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

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 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.