Magnetic Steering and Longitudinal Visualization of Stem Cells for Trabecular Meshwork Therapy in Glaucoma

项目概要/摘要 青光眼是致盲的主要原因，目前的治疗方法不足。青光眼的主要危险因素， 唯一可治疗的危险因素是眼内压升高。目前的降眼压疗法也失败了 通常，因此人们对新型IOP控制策略仍然抱有极大的兴趣。小梁网 (TM)，决定眼压的关键组织，减少了青光眼的细胞构成，这使得许多研究小组， 研究以干细胞为基础的TM疗法。这种疗法的一个障碍是细胞递送：目前的方法 对TM具有低的细胞递送效率和特异性，并且不能将细胞递送到TM的所有部分。 这里提出了一种用于将干细胞“引导”到TM的新技术解决方案，其可以另外地 用于可视化干细胞输送和监测干细胞凋亡。这种方法将在几个 高眼压模型。其关键技术是超顺磁性和光敏纳米粒子。 它们的超顺磁性意味着细胞吸收了这些纳米颗粒后， 通过放置在利姆布斯的磁铁转向TM。它们的光敏性意味着细胞可以通过 超声/光声成像在活体眼睛中的应用。这些纳米颗粒的功能将进一步 通过使用活性半胱天冬酶-3的光敏标记物来监测干细胞凋亡来增强。我们的整体 目的是验证这些技术作为监测和操纵阀杆的安全有效方法 细胞的TM，从而恢复青光眼患者的眼内压（IOP）稳态。 根据我们重要的初步数据，提出了实现这一长期目标的三个具体目标。在aim中 1，将合成和表征用于监测细胞凋亡的新型胱天蛋白酶-3敏感性报告基因， 将优化用于引导干细胞到TM的磁体。在目标2中，提供了一种能够对标记的细胞成像的仪器， 全眼的干细胞，包括干细胞分布和凋亡的纵向监测， 开发在aim 3中，干细胞将被输送到两个青光眼模型中的TM，并且它们恢复青光眼的能力。 将评价IOP稳态。超声/光声成像监测干细胞输送的能力 TM和干细胞凋亡也将得到验证;间充质干细胞（MSC）将被比较 分化的诱导多能干细胞（iPSC-TM）在恢复IOP稳态中的功效。这 该项目具有高度创新性：它是第一项将干细胞作为治疗眼部疾病的一部分进行引导和可视化的研究。 高血压这也是第一次比较MSC与iPSC-TM治疗高眼压症的疗效。 正如我们强有力的初步数据所表明的那样，我们希望发现干细胞可以有效地 通过放置在利姆布斯的简单磁铁选择性地转向TM，持续时间短至15分钟;并且它将 可以准确地监测干细胞在眼睛中的位置和干细胞随时间的凋亡。此外，本发明还 我们预期，如在2个青光眼模型中所测试的，以这种方式操纵的干细胞将改善TM功能。

Project Summary/Abstract Glaucoma is a major cause of blindness and current treatments are insufficient. A major risk factor for glaucoma, and the only treatable risk factor, is elevated intraocular pressure (IOP). Current IOP-lowering therapies fail too often, and thus there continues to be great interest in novel IOP control strategies. The trabecular meshwork (TM), the key tissue determining IOP, has reduced cellularity in glaucoma, which has led a number of groups to study stem cell-based therapies for the TM. An obstacle to such therapies is cell delivery: current approaches have low cell delivery efficiency and specificity for the TM, and cannot deliver cells to all parts of the TM. Here a novel technological solution for “steering” stem cells to the TM is proposed, which can be additionally used to visualize stem cell delivery and to monitor stem cell apoptosis. This approach will be tested in several models of ocular hypertension. The key technology is superparamagnetic and photosensitive nanoparticles. Their superparamagnetic properties mean that cells which have taken up these nanoparticles can be rapidly steered to the TM by a magnet placed at the limbus. Their photosensitivity means that cells can be visualized by ultrasound/photoacoustic imaging in the living eye. The functionality of these nanoparticles will be further enhanced by using a photosensitive marker of active caspase-3 to monitor stem cell apoptosis. Our overall objective is to validate these technologies as a safe and effective approach for monitoring and steering of stem cells to the TM, thereby restoring intraocular pressure (IOP) homeostasis in glaucoma patients. Three specific aims towards this long-term goal are proposed, building on our significant preliminary data. In aim 1, a novel caspase-3-sensitive reporter for monitoring apoptosis will be synthesized and characterized, and magnets for steering stem cells to the TM will be optimized. In aim 2, an instrument capable of imaging of labeled stem cells in whole eyes, including longitudinal monitoring of stem cell distribution and apoptosis, will be developed. In aim 3, stem cells will be delivered to the TM in two glaucoma models, and their ability to restore IOP homeostasis will be evaluated. The ability of ultrasound/photoacoustic imaging to monitor stem cell delivery to the TM and stem cell apoptosis will also be validated; and mesenchymal stem cells (MSCs) will be compared to differentiated induced pluripotent stem cells (iPSC-TMs) for their efficacy in restoring IOP homeostasis. This project is highly innovative: it the first study to steer and visualize stem cells as part of a treatment for ocular hypertension. It is also the first to compare the efficacy of MSCs vs. iPSC-TMs for treating ocular hypertension. We expect, as suggested by our strong preliminary data, to discover that stem cells can be efficiently and selectively steered to the TM by a simple magnet placed at the limbus for as little as 15 minutes; and that it will be possible to accurately monitor the location of stem cells in the eye and stem cell apoptosis over time. Further, we expect that TM function will be improved by stem cells steered in this way, as tested in 2 glaucoma models.