Interrogating Cadherin/Matrix Rigidity Dependent Neural Differentiation and Neuromuscular Junction Formation of Multipotent Stem Cells

PI: Kong， Hyunjoon提案编号：1403491机构：伊利诺伊大学厄巴纳- champaignuniversity标题：询问钙粘蛋白/基质刚性依赖的神经分化和多能干细胞的神经肌肉连接形成理解和重建神经网络代表了一个重大的科学和工程挑战，其影响范围从基础生物学到临床翻译。驻留在骨骼和脂肪组织中的干细胞具有转化为构成神经网络的神经元的潜力。当细胞相互接触并在非基质上培养时，这种细胞转化过程受到刺激。太难了?还是?太软了?，但那是？只是对吧?。这些神经元将有助于控制肌肉组织的运动，从而为基础和应用生物科学研究创造各种生物医学工具。本提案中的研究使用了一种类似凝胶的水凝胶系统，该系统与参与细胞间通信的蛋白质相连，并经过调整以呈现一种？只是对吧?柔软。因此，该项目的成功完成将创建一个先进的细胞培养平台，该平台将广泛用于研究各种干细胞并将其用于神经组织工程。这项工作还将揭示细胞-细胞粘附和基质柔软对干细胞命运的协调作用，包括向神经元的分化。最后，所提出的研究结果将在创造各种神经植入物方面非常有用，比以前更有效，更智能，生物机器人。利用这项研究的结果作为教育模块，将吸引未来的年轻科学家并鼓励他们从事工程事业，从而产生广泛的影响。本提案由化学、生物工程、环境和运输系统部门的生物医学工程项目和材料研究部门的生物材料项目共同资助。在基础和应用神经科学研究中，已经有许多尝试在体外复制神经网络和神经肌肉连接，这些研究使用从多能干细胞分化出来的神经细胞。因此，从分化水平和胶质细胞向神经细胞群的角度出发，以精细的方式调节干细胞的神经分化是至关重要的。本研究旨在系统地了解和调节细胞-细胞粘附和细胞-基质粘附在调节干细胞神经分化中的潜在相互作用。为此，本研究的目标是：(1)通过结合钙粘蛋白（一种细胞-细胞粘附蛋白）和基质硬度的作用来调节多能骨髓基质细胞（BMSCs）的神经分化；(2)利用分化的胆碱能神经元在体外构建功能性的神经肌肉连接。这一目标将通过在与特定数量的重组N-或e -钙粘蛋白化学连接的水凝胶上培养骨髓间充质干细胞来实现，并进一步定制以呈现可控的硬度。我们将分析钙粘蛋白和基质刚性对细胞分化、细胞牵引力和Rac1-GTPase信号激活的综合影响。最后，分化的胆碱能神经元将与骨骼肌母细胞共培养，形成神经肌肉连接，并评估神经对肌肉收缩和松弛的控制。该研究的成功完成预计将对神经科学、神经工程和干细胞技术做出重大贡献。

PI: Kong, Hyunjoon Proposal Number: 1403491Institution: University of Illinois at Urbana-ChampaignTitle: Interrogating Cadherin/Matrix Rigidity Dependent Neural Differentiation and Neuromuscular Junction Formation of Multipotent Stem CellsUnderstanding and recreating neural networks represents a major scientific and engineering challenge with ramifications ranging from fundamental biology to clinical translation. Stem cells residing in bone and fat tissue possess the potential to be converted to neurons that constitute neural networks. This cell conversion process is stimulated when cells contact each other and are cultured on a substrate that is not ?too hard? or ?too soft?, but that is ?just right?. These neurons will help control muscle tissue movement, thus serving to create various biomedical tools used for fundamental and applied bioscience studies. The studies in this proposal use a Jello-like hydrogel system linked with proteins involved in cell-cell communication and tuned to present a ?just right? softness. The successful completion of this project will therefore create an advanced cell culture platform that will be broadly useful for studying a wide array of stem cells and for exploiting this in neural tissue engineering. The proposed work will also uncover the coordinate effects of cell-cell adhesions and matrix softness on stem cell fates, including differentiation into neurons. Finally, the results of the proposed study will be highly useful in creating various neural implants more efficiently than previously possible and intelligent, biological robots. Utilization of the results of this study as an educational module will have broad impacts by attracting future young scientists and encouraging them to pursue engineering careers. This proposal is co-funded by the Biomedical Engineering Program in the Chemical, Bioengineering, Environmental and Transport Systems Division, and by the Biomaterials Program in the Division of Materials Research.There have been numerous attempts to reproduce neural networks and neuromuscular junctions in vitro for both fundamental and applied neuroscience studies that used neuronal cells differentiated from multipotent stem cells. Therefore, it is crucial to regulate neural differentiation of stem cells in an elaborate manner, in terms of differentiation levels and glial-to-neural cell population. The proposed research seeks to systematically understand and modulate the potential interplay between cell-cell adhesion and cell-matrix adhesion in regulating the neural differentiation of stem cells. To this end, the goals of this proposed study are (1) to modulate the neural differentiation of multipotent, bone marrow stromal cells (BMSCs) by combining the effects of cadherin, a cell-cell adhesion protein, and matrix stiffness; and (2) to use the differentiated cholinergic neurons to build a functional, neuromuscular junction in vitro. This goal will be accomplished by culturing BMSCs on a hydrogel chemically linked with a specific number of recombinant N- or E-cadherin, and further tailored to present controlled stiffness. The integrative effects of cadherin and matrix rigidity on cellular differentiation, cell traction force, and Rac1-GTPase signal activation will be analyzed. Finally, differentiated cholinergic neurons will be co-cultured with skeletal myoblasts to form a neuromuscular junction and evaluate neural control over muscular contraction and relaxation. Successful completion of the proposed studies is expected to result in significant contributions in neuroscience and neuroengineering and stem cell technology.