Restoration of Trabecular Meshwork by hMSC and Induced Pluripotent Stem Cells

描述(申请人提供)：原发性开角型青光眼是一种主要的致盲疾病，眼压升高是一个关键的危险因素。小梁网(TM)的细胞负责维持眼压的稳态。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眼压平衡能力。目前的治疗方法帮助很多，但并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效。添加TM样细胞以补充剩余的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/患病的TM细胞的替代品。这两种潜在的自体细胞类型，人间充质干细胞(HMSC)和诱导多能干细胞(iPS细胞)，已被用于重新填充其他组织。这些置换的目的将是恢复正常的眼压稳态。在这项提议中，中心假设是一种或两种类型的干细胞都可以被诱导生长、扩增和取代患病的TM细胞。我们认为，它们将采用许多或全部结构、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，我们将通过移植这两种干细胞作为疾病TM细胞的替代品，来研究这两种干细胞在模型眼灌流系统中的治疗潜力。在目标1中，我们将TM细胞生物标记物与hMSC、iPS细胞和分化的干细胞的生物标记物进行比较。通过跟踪这些标记的进展，我们将检验工作假设，即我们可以使用它们来评估这两种干细胞类型与TM细胞的相似性。通过用房水、细胞外基质成分、不同的介质制剂和/或其他试剂培养，我们将改变干细胞的局部环境。有了这些标记物的部分指导，干细胞向TM样细胞的分化将通过免疫组织化学、共聚焦显微镜、qRT-PCR和Western免疫印迹进行跟踪。使用这种方法，我们可以确定在TM中进行分化和再生治疗的最佳干细胞候选。在目标2中，我们将评估的功能 从未分化和分化的hMSC和iPS细胞中移植细胞。我们将在实验中部分剥离人眼的TM细胞，以验证我们可以模拟青光眼TM细胞减少的工作假设，然后在一个模型眼血流系统中评估移植的替代细胞的功能。将分析替代细胞恢复TM样眼压动态平衡的能力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条新路，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.