ******During cellular reprogramming, cellular metabolism shifts from oxidative phosphorylation (i.e. mitochondrial) to aerobic glycolysis (non-mitochondrial). Recently β-catenin has also been shown to promote this so called Warburg effect in cancer cells by upregulating glycolytic genes. ******OBJECTIVES. Using the following objectives, we will test if fluid shear promotes mESC pluripotency and cellular reprograming in the bioreactor via mechanotransduction: ******1. β-catenin Mechanotransduction. Using flow chambers and molecular assays, in combination with pharmacological and genetic approaches, we will determine if β-catenin is released from E-cadherin and translocates into the nucleus in reponse to shear. ******2. E-cadherin Mechanoreception. Using magnetic twisting cytometry and molecular assays, in combination with pharmacological and genetic approaches, we will determine if shear applied to E-cadherin results in β-catenin nuclear translocation. ******3. Pluripotency and Warburg Effect. Using pharmacological and genetic approaches, we will determine if β-catenin mechanotransduction promotes pluripotency and aerobic glycolysis during reprogramming and expansion in the bioreactor. ******IMPACT. Government investment in pluripotent stem cell (PSC) biobanking suggests bioreactors will become a future application standard. As leaders in the bioreactor expansion of PSCs, we are positioned to make a significant impact. Understanding the mechanism behind “mechano-pluripotency” will enable the development of more efficient and affordable bioprocesses for producing PSCs and their cell/tissue derivatives. We will continue to train students and postdocs in the research process, while simultaneously helping them to develop leadership and professional skills through experiential learning and knowledge translation.