在眼睛里,视网膜色素上皮(RPE)粘附在一种称为布鲁赫膜(BrM)的复杂蛋白质基质上。这项研究的目的是通过生产BrM样基质为RPE细胞培养提供富集条件。我们的假设是,人类RPE细胞系会沉积类似BrM的细胞外基质(ECM)。表征了由ARPE19细胞沉积的ECM(ARPE19-ECM)的组成和结构。为了生产ARPE19-ECM,在硫酸葡聚糖存在下培养ARPE19细胞。使用脱氧胆酸盐脱细胞化ARPE19-ECM,并通过免疫染色和蛋白质印迹分析表征。将原代人RPE和诱导的多能干细胞接种到ARPE19-ECM或geltrex包被的表面上,并通过显微镜或RT-PCR检查。用硫酸葡聚糖培养ARPE19细胞可促进SOX2的核定位,紧密连接的形成和ECM的沉积。ARPE19细胞沉积了在BrM内层发现的ECM蛋白,包括纤连蛋白,玻连蛋白,胶原蛋白IV和V以及层粘连蛋白-α-5,但不是在中间弹性层(弹性蛋白)或外层(胶原蛋白VI)中发现的那些。ARPE19-ECM促进人RPE和多能干细胞培养物中的色素沉着。在分化的多能干细胞培养物中,与geltrex相比,RPE65在ARPE19-ECM上的表达显着增加。ARPE19细胞在视网膜中沉积具有类似于BrM的组成和结构的ECM。存在于ARPE19-ECM中的分子线索促进RPE表型的获得和维持。一起,这些结果证明了产生用于富集RPE细胞培养物的BrM样表面的简单方法。
In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch\'s membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.