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

项目摘要/摘要 青光眼是导致失明的主要原因，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压升高。目前的降眼压疗法也失败了 因此，人们对新的眼压控制策略仍然非常感兴趣。小梁网 (TM)，决定眼压的关键组织，已经减少了青光眼的细胞密度，这导致了一些小组 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输送：目前的方法 TM的细胞输送效率和特异性较低，并且不能将细胞输送到TM的所有部分。 这里提出了一种新的技术解决方案，将干细胞“引导”到TM，该方案可以另外 用于可视化干细胞输送和监测干细胞凋亡。这一方法将在几个方面进行测试 高眼压模型。关键技术是超顺磁性和光敏纳米颗粒。 它们的超顺磁性意味着摄取这些纳米颗粒的细胞可以迅速 通过放置在边缘的磁铁引导到TM。它们的光敏性意味着细胞可以通过 活眼的超声/光声成像。这些纳米粒子的功能将进一步 通过使用活性caspase-3的光敏标记物来监测干细胞凋亡而增强。我们的整体 目的是验证这些技术是安全和有效的监测和转向阀杆的方法 Tm，从而恢复青光眼患者的眼压(IOP)动态平衡。 在我们重要的初步数据的基础上，提出了实现这一长期目标的三个具体目标。在AIM 1，将合成并表征一种新型的caspase-3敏感的细胞凋亡监测报告，以及 引导干细胞进入TM的磁铁将得到优化。在目标2中，能够对标记物进行成像的仪器 全眼干细胞，包括对干细胞分布和凋亡的纵向监测，将是 发展起来的。在AIM 3中，干细胞将在两个青光眼模型中被输送到TM，并恢复它们的能力 将评估眼压动态平衡。超声/光声成像监测干细胞输送的能力 还将验证TM和干细胞的凋亡；并将比较间充质干细胞(MSCs) 目的探讨分化诱导多能干细胞(IPSC-TMS)恢复眼压稳态的效果。这 该项目极具创新性：它是将干细胞作为眼科治疗的一部分并将其可视化的第一项研究 高血压。这也是第一次比较骨髓间充质干细胞和IPSC-TMS治疗高眼压的疗效。 我们希望，正如我们强有力的初步数据所表明的那样，干细胞可以有效地和 通过在边缘放置一块简单的磁铁，选择性地转向TM，时间短至15分钟；它将 随着时间的推移，可以准确监测干细胞在眼睛中的位置和干细胞的凋亡。此外， 我们希望通过这种方式引导干细胞，TM的功能将得到改善，正如在2个青光眼模型中所测试的那样。

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.