CAREER: Biophysical Control of Cardiac Differentiation in Patient Specific Cardiac Stem Cells

PI：Black，Lauren D.Proposal编号：1351241标题：Career：BiPhysialControl of Different Stem Cellers从患者特定的干细胞中获得功能心肌细胞的现有方法效率较低，部分原因是它们仅模拟体内存在的细胞环境的化学信号方面。这项建议中描述的研究旨在通过了解细胞外部的生物物理和生化环境如何影响心脏内驻留干细胞转化为心肌细胞的能力，来加强我们对患者特定干细胞如何被影响分化为心肌细胞的理解。这项工作特别重要，因为目前许多干细胞分化为心肌细胞的方法都涉及到生长因子的使用，效率非常低。如果成功，拟议中的研究将在推进基础科学方面产生影响，并在使医学应用更接近临床方面具有巨大潜力。教育和外联活动与拟议的研究很好地结合在一起，涉及当地和市中心的高中以及为本科生和研究生编制的课程。最近，cKit+心脏前体细胞(CPC)因其相对丰富、易于分离以及能够分化为心脏中的三种主要细胞类型：平滑肌细胞、内皮细胞和心肌细胞(CMS)而被选为最有前途的心脏修复细胞来源之一。用标准的基于可溶性因子的方法将CPC分化为功能性CMS的效率一直较低，但与天然CMS共培养或体内移植显示了增强的效果，表明组织微环境中的其他因素可能对促进CPC的心脏分化起重要作用。这一建议的总体假设是，整合底物特性和生物电信号将导致儿科患者来源的CPC功能分化的增强。这一假设将通过以下几个方面得到验证：1)使用生长因子或基于小分子的分化方案，建立来自儿童患者的cKit+CP克隆群体心脏分化的基线；2)评估底物硬度、底物组成和静息膜电位对CPC克隆心脏分化的个体影响，并阐明具体的机械转导和分化途径；以及3)确定生物物理和生物电信号在促进cKit+CPC分化为功能性CMS过程中的交互作用。为了实现这些目标，PI实验室以前开发的技术将用于分离/表征心脏细胞外基质，创建具有不同机械性能的底物，并控制细胞的静息膜电位。细胞的功能分化将通过基因和蛋白质表达以及钙瞬变成像和动态牵引力显微镜测量来评估。

PI: Black, Lauren D.Proposal Number: 1351241Title: CAREER: Biophysical Control of Cardiac Differentiation in Patient Specific Cardiac Stem CellsCurrent methods for deriving functional cardiac muscle cells from patient-specific stem cells have resulted in low efficiency in part because they only mimic the chemical signaling aspect of the environment of the cell present in the body. The studies described in this proposal seek to enhance our understanding of how patient-specific stem cells can be influenced to differentiate to cardiac muscle cells by understanding how the biophysical and biochemical environment external to the cells affects the ability of resident stem cells in the heart to turn into cardiac muscle. This work is particularly relevant because many of the current methods for differentiation of stem cells to cardiac muscle cells involve the use of growth factors and are highly inefficient. If successful, the proposed studies will be impactful in terms of advancing fundamental science, and for having significant potential in bringing medical applications closer to the clinic. Education and outreach activities are well integrated with the proposed research and involve local and inner city high schools as well as curriculum development for undergraduate and graduate students. Recently, ckit+ cardiac progenitor cells (CPCs) have been singled out as one of the most promising sources of cells for cardiac repair because of their relative abundance, ease of isolation and their ability to differentiate into the three major cells types in the heart: smooth muscle cells endothelial cells and cardiomyocytes (CMs). The efficiency of differentiation of CPCs to functional CMs with standard soluble factor-based protocols has been low, but co-culture with native CMs or implantation in vivo have demonstrated an enhanced effect, indicating that other factors in the tissue microenvironment may be important for promoting cardiac differentiation of CPCs. The overall hypothesis of this proposal is that integrating substrate properties and bioelectric signaling will lead to enhancements in functional differentiation of pediatric patient derived CPCs. This hypothesis will be tested by: 1) establishing a baseline of cardiac differentiation of clonal populations of ckit+ CPCs derived from pediatric patients using growth factor or small molecule based differentiation protocols; 2) assessing the individual effects of substrate stiffness, substrate composition and resting membrane potential on cardiac differentiation of CPC clones and elucidate the specific mechanotransductive and differentiation pathways involved; and 3) determining the interactive effects of biophysical and bioelectric signaling in promoting the development of functional CMs from ckit+ CPCs. To carry out these aims, techniques previously developed in the PI's lab will be used to isolate/ characterize cardiac extracellular matrix, create substrates with different mechanical properties, and control resting membrane potential of cells. Functional differentiation of cells will be assessed via gene and protein expression, as well as calcium transient imaging and contraction force measurements via dynamic traction force microscopy.