Liang Lina, Yan Run-Tao, Ma Wenxin, Zhang Huanmin, Wang Shu-Zhen
Department of Ophthalmology, University of Alabama at Birmingham, 700 South 18th Street, Birmingham, AL 35233, USA.
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):5066-74. doi: 10.1167/iovs.06-0515.
To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye.
RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed.
Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina.
Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.
通过检查移植到发育中的鸡眼中的细胞分化情况，研究通过对视网膜色素上皮（RPE）转分化进行编程来生成光感受器的可能性。
分离RPE，将细胞解离、培养，并用表达NeuroD的逆转录病毒（RCAS-NeuroD）感染来引导其转分化，使用RCAS-绿色荧光蛋白（GFP）作为对照。然后收获细胞并显微注射到第5至7天鸡胚的发育眼中，分析其发育和整合情况。
对照培养的细胞整合到宿主RPE中。当移植大量细胞时，形成了多层RPE样组织，额外的组织由移植细胞和宿主细胞组成。对照培养的细胞均未表达光感受器特异性基因。相比之下，来自RCAS-NeuroD感染培养物的大多数细胞仍无色素沉着。其中大量细胞表达了光感受器特异性基因，如视锥蛋白和视蛋白。抗红色视蛋白抗体标记了移植的转分化细胞的顶端和细胞体。在视网膜下间隙中，视锥蛋白阳性细胞沿着RPE或RPE样结构排列。当整合到宿主外核层时，移植细胞向外丛状层发出精细的轴突分支。
培养的RPE细胞保留了其显著的再生能力。通过NeuroD引导沿光感受器途径转分化的细胞形成了高度有序的细胞结构，并可整合到外核层中。这些数据表明，通过基因编程，RPE细胞可能是光感受器细胞的潜在来源。
