This proposal is responsive to the Area of Scientific Priority """"""""Development of tools to facilitate research on the basic biology of aging"""""""" of the National Institute on Aging. Accurate, fast and unbiased methods to detect rare cells in cell suspensions for regenerative medicine are in much need and in short supply. Rapid detection of rare cells in blood and other body fluids is critical for the diagnosis of a variety of age-dependent pathological conditions, and for screening potential donors for cell therapy. Current methods rely on sophisticated, multi- parameter analysis of cell surface markers of uncertain relevance, making them impractical for reliable diagnosis. Long-term goal. Using a combination of advanced protein engineering, materials science, molecular biology of cell signaling and bioengineering, we propose a novel platform for a faster, unbiased and efficient method for rare cell capture, analysis and expansion. The principles of this platform are generally applicable to circulating progenitor cells (for endothelial, smooth muscle, or fibroblast progenitors), or tumor cells, etc. For proof of principle we will focus on 'endothelial progenitor cells'(EPC), a class of circulating adult stem cells with a central role in maintaining a functional endothelial layer and in adult neovascularization, both deteriorating with age. Using a phage display technology, our team has identified a number of peptides with specific binding to EPC. These peptides were incorporated in a terpolymer while still maintaining their affinity for EPC. Moreover, using rational peptide design based on crystallographic data, our team also synthesized a biotinylated peptide binding to the endothelial-type VEGF receptor 2, also present on EPC.
Specific Aim 1. Platform assembly. In this step we will synthesize the capture peptides with affinity for the cell surface, and will deposit the peptide-enriched attachment matrix in tissue culture wells.
Specific Aim 2. Assay optimization. In order to obtain the best discrimination between target cells and bystanders, we will test the number and composition of cell colonies derived from suspensions of known composition, as dependent on layer thickness, fiber density, polymer and peptide composition of the scaffold, as well as on the cell attachment time, washing strength and duration.
Specific Aim 3. Assay validation. We will validate our assay on a human population stratified by age, by comparing the results obtained by this method with the traditional endothelial cell colony assay, and with flow cytometry. Our proposal has both short and long term economic and educational benefits. Its implementation will immediately create several research positions, and help maintaining more others. Moreover, the proprietary technology developed may enable the formation of a start-up company, creating additional employment opportunities relating to biotechnology, material sciences and administration.
This application is in response to the Area of Scientific Priority """"""""Development of tools to facilitate research on the basic biology of aging"""""""" of the National Institute on Aging. Based on multiple collaborations and on solid preliminary data, we propose to develop a novel assay, and the corresponding instrument, for collecting from blood, analyzing and multiplying circulating stem/progenitor cells (for endothelium, vascular muscle, neurons, cardiomyocytes, etc., tumor cells). Among these of outmost importance are the endothelial progenitor cells (EPC), the focus of our proposal. Circulating levels of EPC and their endothelial differentiation capacity were associated with aging, gender, and with multiple cardiovascular pathologies and risk factors, such as atherosclerosis, heart failure, diabetes, hypertension, obesity, smoking, etc. EPC are also a major cell type intended to be used for cell therapy for heart, lung and blood diseases. Therefore, both diagnosis and cell therapy protocols will substantially benefit from this improved tool of progenitor cells detection in circulation, in bone marrow aspirates, or after multiplication in tissue culture. The project will have both short term economic impact and long term sustainability by creation of a company for commercialization of the assay kit.
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