Ultrasound-mediated Directed Osteogenic Differentiation of Mesenchymal Stem Cells

描述（由申请人提供）：本研究的长期目标是开发一种跨学科的方法来增强体外人间充质干细胞（hMSC）的成骨分化，以推进退行性骨疾病（如骨质疏松症）的细胞治疗。 人类间充质干细胞（hMSCs）是前体细胞，形成和愈合几乎所有的机械组织在人类，包括骨。目前正在从成人中分离hMSCs，以探索这些细胞是否可以在体外分化为成骨细胞 并作为细胞疗法重新植入以阻止甚至逆转退行性骨疾病如骨质疏松症。虽然已经取得了一些初步的有希望的进展，在证明hMSC成骨的基质刚度和外部机械力的机械响应性调节，在体外细胞培养条件的hMSC成骨仍然是次优的。 我们假设，一个通用的体外细胞培养系统，允许快速和可逆的 细胞培养环境的动态机械调节将能够改善对hMSC的成骨分化的控制。为了增强体外hMSC成骨并进一步帮助hMSC中的机械转导系统的机制研究，我们提出开发一种新的跨学科方法，该方法结合了从该提案的两个co-PI的实验室开发的两种新的微工程技术，以精确地调节动态亚细胞机械力，同时测量hMSC的细胞骨架收缩性的活细胞亚细胞响应。这些新工具包括1）弹性体微柱阵列的标准化库，以精确地调节基底刚性，并作为非破坏性活细胞牵引力传感器用于细胞骨架收缩性的亚细胞定量;和2）新型“声学镊子”，其能够对细胞膜上的特定粘附受体产生受控的机械力，作为施加影响细胞骨架收缩性的外力的独特策略。 我们的具体目标是：1）表征超声镊子诱导hMSC细胞骨架CSK收缩性的时空变化; 2）表征超声镊子如何施加机械扰动以调节hMSC的成骨分化;和3）表征RhoA/ROCK/肌球蛋白信号轴如何参与超声镊子在hMSC中的胞内力传递。这项研究的结果预计将推进我们目前对hMSC机械传导的了解，为增强其成骨分化提供关键基础。对hMSC中的机械转导系统的进一步理解可以为hMSC生物学提供基本的见解，以及改善hMSC体外分化以用于治疗骨疾病的基于细胞的治疗应用的实用方法。

DESCRIPTION (provided by applicant): The long term objective of this research is to develop an interdisciplinary approach to enhance Human mesenchymal stem cell (hMSC) osteogenic differentiation in vitro, in order to advance cell-based therapies for degenerative bone diseases such as osteoporosis. Human mesenchymal stem cells (hMSCs) are precursor cells that form and heal nearly all of the mechanical tissues in humans, including bone. hMSCs are now being isolated from adults to explore whether these cells can be differentiated into osteoblasts in vitro and re-implanted as a cellular therapy to arrest or even reverse degenerative bone diseases such as osteoporosis. While some initial promising progress has been made in demonstrating the mechanoresponsive regulation of hMSC osteogenesis by matrix rigidity and external mechanical forces, in vitro cell culture conditions for hMSC osteogenesis still remain suboptimal. We hypothesize that a versatile in vitro cell culture system that allows for a rapid and reversible dynamic mechanical regulation of cell culture environment will enable an improved control of osteogenic differentiation of hMSCs. To enhance in vitro hMSC osteogenesis and further aid in mechanistic investigation of the mechanotransductive system in hMSCs, we propose to develop a novel, interdisciplinary approach combining two novel microengineering techniques developed from laboratories of the two co-PIs of this proposal, to precisely modulate dynamic subcellular mechanical forces while simultaneously measuring live- cell subcellular responses of cytoskeleton contractility of hMSCs. These novel tools include 1) A standardized library of elastomeric micropost arrays to precisely regulate substrate rigidity and as non-destructive live-cell traction force sensors for subcellular quantification of cytoskeleton contractility; and 2) A novel "acoustic tweezer" capable of generating controlled mechanical forces to specific adhesion receptors on cell membrane, as a unique strategy to apply external forces affecting cytoskeleton contractility. Our specific aims are: 1) To characterize spatiotemporal changes of hMSC cytoskeCSK contractility induced by ultrasound tweezers; 2) To characterize how ultrasound tweezers exert mechanical perturbations to regulate osteogenic differentiation of hMSCs; and 3) To characterize how RhoA/ROCK/myosin signaling axis is involved in intracellular force transduction from ultrasound tweezers in hMSCs. Results from this research are expected to advance our current understanding of mechanotransduction in hMSCs to provide a pivotal foundation for enhancing their osteogenic differentiation. Improved understanding the mechanotransduction system in hMSCs may provide fundamental insights into hMSC biology, as well as practical approaches to improve hMSC differentiation in vitro for cell-based therapeutic applications for treating bone diseases.