Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
Invest Ophthalmol Vis Sci. 2012 Sep 14;53(10):6282-90. doi: 10.1167/iovs.12-9721.
Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).
We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of α(v)β₅ integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.
Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.
Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.
视网膜色素上皮 (RPE) 自体移植物可从诱导多能干细胞 (iPS) 细胞中轻易获得。使用标准化和可量化的方法严格表征 iPS-RPE 非常重要，以确保在临床环境中使用它们之前，它们是安全且足够的疾病 RPE 替代物。一个重要且必需的功能是 iPS-RPE 吞噬光感受器外节 (POS)。
我们开发了一种基于流式细胞术的测定法来监测 FITC 标记的 POS 与 ARPE-19、人胎儿 RPE (hfRPE) 和两种类型的 iPS-RPE 的结合和内化。比较了吞噬作用所需的α(v)β₅整联蛋白、CD36 和 MerTK 受体的表达和密度。
用胰蛋白酶处理 RPE 细胞可导致结合的 POS 释放。使用流式细胞术测量释放的 POS 数量、内化 POS 的细胞百分比、细胞内 FITC 信号的亮度以及单个 RPE 细胞上的吞噬受体的表面密度。这些测定表明受体密度在分化过程中是动态的，这会影响 RPE 细胞的结合和内化动力学。高度分化的 iPS-RPE 比 hfRPE 更有效地吞噬 POS。
在证明它们完全功能之前，应谨慎使用 RPE 移植物。吞噬受体的密度是动态的，可作为预测 iPS-RPE 细胞在体内功能的指标。在 iPS-RPE 和原代 RPE 之间观察到的吞噬动力学非常令人鼓舞，并增加了越来越多的证据表明 iPS-RPE 可能是人类患者中功能失调或死亡的 RPE 的可行替代品。
