Development of an In Vivo Stem Cell Tracking Method Using Ultrasound Imaging

描述（由申请人提供）：该申请涉及广泛的挑战领域（14）干细胞，以及特定的挑战主题14- gb -102：成像干细胞迁移和分化。在西方国家，由心肌梗死和随后的不良左心室重构引起的充血性心力衰竭是发病率、死亡率和医疗保健支出的主要原因。尽管心力衰竭的治疗取得了进展，但目前的药物和机械治疗并不能解决主要的潜在问题，即心肌细胞的死亡。心脏自身有限的自我修复能力进一步加剧了当代治疗方法的局限性。这一困境激发了对心脏修复治疗的探索，并导致了干细胞治疗性输送到损伤心肌的探索。在急性心肌梗死的动物研究中，细胞制剂如未分离的成人骨髓或骨髓间充质干细胞（MSCs）的递送可以改善灌注和心室功能指标。有益作用的机制尚不完全清楚，部分原因是对分娩后干细胞命运的连续跟踪方法有限。最近的数据支持这样的假设：MSCs的旁分泌功能主要是促进心肌愈合、血管生成、内源性自我修复和减轻炎症。令人鼓舞的临床前结果迅速导致了急性梗死细胞治疗的临床试验，这就产生了对安全、无创、易于获得的方法的迫切需求，用于追踪和定量输送后的干细胞。事实上，治疗方案的优化和对治疗性细胞递送机制的了解，与连续跟踪干细胞生物分布和体内活力的能力有关。目前具有临床潜力的干细胞可视化成像方法利用磁共振或核成像探针。这些技术在辐射暴露、空间分辨率、对干细胞基因修饰的要求、技术复杂性以及由此产生的连续成像障碍等方面都有局限性。因此，为了解决这一成像技术上的差距，目前的研究应用将开发一种基于超声的技术，用于在体内观察外源性干细胞。声学活性充满气体的聚合物微泡将被开发为造影剂，用于在治疗前对MSCs进行体外标记。要测试的总体假设是，MSC对微泡的摄取将使临床可用的超声扫描仪能够在体内可视化干细胞运输。该项目将从体外验证和优化微泡化学、声学参数和细胞毒性测试开始；在小动物身上进行概念的体内验证；然后将成像技术应用于具有临床意义的MSC治疗大动物模型。这些研究将最终发展出一种安全、无创、便携、高分辨率、活体、实时的超声方法，用于观察治疗输送的间充质干细胞。预计干细胞的超声造影增强成像将在评估细胞疗法以减轻人类缺血性心脏病和充血性心力衰竭负担方面发挥关键作用。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells, and specific Challenge Topic 14-GB-102: Imaging Stem Cell Migration and Differentiation. Congestive heart failure resulting from myocardial infarction and subsequent adverse left ventricular remodeling is a major cause of morbidity, mortality and health care spending in Western nations. Despite advances in the treatment of heart failure, current pharmacologic and mechanical therapies do not resolve the principal underlying issue, which is the death of cardiomyocytes. The limitations of contemporary therapies are further exacerbated by the heart's own limited ability for self-repair. This dilemma has motivated a search for reparative treatments for the heart, and led to the exploration of therapeutic delivery of stem cells to injured myocardium. In animal studies of acute myocardial infarction, delivery of cell preparations such as unfractionated adult bone marrow or marrow-derived mesenchymal stem cells (MSCs) has improved perfusion and indices of ventricular function. The mechanisms of beneficial effect are incompletely understood, partly because methods are limited for serial tracking of stem cell fate after delivery. Recent data favor the hypothesis that paracrine functions of MSCs predominate to facilitate myocardial healing, angiogenesis, endogenous self-repair, and mitigate inflammation. Encouraging pre-clinical results have rapidly led to clinical trials of cell therapy in acute infarction, which has created a critical need for safe, non-invasive, readily available methods for tracking and quantifying stem cells after delivery. Indeed, the optimization of treatment protocols and acquisition of mechanistic insights into therapeutic cell delivery, are linked to the ability to serially track stem cell biodistribution and viability in vivo. Current imaging methods with clinical potential for visualizing stem cells utilize probes for magnetic resonance or nuclear imaging. These have limitations in terms of radiation exposure, spatial resolution, the requirement for genetic modification of stem cells, technical complexity, and resulting impediments to serial imaging. Accordingly, to address this gap in imaging technology, the current research application will develop an ultrasound-based technology for visualizing exogenously delivered stem cells in vivo. Acoustically active gas-filled polymer microbubbles will be developed as contrast agents for ex vivo labeling of MSCs prior to therapeutic delivery. The overall hypothesis to be tested is that MSC uptake of the microbubbles will enable in vivo visualization of stem cell trafficking using clinically available ultrasound scanners. The project will begin with in vitro validation and optimization of microbubble chemistry, testing of acoustic parameters and cytotoxicity; proceed to in vivo proof of concept in small animals; and then apply the imaging technology to a clinically relevant large animal model of MSC therapy. These studies will culminate in the development of a safe, non-invasive, portable, high resolution, in vivo, real time ultrasound method for visualizing therapeutically delivered MSCs. It is anticipated that contrast- enhanced ultrasound imaging of stem cells will play a pivotal role in the evaluation of cell-based therapies for ameliorating the burden of ischemic heart disease and congestive heart failure in human populations. PUBLIC HEALTH RELEVANCE: Cell-based therapies, such as administration of mesenchymal stem cells (MSCs), are a promising new approach to repair the heart after myocardial infarction. Successful implementation of this strategy requires a greater insight into the mechanism by which MSCs confer therapeutic benefit, which in turn requires an understanding of the distribution and fate of MSCs after local or systemic delivery. This proposal aims to develop an ultrasound-based approach for visualizing MSCs that can be used in patients. This new imaging technology may facilitate the clinical application of MSC therapy, which may ultimately decrease death and disability from myocardial infarction and congestive heart failure.