Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.
Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.
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DOI：
10.1007/978-1-4939-7553-2_15
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Bharti K
中科院分区：
文献类型：
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作者：
Khristov V;Wan Q;Sharma R;Lotfi M;Maminishkis A;Bharti K
位于顶端和基底质膜上的表面蛋白是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所需的。在极化细胞中，表面蛋白质差异地定位于细胞的顶侧或基底侧。视网膜色素上皮（RPE）是极化细胞的一个实例，其执行依赖于其极化状态的多种功能，包括跨RPE单层的离子、流体和代谢物的运输。这些功能对于维持相邻光感受器（视网膜的感光细胞）的健康和完整性至关重要。在这里，我们提出了一系列的方法来识别和验证培养的原代人RPE细胞的极化状态，使用免疫染色的RPE顶侧/基底侧标志物，极化细胞因子分泌，电生理学，流体运输，吞噬作用，并通过细胞表面捕获技术的质膜蛋白的识别。这些方法目前被用于验证人诱导多能干（iPS）细胞衍生的RPE细胞的极化状态和上皮表型。这项工作为开发使用患者特异性iPS细胞衍生的RPE的年龄相关性黄斑变性的自体细胞疗法提供了基础。
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.