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）生物标志物的发现和验证以及特定的挑战主题，03- hl -101：确定和验证血液，血管，心脏和呼吸功能障碍的诊断和治疗反应的临床相关，可量化的生物标志物。干细胞移植的未来取决于非侵入性生物标志物在移植前表征细胞和干细胞聚集体。表征是区分干细胞和分化细胞的最低要求，理想情况下是预测最适合形成特定组织类型的细胞。这种生物标记物的非侵入性至关重要，因为对干细胞的操作可能会无意中诱导分化。许多生物标志物已被鉴定和验证；然而，大多数与蛋白质表达相对应，因此需要在分析之前侵入性地对细胞进行外部标记。我们假设并有初步数据表明，当使用基于多光子光学的方法检测时，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，因为它能够深入探测多细胞聚集体（如干细胞），与其他先进的光学方法（如二次谐波成像（SHG）和荧光寿命成像（FLIM））兼容，并且能够在广泛的激发波长范围内进行调谐。我们最近将多光子光学耦合到流式细胞术系统中，该系统可以以高通量的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）对细胞群进行分类的潜力。因此，我们有可能严格定义干细胞的内在特征，并在这些特征的基础上纯化群体，创造一种理想的移植移植物。我们建议通过以下目标采取实质性步骤实现这一目标：目标1，确定预测人类胚胎干细胞（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，并确定这些分类群体的心源分化潜力和功能能力。如果成功，我们将满足长期未满足的临床干细胞移植和基础干细胞生物学的需求。也许这个项目最直接的捐助者是每年超过50,000名骨髓、脐带血或浓缩外周血移植受者，他们目前接受的是异质的单个核细胞。如果存在一种能够在没有外部标记的情况下区分细胞并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-这两种可能性都是本建议成功的可能性。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用于治疗包括心脏病在内的广泛疾病的细胞疗法将潜在地受益于这种检测和细胞分选策略。

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.