CAREER: Mechanical Biomarkers and Mesenchymal Stem Cell Differentiation

职业：机械生物标志物和间充质干细胞分化

• 批准号: 1253189
• 负责人: Eric Darling
• 金额: $ 40.18万
• 依托单位: Brown University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1253189&HistoricalAwards=false
• 关键词: CAREER; Mechanical; Biomarkers; Mesenchymal; Stem

1253189DarlingMesenchymal stem cells (MSCs) pose exciting possibilities for repairing tissues damaged by injury or disease. Their relative abundance and capability to become many different cell types make them attractive as an alternative autologous cell source. However, the cellular heterogeneity present in MSC harvests complicates their therapeutic application since not all cells will behave in the same manner. Individual stem cells respond to biochemical and mechanical stimuli in their local microenvironment, which can direct differentiation along specific pathways/lineages. While substrate compliance has been extensively investigated in recent years as it relates to stem cell differentiation, little attention has been given to how the mechanical properties of individual cells can influence this process for themselves and their neighbors. Findings from the PI?s laboratory indicate cellular mechanical properties correlate with biological characteristics, such as gene expressions, and play an important role in determining the differentiation fate of stem cells. Not all MSCs behave uniformly, and this heterogeneity can dramatically impact the overall response of a sample. Understanding how the biological and mechanical characteristics of local cell populations relate to the larger group can provide clues to optimizing future regenerative therapies. The proposed project will investigate the heterogeneity of MSC samples at the single-cell, neighboring (~101 cells), and population (~103+ cells) levels using two complimentary strategies. First, live-cell, gene expression-based markers will be used to identify differentiating and non-differentiating MSCs that have been chemically induced for osteogenesis and adipogenesis. Second, single-cell, elastic and viscoelastic testing will assess the mechanical heterogeneity in undifferentiated, ?partially? differentiated, and fully differentiated stem cell cultures. In addition to expanding knowledge of stem cell mechanics, this research program has the potential to vastly improve experimental approaches that strive to direct stem cell differentiation and improve regenerative responses following implantation. Intellectual Merit: The proposed project seeks to investigate the inherent heterogeneity present in mesenchymal stem cell populations. MSCs are typically investigated at the population level, which can obscure variations that exist among individual cells. Little is known about how single-cell, mechanical properties change during differentiation, but this knowledge is critical for fully understanding cell-substrate and cell-cell behavior. Likewise, elucidating the spatiotemporal patterns of gene expression in MSC samples can help determine optimal times and locations for biochemical stimulation. To pursue these goals, this project will use custom-designed molecular beacons to visualize mRNA molecules in live, differentiating MSCs. Atomic force microscopy will be used to mechanically characterize the elastic and viscoelastic properties of individual cells. Focus will be given to how local cell populations behave, biochemically and mechanically, in relation to the overall sample. Findings will be applicable to many fields, including stem cell biology, cell mechanics, and tissue engineering. The PI is a pioneer in the area of single-cell, mechanical biomarkers, an exciting new field that provides phenotypic characterization akin to gene and protein expression. The proposed research furthers the maturation of these transformative approaches by investigating the role of mechanical biomarkers in stem cell differentiation.Broader Impact: As a biomedical engineer conducting research at the cross-section of math, biology, chemistry, and engineering, the PI is well-suited to provide an environment that highlights the need for interdisciplinary understanding. The outreach effort proposed here will provide primary and secondary school educators at the Providence Public School District the opportunity to conduct team-based research during the summer. Teachers specializing in different subjects will work together on projects that incorporate multiple areas of expertise. The goal of this project is to facilitate interaction among teachers so that they can develop interdisciplinary examples and experiments for their own classes. Students take many math and science courses throughout their education, encompassing algebra, geometry, biology, chemistry, and physics. However, these courses are almost always taught as isolated subjects, whereas real-world applications are increasingly interdisciplinary. The proposed project will be conducted as an extension of Brown University?s established GK-12 program. In addition to this outreach effort, the PI will refine his recently designed course on stem cell engineering for graduate and undergraduate students that covers areas of stem cell biology, regenerative medicine, and hands-on laboratory exercises. Experimental findings from the research portion of this proposal will be directly applicable to this course, which is the first of its kind at Brown University.

1253189达令间充质干细胞（MSC）为修复因损伤或疾病而受损的组织提供了令人兴奋的可能性。它们的相对丰度和成为许多不同细胞类型的能力使它们作为替代自体细胞来源具有吸引力。然而，MSC收获物中存在的细胞异质性使其治疗应用复杂化，因为并非所有细胞都以相同的方式表现。个体干细胞在其局部微环境中对生物化学和机械刺激做出反应，这可以沿着沿着特定途径/谱系指导分化。近年来，虽然基质顺应性与干细胞分化有关，但很少有人关注单个细胞的机械特性如何影响其自身及其邻居的这一过程。PI的发现？的实验室研究表明，细胞的力学特性与基因表达等生物学特性相关，并在决定干细胞的分化命运方面发挥重要作用。并非所有MSC的行为都是一致的，这种异质性会显著影响样品的整体响应。了解局部细胞群的生物学和机械特性如何与更大的群体相关，可以为优化未来的再生疗法提供线索。拟议的项目将使用两种互补策略在单细胞、相邻细胞（~101个细胞）和群体（~103+细胞）水平上研究MSC样本的异质性。首先，活细胞，基因表达为基础的标志物将被用来识别分化和非分化的MSC已被化学诱导成骨和脂肪生成。其次，单细胞，弹性和粘弹性测试将评估未分化，？一部分吗分化的和完全分化的干细胞培养物。除了扩大干细胞力学的知识，这项研究计划有可能大大改善实验方法，努力指导干细胞分化和改善植入后的再生反应。 智力优势：该项目旨在研究间充质干细胞群体中存在的固有异质性。MSC通常在群体水平上进行研究，这可能会掩盖个体细胞之间存在的变化。关于单细胞在分化过程中机械特性如何变化的知之甚少，但这些知识对于充分理解细胞-基质和细胞-细胞行为至关重要。同样，阐明MSC样品中基因表达的时空模式可以帮助确定生化刺激的最佳时间和位置。为了实现这些目标，该项目将使用定制设计的分子信标来可视化活的分化MSC中的mRNA分子。原子力显微镜将用于机械表征单个细胞的弹性和粘弹性。重点将给予如何当地的细胞群体的行为，生物化学和机械，相对于整体样品。研究结果将适用于许多领域，包括干细胞生物学，细胞力学和组织工程。PI是单细胞机械生物标志物领域的先驱，这是一个令人兴奋的新领域，提供类似于基因和蛋白质表达的表型表征。通过研究机械生物标志物在干细胞分化中的作用，进一步推动这些变革性方法的成熟。更广泛的影响：作为一名生物医学工程师，PI在数学，生物学，化学和工程学的交叉领域进行研究，非常适合提供强调跨学科理解的环境。这里提出的推广工作将为普罗维登斯公立学区的中小学教育工作者提供在夏季进行团队研究的机会。专攻不同学科的教师将共同致力于融合多个专业领域的项目。该项目的目标是促进教师之间的互动，使他们能够为自己的班级开发跨学科的例子和实验。学生在整个教育过程中学习许多数学和科学课程，包括代数，几何，生物，化学和物理。然而，这些课程几乎总是作为孤立的学科教授，而现实世界的应用越来越多地跨学科。拟议的项目将作为布朗大学的延伸进行？建立GK-12计划。除了这项推广工作，PI将完善他最近为研究生和本科生设计的干细胞工程课程，涵盖干细胞生物学，再生医学和动手实验室练习等领域。本提案研究部分的实验结果将直接适用于本课程，这是布朗大学的第一个此类课程。