Table_5_661W Photoreceptor Cell Line as a Cell Model for Studying Retinal Ciliopathies.XLSX

The retina contains several ciliated cell types, including the retinal pigment epithelium (RPE) and photoreceptor cells. The photoreceptor cilium is one of the most highly modified sensory cilia in the human body. The outer segment of the photoreceptor is a highly elaborate primary cilium, containing stacks or folds of membrane where the photopigment molecules are located. Perhaps unsurprisingly, defects in cilia often lead to retinal phenotypes, either as part of syndromic conditions involving other organs, or in isolation in the so-called retinal ciliopathies. The study of retinal ciliopathies has been limited by a lack of retinal cell lines. RPE1 retinal pigment epithelial cell line is commonly used in such studies, but the existence of a photoreceptor cell line has largely been neglected in the retinal ciliopathy field. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration, but not described as a model for studying retinal ciliopathies such as retinitis pigmentosa. Here, we characterize the 661W cell line as a model for studying retinal ciliopathies. We fully characterize the expression profile of these cells, using whole transcriptome RNA sequencing, and provide this data on Gene Expression Omnibus for the advantage of the scientific community. We show that these cells express the majority of markers of cone cell origin. Using immunostaining and confocal microscopy, alongside scanning electron microscopy, we show that these cells grow long primary cilia, reminiscent of photoreceptor outer segments, and localize many cilium proteins to the axoneme, membrane and transition zone. We show that siRNA knockdown of cilia genes Ift88 results in loss of cilia, and that this can be assayed by high-throughput screening. We present evidence that the 661W cell line is a useful cell model for studying retinal ciliopathies.

视网膜包含多种纤毛细胞类型，包括视网膜色素上皮（retinal pigment epithelium, RPE）细胞与感光细胞。感光细胞纤毛是人体中高度特化的感觉纤毛之一。感光细胞的外节属于结构极为复杂的初级纤毛（primary cilium），其膜层堆叠或折叠的区域即为光色素分子（photopigment molecules）的定位位点。不出所料，纤毛缺陷常引发视网膜表型（retinal phenotypes），既可作为累及其他器官的综合征表现之一，也可单独出现，即所谓的视网膜纤毛病（retinal ciliopathies）。视网膜纤毛病的研究长期受限于视网膜细胞系的匮乏。RPE1视网膜色素上皮细胞系常被用于此类研究，但感光细胞系的开发在视网膜纤毛病研究领域长期被忽略。661W锥感光细胞系源自小鼠，已被广泛用作黄斑变性研究的模型，但尚未被作为视网膜纤毛病（如色素性视网膜炎retinitis pigmentosa）研究的模型加以报道。本研究将661W细胞系表征为视网膜纤毛病研究的细胞模型。我们通过全转录组RNA测序（whole transcriptome RNA sequencing）全面解析了该细胞的表达谱，并将相关数据上传至基因表达综合数据库（Gene Expression Omnibus, GEO），以服务于全球科研社群。我们证实该细胞表达绝大多数锥细胞起源的标志物。通过免疫染色（immunostaining）、共聚焦显微镜（confocal microscopy）及扫描电子显微镜（scanning electron microscopy）观察，我们发现该细胞可生长出细长的初级纤毛，其形态与感光细胞外节相似，且众多纤毛蛋白定位于轴丝（axoneme）、细胞膜及过渡区（transition zone）。我们证实，通过siRNA干扰纤毛基因Ift88可导致纤毛丢失，且该表型可通过高通量筛选进行检测。综上，我们证明661W细胞系是用于视网膜纤毛病研究的优质细胞模型。