Intrinsic fluorescence to guide characterization and purification of stem cells

描述(由申请人提供)：本申请涉及广泛的挑战领域(03)生物标记物的发现和验证以及特定的挑战主题，03-HL-101：识别和验证血液、血管、心脏和呼吸功能障碍的诊断和治疗反应的临床相关、可量化的生物标记物。干细胞移植的未来依赖于移植前非侵入性生物标记物来表征细胞和干细胞聚集体。为了区分干细胞和分化的细胞，最小限度地需要定性，理想的情况是预测哪些细胞最有能力对特定的组织类型做出贡献。这些生物标记物的非侵入性是必不可少的，因为对干细胞的操纵可能会无意中诱导分化。许多生物标记物已经被识别和验证；然而，大多数与蛋白质表达相对应，因此需要在分析之前对细胞进行侵入性的标记。我们假设并有初步数据表明，当用多光子光学方法检测干细胞时，内在代谢特征(即NADH、FAD)可以作为干细胞的非侵入性生物标记物。与其他荧光显微镜方法相比，内在生物标志物的多光子成像分析具有优势，因为它能够深入探测多细胞聚集体(干细胞通常是这种情况)，与其他先进的光学方法兼容，如二次谐波成像(SHG)和荧光寿命成像(FLIM)，并能够在广泛的激发波长范围内进行调节。我们最近将多光子光学系统与流式细胞仪系统相结合，该系统可以高通量地分析单细胞和多细胞聚集体，并有可能根据光学输出(包括本征荧光)对细胞群体进行分类。因此，我们有可能严格定义干细胞的固有特征，并基于这些特征来纯化群体，创造出理想的移植器官。我们建议通过以下目标朝着这一目标迈出实质性的一步：目标1，识别预测人胚胎干细胞(HES)心肌细胞分化的强大内在特征；目标2，基于内在特征纯化类胚体，并确定此类分选群体的心脏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得到满足的需求。也许该项目最直接的受益者是每年超过50,000名骨髓、脐带血或浓缩外周血移植接受者，他们目前接受的是不同种类的单核细胞。如果存在一种能够在没有外在标记的情况下区分细胞并能够在鉴定后对细胞进行分类的仪器，这些移植的构成可以得到优化--这两种方法都是这一提议成功的可能性。我们将在这里特别关注心肌细胞分化，因为未来正在开发的治疗包括心脏病在内的广泛疾病的细胞疗法可能会从这种检测和细胞分选策略中受益。 与公共卫生相关：全世界每年进行数以万计的骨髓、脐带血或浓缩外周血移植，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记物，以及能够基于这些生物标记物区分和分选异种细胞的仪器，则可以筛选和优化这些移植的构成。如果成功，该项目的目标将满足这些需求。

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.