Khristov Vladimir, Wan Qin, Sharma Ruchi, Lotfi Mostafa, Maminishkis Arvydas, Bharti Kapil
Section on Epithelial and Retinal Physiology and Disease, National Eye Institute, National Institutes of Health, Bethesda, MD, USA.
Unit on Ocular and Stem Cell Translational Research, National Eye Institute, National Institute of Health, Bethesda, MD, USA.
Methods Mol Biol. 2018;1722:223-247. doi: 10.1007/978-1-4939-7553-2_15.
Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.
位于顶端和基底质膜上的表面蛋白对于细胞感知其环境并将离子、细胞因子、趋化因子和激素水平的变化传递到细胞内部是必需的。在极化细胞中，表面蛋白在细胞的顶端或基底外侧差异定位。视网膜色素上皮（RPE）是极化细胞的一个例子，它执行多种依赖于其极化状态的功能，包括离子、液体和代谢物跨RPE单层的运输。这些功能对于维持相邻光感受器（视网膜的感光细胞）的健康和完整性绝对至关重要。在这里，我们展示了一系列方法，通过对RPE顶端/基底外侧标记物进行免疫染色、极化细胞因子分泌、电生理学、液体运输、吞噬作用以及通过细胞表面捕获技术鉴定质膜蛋白，来识别和验证培养的原代人RPE细胞的极化状态。这些方法目前正用于验证人诱导多能干细胞（iPS）来源的RPE细胞的极化状态和上皮表型。这项工作为使用患者特异性iPS细胞来源的RPE开发用于年龄相关性黄斑变性的自体细胞疗法提供了基础。
