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Intrinsic fluorescence to guide characterization and purification of stem cells

内在荧光指导干细胞的表征和纯化

基本信息

批准号：
7815748
负责人：
Brenda M Ogle
金额：
$ 100万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-07 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7815748
关键词：
Activities of Daily Living Address Area Biological Markers Biological Process Blood Blood Vessels Bone Marrow Cardiac Cardiac Myocytes Cell Separation Cells Clinical Coupled Cues Data Detection Diagnostic Disease Down-Regulation Event Exhibits Flavin-Adenine Dinucleotide Flow Cytometry Fluorescence Fluorescence Microscopy Functional disorder Future Generations Goals Heart Diseases Hematopoietic Neoplasms Histocompatibility Testing Image Image Analysis Label Metabolic Metabolism Methods Microscopy Mitochondria Molecular Mononuclear NADH Nicotinamide adenine dinucleotide Optical Methods Optics Output Population Population Heterogeneity Property Reporter Reporting Sorting - Cell Movement Stem cell transplant Stem cells System Therapeutic Transplant Recipients Transplantation Umbilical Cord Blood Validation base clinically relevant fluorescence imaging fluorophore human embryonic stem cell induced pluripotent stem cell instrument instrumentation oxidation peripheral blood population based promoter protein expression public health relevance respiratory response second harmonic stem cell biology stem cell differentiation stem cell fate stem cell population success therapy development

项目摘要

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03) Biomarkers Discovery and Validation and specific Challenge Topic, 03-HL-101: Identify and validate clinically relevant, quantifiable biomarkers of diagnostic and therapeutic responses for blood, vascular, cardiac and respiratory dysfunction. The future of stem cell transplantation is dependent on noninvasive biomarkers to characterize cells and stem cell aggregates prior to transplantation. Characterization is needed minimally to distinguish stem cells from differentiated cells and ideally to predict the cells best poised to contribute to a specific tissue type. The noninvasive quality of such biomarkers is essential, since manipulation of stem cells can unintentionally induce differentiation. Many biomarkers have been identified and validated; however most correspond to protein expression and so require the invasive application of extrinsic labels to cells prior to analysis. We hypothesize and have preliminary data to suggest that intrinsic metabolic signatures (i.e., NADH, FAD) can serve as noninvasive biomarkers of stem cells when detected with multiphoton optical based approaches. Multiphoton imaging analysis of intrinsic biomarkers is advantageous over other fluorescence microscopy methods due to its ability to probe deeply within multicellular aggregates (as is frequently the case with stem cells), compatibility with other advanced optical methods such second harmonic generation imaging (SHG) and fluorescence lifetime imaging (FLIM) and ability to tune over a broad range of excitation wavelengths. We have recently coupled multiphoton optics to a flow cytometry system that can analyze single cell and multicell aggregates in a high throughput manner, with potential to sort cell populations based on optical outputs, including intrinsic fluorescence. Thus we have the potential to rigorously define intrinsic signatures of stem cells and to purify populations based on these signatures, creating an ideal graft for transplantation. We propose to take substantial steps toward this goal via the following aims: Aim 1, to identify robust intrinsic signatures predictive of cardiomyocyte differentiation of human embryonic stem cells (hES) and Aim 2, to purify embryoid bodies based on intrinsic signatures and determine the cardiogenic differentiation potential and functional capacity of such sorted populations. If successful, we will have fulfilled a long unmet need for clinical stem cell transplantation and basic stem cell biology. Perhaps the most immediate benefactors of this project are the over 50,000 bone marrow, cord blood or enriched peripheral blood transplant recipients each year who currently receive a heterogeneous population of mononuclear cells. The composition of these transplants could be optimized if there existed an instrument capable of distinguishing cells without extrinsic markers and capable of sorting cells after identification - both are possibilities with success of this proposal. We will look specifically at cardiomyocyte differentiation here as future cellular therapies under development for the treatment of a broad spectrum of diseases including heart disease will potentially benefit from such detection and cell sorting strategies. PUBLIC HEALTH RELEVANCE: Tens of thousands of bone marrow, cord blood or enriched peripheral blood transplants are performed each year worldwide to treat a myriad of blood cancers. The composition of these transplants could be screened and optimized if there existed non-invasive biomarkers capable of predicting stem cell fate and instrumentation capable of distinguishing and sorting heterogeneous cells based on such biomarkers. If successful, the aims of this project will fulfill these needs.

描述（由申请人提供）：本申请涉及广泛的挑战领域（03）生物标志物发现和验证以及特定的挑战主题03-HL-101：鉴别和验证血液、血管、心脏和呼吸功能障碍诊断和治疗反应的临床相关、可量化的生物标志物。干细胞移植的未来取决于在移植前表征细胞和干细胞聚集体的非侵入性生物标志物。最低限度地需要表征来区分干细胞和分化的细胞，并且理想地预测最好准备贡献于特定组织类型的细胞。这种生物标志物的非侵入性质量是必不可少的，因为干细胞的操作可能无意中诱导分化。许多生物标志物已被鉴定和验证;然而，大多数对应于蛋白质表达，因此需要在分析之前将外源性标记侵入性地应用于细胞。我们假设并有初步数据表明，内在代谢特征（即，NADH、FAD）在用基于多光子光学的方法检测时可以用作干细胞的非侵入性生物标志物。内在生物标志物的多光子成像分析优于其他荧光显微镜方法，这是由于其能够深入探测多细胞聚集体（如干细胞的情况下），与其他先进的光学方法，如二次谐波产生成像（SHG）和荧光寿命成像（FLIM）的兼容性，并能够在宽范围内调谐激发波长。我们最近将多光子光学器件耦合到流式细胞术系统，该系统可以以高通量方式分析单细胞和多细胞聚集体，具有基于光学输出（包括固有荧光）对细胞群进行分选的潜力。因此，我们有可能严格定义干细胞的内在特征，并根据这些特征纯化细胞群，为移植创造理想的移植物。我们建议通过以下目标朝着这一目标采取实质性步骤：目标1，鉴定预测人胚胎干细胞（hES）心肌细胞分化的强大内在特征，目标2，基于内在特征纯化胚状体，并确定此类分选群体的心源性分化潜力和功能能力。如果成功，我们将满足临床干细胞移植和基础干细胞生物学长期未满足的需求。也许这个项目最直接的受益者是每年超过50，000名骨髓、脐带血或富集外周血移植接受者，他们目前接受的是异质性单核细胞群体。如果存在能够在没有外部标记的情况下区分细胞并且能够在鉴定后分选细胞的仪器，则可以优化这些移植物的组成-这两者都是该提议成功的可能性。我们将特别关注心肌细胞分化，因为未来正在开发的用于治疗包括心脏病在内的广泛疾病的细胞疗法将可能受益于这种检测和细胞分选策略。公共卫生相关性：全世界每年进行数以万计的骨髓、脐带血或富集外周血移植，以治疗无数的血癌。如果存在能够预测干细胞命运的非侵入性生物标志物和能够基于这些生物标志物区分和分选异质细胞的仪器，则可以筛选和优化这些移植物的组成。如果成功，该项目的目标将满足这些需求。