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

用于胚胎干细胞实时研究的光稳定多重纳米测定

• 批准号: 9132292
• 负责人: X. Nancy Xu
• 金额: $ 19.38万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9132292
• 关键词: Affinity; Binding; Biological; Biological Assay; Biological Markers; Biosensor; Blinking; CXCR4 Receptors; CXCR4 gene; Cardiac; Cardiac Death; Cardiac Myocytes; Cause of Death; Cell Lineage; Cell Therapy; Cells; Cessation of life; Characteristics; Color; Complex; Controlled Study; Detection; Disease; Down-Regulation; Event; Fluorescence; Fluorescence Microscopy; Foundations; Gene Expression; Growth; Health; Heart Diseases; Heart failure; Hour; Image; Imaging Device; Individual; Kinetics; Lasers; Lead; Life; Ligands; Link; Maps; Methods; Molecular; Monitor; Monoclonal Antibodies; Mus; Nanoscopy; Noise; Optics; Pathway interactions; Play; Process; Proteins; Research; Resolution; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Specificity; TNFRSF1A gene; Time; Time Study; Transplantation; Undifferentiated; Up-Regulation; Woman; base; biomaterial compatibility; bone morphogenetic protein 4; cell type; cooperative study; design; embryonic stem cell; fluorophore; imaging probe; innovation; insight; instrumentation; live cell imaging; loss of function; men; nanoassay; nanoparticle; plasmonics; receptor; self-renewal; sensor; single molecule; tool

 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.

 描述（由适用提供）：心脏细胞至关重要，因为心脏病是男女死亡的主要原因。在美国，大约有60万人死于心脏病，每4人死亡。心力衰竭是由功能丧失和心脏细胞死亡引起的。这是对胚胎干细胞（ESC）分化的功能和健康心脏细胞的移植提供了潜在的心脏病治疗方法。研究表明，骨形态发生蛋白4（BMP4）在心脏分化中起关键作用。仅几个BMP4分子与其受体（BMPR）在单小鼠ESC（MESC）上的结合可以诱导它们的分化，从而分别触发少数受体分子（SSEA1和CXCR4）的调节。但是，它们相关的分子机制以及如何精确控制其特定的心脏分化仍然很大未知。我们假设，如果我们可以以单分子（SM）敏感性和实时调整诱导的诱导诱导的诱导，则可以更精确地理解并将其分化为心脏细胞。当前，大多数分化研究是通过与诱导剂的聚类ESC进行的，观察ESC的形态特征，使用PCR探测基因表达，或使用免疫染色的ASSA检测固定死细胞上的生物标志物。这些方法需要几个步骤，这些步骤无法实时研究它们在活细胞中的动态分化或指导其分化。 EC的差异通常需要几天。当前，使用荧光项目的荧光显微镜是实用细胞成像的主要工具。不幸的是，荧光会在几秒钟内进行摄影。那就无法在数小时和几天内连续捕获活细胞的动态事件。在该提议中，我们旨在开发可光稳定的单分子纳米颗粒光学生物传感器（SMNOB）和远场光稳定的光学光学纳米镜检查（光子），并使用它们用于研究BMP4与BMP4的MANESC的结合，并研究MESC的Cardiac特异性分化的分子机制和动力学，并将其用于bmp4的结合，并研究其。 SSEA1（未分化）。生物标志物受体）和CXCR4（分化的生物标志物受体）在单个活MESC上实时实时sm敏感性以监测其分化。这是拟议的研究将提供创新的工具，以指导，控制和研究单个Live ESC的分化为特定的细胞谱系，并以SM敏感性实时描述其相关的分子机制和途径。该研究还将为理性地指导MESC分化为心脏细胞的新见解，并为设计潜在的基于ESC的治疗以治疗心脏病的疗法奠定基础。