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

 描述(由申请者提供)：心脏细胞至关重要，因为心脏病是美国男性和女性的主要死亡原因。在美国，每年约有60万人死于心脏病--即每4人中就有1人死于心脏病。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(ESCs)分化而来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(BMP4)在心脏分化中起关键作用。只有少数BMP4分子与单个小鼠胚胎干细胞(MESCs)上的其受体(BMPR)结合可以诱导其分化，分别触发几个受体分子(SSEA1和CXCR4)的下调和上调。然而，它们的相关分子机制以及如何精确控制它们的特异性心脏分化在很大程度上仍不清楚。我们假设，如果我们能够以单分子(SM)敏感性和实时调整诱导剂，我们可能能够更准确地了解并指导它们向心肌细胞分化。目前，大多数分化研究是通过在诱导剂中培养ESCs，观察ESCs的形态特征，用PCR检测基因表达，或用免疫染色方法检测固定死亡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，也无法在发生时指导它们的分化。内皮细胞的分化通常需要几天的时间。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漂白。因此，它们不能连续捕捉活细胞数小时和数天的动态事件。在这个方案中，我们的目标是发展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)，并利用它们来研究单个活的MESCs上BMP4与BMPR结合诱导MESCs心脏特异性分化的分子机制和动力学，同时以SM敏感性实时定量成像单个活的MESCs上的单个SSEA1(未分化生物标记受体)和CXCR4(差异生物标记受体)以监测其分化。因此，这项研究将为指导、控制和研究单个活的ESCs分化为特定的细胞谱系提供创新的工具，并以SM敏感性实时描述其相关的分子机制和途径。这项研究还将为合理引导mESCs分化为心肌细胞提供新的见解，并为设计潜在的基于ESC的治疗心脏病奠定基础。

 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.