Restoration of Trabecular Meshwork by hMSC and Induced Pluripotent Stem Cells

描述（由申请人提供）：原发性开角型青光眼是一种主要致盲疾病，眼内压（IOP）升高是一个关键风险因素。小梁网（TM）细胞负责维持IOP稳态。在青光眼中，由于遗传和/或环境因素导致的TM细胞减少可能会损害IOP稳态能力。目前的治疗方法帮助了许多人，但不是所有的患者都对它们有适当的反应，或者只在有限的时间内有反应。添加TM样细胞来补充剩余的TM细胞可能有助于功能的恢复。我们建议评估两种候选干细胞类型作为缺失/患病TM细胞的替代品。这两种潜在的自体细胞类型，人类间充质干细胞（hMSC）和诱导多能干细胞（iPS细胞），已被用于重建其他组织。这些置换的目的是恢复正常的IOP稳态。在这个提议中，核心假设是一种或两种干细胞类型可以被诱导生长，扩增和取代患病的TM细胞。我们认为，它们将采用许多或所有的结构、生理、生化和功能属性， 在适当的微环境中。通过多管齐下的方法，我们将研究两种干细胞类型在模型眼灌注系统中的治疗潜力，通过移植它们作为患病TM细胞的替代品。在目标#1中，我们将比较TM细胞生物标志物与hMSC、iPS细胞和分化的干细胞的生物标志物。通过跟踪这些标记物的进展，我们将测试工作假设，即我们可以使用它们来评估两种干细胞类型与TM细胞的相似性。通过与房水、ECM组分、不同培养基制剂和/或其他试剂一起培养，我们将改变干细胞的局部环境。以标记物作为部分指导，将通过免疫组织化学、共聚焦显微镜、qRT-PCR和Western免疫印迹跟踪干细胞向TM样细胞的分化。使用这种方法，我们可以确定最好的干细胞候选人的分化和再生治疗TM。在目标#2中，我们将评估 来自未分化和分化的hMSC和iPS细胞的移植细胞。人眼将实验性地部分去除TM细胞，以测试我们可以模拟昏迷眼的TM细胞减少的工作假设，然后评估移植的替代细胞在模型眼灌注系统中的功能。将分析替代细胞恢复TM样IOP稳态和吞噬消化碎片的能力。这种方法作为青光眼患者进入再生医学时代的一种新方法，具有相当大的前景。

DESCRIPTION (provided by applicant): Primary open angle glaucoma is a major blinding disease, with elevated intraocular pressure (IOP) as a key risk factor. Cells of the trabecular meshwork (TM) are responsible for maintaining IOP homeostasis. In glaucoma, reduced TM cellularity due to genetic and/or environmental factors may compromise IOP homeostatic capabilities. Current treatments aid many, but not all patients respond appropriately to them, or respond only for a limited time. Adding TM-like cells to replenish the remaining TM cells may facilitate restoration of function. We propose to evaluate two candidate stem cell types as replacements for absent/diseased TM cells. These two potentially autologous cell types, human mesenchymal stem cells (hMSC), and induced pluripotent stem cells (iPS cells), have been used to repopulate other tissues. The purpose of these replacements would be to restore normal IOP homeostasis. In this proposal, the central hypothesis is that one or both stem cell types can be induced to grow, expand, and replace diseased TM cells. We contend that they will adopt many or all of the structural, physiological, and biochemical, and functional attributes of TM in the proper microenvironment. With a multi- pronged approach, we will investigate the therapeutic potential of both stem cell types in the model ocular perfusion system by transplanting them as substitutes for diseased TM cells. In Aim #1 we will compare TM cell biomarkers to those of hMSC, iPS cells, and the differentiated stem cells. By following the progress of these markers, we will test the working hypothesis that we can use them to assess the similarities of the two stem cell types to TM cells. By culturing with aqueous humor, ECM components, different media preparations, and/or other agents, we will alter the local milieu of the stem cells. With the markers as a partial guide, differentiation of the stem cells to TM-like cells will be tracked by immunohistochemistry, confocal microscopy, qRT-PCR, and Western immunoblots. Using this approach, we can determine the best stem cell candidate for differentiation and for regenerative therapy in TM. In Aim #2 we will evaluate the functionality of transplanted cells from undifferentiated and differentiated hMSC and iPS cells. Human eyes will be experimentally partially denuded of TM cells, to test the working hypothesis that we can mimic the decreased TM cellularity of the glaucomatous eye, and then assess function of transplanted replacement cells in a model ocular perfusion system. Replacement cells will be analyzed for the capacity to restore TM-like IOP homeostasis and for phagocytotic digestion of debris. This approach has considerable promise as a novel inroad for glaucoma patients to an era of regenerative medicine.