CellTrap: A novel solid phase platform for analysis of stem/progenitor cells

CellTrap：用于分析干细胞/祖细胞的新型固相平台

• 批准号: 7942924
• 负责人: STUART L COOPER
• 金额: $ 65.84万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942924
• 关键词: Adhesions; Adult; Affinity; Age; Aging; Antibodies; Area; Aspirate substance; Atherosclerosis; Binding; Biological Assay; Biology of Aging; Biomedical Engineering; Biotechnology; Biotinylation; Blood; Blood Circulation; Blood Vessels; Body Fluids; Bone Marrow; Cardiac Myocytes; Cardiovascular Pathology; Cell Adhesion; Cell Therapy; Cell surface; Cell-Matrix Junction; Cells; Chemical Engineering; Child; Collaborations; Collecting Cell; Collection; Communities; Computer Simulation; Consensus; Data; Deposition; Detection; Development; Diabetes Mellitus; Diagnosis; Discrimination; Economics; Employment Opportunities; Endothelial Cells; Fiber; Fibroblasts; Figs - dietary; Flow Cytometry; Funding; Gender; Goals; Health; Heart; Heart failure; Hematological Disease; Human; Hypertension; In Situ; In Vitro; Label; Laser Scanning Cytometry; Liquid substance; Lung; Magnetism; Medicine; Methodology; Methods; Modeling; Molecular Biology; Muscle; National Institute on Aging; Nature; Neurons; Obesity; Patients; Peptides; Phage Display; Phase; Phase-Contrast Microscopy; Phenotype; Polymers; Population; Positioning Attribute; Precipitation; Process; Protein Engineering; Protocols documentation; Regenerative Medicine; Research; Risk Factors; Role; Science; Scientist; Screening procedure; Signal Transduction; Smoking; Smooth Muscle; Solid; Specificity; Stem cells; Streptavidin; Surface; Suspension substance; Suspensions; Technology; Testing; Thick; Time; Tissues; Validation; Vascular Endothelial Growth Factor Receptor; Vascular Endothelium; adult stem cell; age related; aldehyde dehydrogenases; base; cell type; commercialization; data mining; density; design; economic impact; improved; instrument; magnetic beads; neoplastic cell; neovascularization; novel; progenitor; public health relevance; rapid detection; repaired; response; scaffold; stem; tissue culture; tool; tool development

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.

描述（由申请人提供）：本提案响应国家老龄问题研究所的科学优先领域“开发工具以促进老龄化基础生物学研究”。准确，快速和公正的方法来检测再生医学细胞悬浮液中的稀有细胞是非常需要和短缺的。快速检测血液和其他体液中的稀有细胞对于诊断各种年龄依赖性病理状况以及筛选细胞治疗的潜在供体至关重要。目前的方法依赖于不确定相关性的细胞表面标志物的复杂的多参数分析，使得它们对于可靠的诊断是不切实际的。 长期目标。利用先进的蛋白质工程，材料科学，细胞信号传导的分子生物学和生物工程的组合，我们提出了一个新的平台，用于更快，无偏见和有效的方法，用于稀有细胞的捕获，分析和扩增。该平台的原理通常适用于循环祖细胞（内皮、平滑肌或成纤维细胞祖细胞）或肿瘤细胞等。为了证明原理，我们将重点关注“内皮祖细胞”（EPC），一类循环成体干细胞，在维持功能性内皮层和成体新血管形成中发挥核心作用，两者均随年龄而恶化。利用噬菌体展示技术，我们的团队已经鉴定了许多与EPC特异性结合的肽。将这些肽掺入到载体中，同时仍保持其对EPC的亲和力。此外，使用基于晶体学数据的合理肽设计，我们的团队还合成了与内皮型VEGF受体2结合的生物素化肽，该受体也存在于EPC上。 具体目标1。平台组装。在该步骤中，我们将合成对细胞表面具有亲和力的捕获肽，并将富集肽的附着基质存款在组织培养威尔斯孔中。 具体目标2。测定优化。为了获得靶细胞和旁观者之间的最佳区分，我们将测试源自已知组成的悬浮液的细胞集落的数量和组成，这取决于支架的层厚度、纤维密度、聚合物和肽组成，以及细胞附着时间、洗涤强度和持续时间。 具体目标3。含量测定验证。我们将通过比较该方法与传统的内皮细胞集落测定法和流式细胞术获得的结果，在按年龄分层的人群中验证我们的测定法。 我们的建议有短期和长期的经济和教育效益。它的实施将立即创造几个研究职位，并有助于维持更多的其他职位。此外，所开发的专利技术可能有助于成立一家新公司，创造与生物技术、材料科学和行政管理有关的额外就业机会。 公共卫生相关性：本申请是为了响应国家老龄问题研究所的科学优先领域“开发工具以促进对老龄化基础生物学的研究”。基于多方合作和扎实的初步数据，我们建议开发一种新型检测方法和相应的仪器，用于从血液中收集、分析和繁殖循环干/祖细胞（用于内皮细胞、血管肌肉、神经元、心肌细胞等），肿瘤细胞）。其中最重要的是内皮祖细胞（EPC），我们的建议的重点。EPC的循环水平及其内皮分化能力与年龄、性别以及多种心血管病理和危险因素相关，如动脉粥样硬化、心力衰竭、糖尿病、高血压、肥胖、吸烟等。EPC也是用于心脏、肺和血液疾病细胞治疗的主要细胞类型。因此，诊断和细胞治疗方案都将大大受益于这种在循环中、骨髓抽吸物中或在组织培养物中增殖后检测祖细胞的改进工具。该项目将通过创建一家公司来实现检测试剂盒的商业化，从而产生短期经济影响和长期可持续性。