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（由申请人提供）：心肌细胞至关重要，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 个人中就有 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，移植由胚胎干细胞（ESC）分化而来的功能性健康心脏细胞为心脏病提供了潜在的治疗方法。形态发生蛋白 4 (BMP4) 在心脏分化中发挥关键作用，仅少数 BMP4 分子与其受体 (BMPR) 在单个小鼠 ESC (mESC) 上的结合即可诱导其分化，从而触发少数受体分子的下调和上调。然而，它们的相关分子机制以及如何精确控制它们的特定心脏分化仍然很大程度上未知，如果我们能够的话，我们也许能够更精确地理解并指导它们分化为心肌细胞。以单分子 (SM) 灵敏度实时调整诱导剂 目前，大多数分化研究是通过用诱导剂培养 ESC、观察 ESC 的形态特征、使用 PCR 探测基因表达或使用免疫染色检测来进行。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指导它们的分化，目前，使用荧光显微镜进行分化。不幸的是，荧光探针在几秒钟内就会发生光漂白，因此它们无法在数小时和数天内连续捕获活细胞的动态事件。纳米颗粒光学生物传感器（SMNOBS）和远场光稳定光学纳米镜（PHOTON），并利用它们研究单细胞上 BMP4 与 BMPR 结合诱导的 mESC 心脏特异性分化的分子机制和动力学。活的 mESCs，同时对单个活的 mESCs 上的单个 SSEA1（未分化的生物标志物受体）和 CXCR4（分化的定量生物标志物受体）进行实时成像，并具有 SM 敏感性，以监测其分化。因此，拟议的研究将提供指导、控制其分化的创新工具。研究单个活ESC向特定细胞谱系的分化，并利用SM敏感性实时描述其相关的分子机制和途径。该研究还将为合理引导mESC向特定细胞谱系的分化提供新的见解。心脏细胞，并为设计潜在的基于 ESC 的心脏病疗法奠定基础。