Photostable Multiplexing NanoAssays for Real-Time Study of Embryonic Stem Cells

 描述（由申请人提供）：心脏细胞至关重要，因为心脏病是美国男性和女性死亡的主要原因。在美国，每年约有60万人死于心脏病，即每4例死亡中就有1例。心力衰竭是由心脏细胞功能丧失和死亡引起的。因此，从胚胎干细胞（ESC）分化的功能和健康的心脏细胞的移植为心脏病提供了潜在的治疗方法。研究表明骨形态发生蛋白4（BMP-4）在心脏分化中起关键作用。在单个小鼠胚胎干细胞（mESC）上仅几个BMP 4分子与其受体（BMPR）的结合可以诱导它们的分化，分别触发几个受体分子（SSEA 1和CXCR 4）的下调和上调。然而，其相关的分子机制以及如何精确控制其特异性心脏分化仍然是未知的。我们假设，如果我们能够以单分子（SM）灵敏度和实时调节诱导剂，我们可能能够更精确地理解和指导它们分化为心肌细胞。目前，大多数分化研究是通过用诱导剂培养ESCs，观察ESCs的形态学特征，使用PCR探针基因表达，或使用免疫染色法检测固定死细胞上的生物标志物来进行的。这些方法需要几个步骤，无法实时研究它们在活细胞中的动态分化或在分化发生时指导它们的分化。EC的分化通常需要数天。目前，使用荧光探针的荧光显微镜是活细胞成像的主要工具。不幸的是，荧光探针在几秒钟内就会褪色。因此，它们不能在数小时和数天内连续捕获活细胞的动态事件。在这项提议中，我们的目标是开发光稳定的多色单分子纳米颗粒光学生物传感器（SMNOBS）和远场光稳定光学纳米显微镜（PHOTON），并使用它们来研究单个活mESCs上BMP 4与BMPR结合诱导mESCs心脏特异性分化的分子机制和动力学，同时对单个SSEA 1进行定量成像，（未分化生物标志物受体）和CXCR 4图1示出了具有SM灵敏度的真实的时间的单个活mESC上的分化生物标志物受体（分化生物标志物受体）的细胞模型，以监测它们的分化。因此，本研究将为指导、控制和研究单个活ESC分化为特定细胞谱系提供创新工具，并在具有SM敏感性的情况下真实的时间描绘其相关分子机制和途径。该研究还将为合理引导mESC分化为心脏细胞提供新的见解，并为设计潜在的基于ESC的治疗心脏疾病的疗法奠定基础。

 DESCRIPTION (provided by applicant): Cardiac cells are critically important, because heart disease is the leading cause of death in both men and women is US. About 600,000 people die of heart disease in the US every year-that's 1 in every 4 deaths. Heart failure is caused by loss of function and death of cardiac cells. Thus, transplantation of functional and healthy cardiac cells differentiated from embryonic stem cells (ESCs) offers potential treatments for heart disease. Studies have showed that bone morphogenetic protein-4 (BMP4) plays key roles in cardiac differentiation. The binding of just a few BMP4 molecules with its receptors (BMPR) on single mouse ESCs (mESCs) can induce their differentiation, triggering down and up regulation of a few receptor molecules (SSEA1 and CXCR4), respectively. However, their related molecular mechanisms and how to precisely control their specific cardiac differentiation remain largely unknown. We hypothesize that we may be able to more precisely understand and direct their differentiation into cardiac cells if we can tune the inducer with single-molecule (SM) sensitivity and in real-time. Currently, most differentiation studies were performed by culturing ESCs with inducing agents, observing morphological characteristics of ESCs, using PCR to probe gene expression, or using immunostaining assays to detect biomarkers on fixed dead cells. These methods require several steps, which are unable to real-time study their dynamic differentiation in live cells or guide their differentiation as it occurs. The differentiation of ECs often takes days. Currently, fluorescence microscopy using fluorescence probes is the primary tool for live cell imaging. Unfortunately, fluorescence probes photo-bleach within seconds. Thus, they cannot continuously capture the dynamic events of live cells over hours and days. In this proposal, we aim to develop photostable multicolored single-molecule nanoparticle optical biosensors (SMNOBS), and far-field photostable optical nanoscopy (PHOTON) and use them to study molecular mechanisms and kinetics of cardiac specific differentiation of mESCs induced by the binding of BMP4 with BMPR on single live mESCs, while simultaneously and quantitatively imaging individual SSEA1 (undifferentiated biomarker receptor) and CXCR4 (differentiated biomarker receptor) on single live mESCs in real time with SM sensitivity to monitor their differentiation. Thus, the proposed study will offer innovative tools to direct, contol and study the differentiation of single live ESCs into a specific cell lineage, and depict their related molecular mechanisms and pathways in real time with SM sensitivity. The study will also offer new insights into rationally directing differentiation of mESCs into cardiac cells, and lay down the foundation for design of potential ESC-based therapies to treat heart diseases.