Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland.
Department of Biomedicine, University Hospital Basel & University Basel, Hebelstr. 20, 4031, Basel, Switzerland.
Sci Rep. 2020 Jun 1;10(1):8890. doi: 10.1038/s41598-020-65657-y.
The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level.
视网膜色素上皮（RPE）是一层鹅卵石样的上皮细胞，完成视网膜的关键功能。已经发表了几种方案将多能干细胞分化为适合疾病建模和治疗开发的 RPE 细胞。在我们的研究中，广泛表征了人诱导多能干细胞（hiPSC）衍生的 RPE（iRPE）的 RPE 特性，然后用于测试慢病毒介导的 RPE65 基因增强治疗。慢病毒载体的剂量研究显示，载体对 RPE65 mRNA 水平具有剂量依赖性影响。在 iRPE（100 倍）以及源自另一种 hiPSC 来源和源自胎儿人 RPE 的实验设置中，也观察到 RPE65 mRNA 的显著增加。尽管 iRPE 表现出与真正 RPE 非常接近的特征，但根据蛋白质检测方法，观察到 RPE65 蛋白水平没有或适度增加。其他两种细胞系也观察到了类似的结果。RPE65 蛋白调节的机制仍有待阐明，但目前的工作表明，高载体表达不会产生过多的正常 RPE65 蛋白水平。
