Schikora Juliane, Nickel Antonia, Bergert Jasmin, Hähnel Rike, Dort Aaron, King Ben C, Schayan-Araghi Stella Y, Banerjee Pratiti, Wolf Hannah N, May-Simera Helen, Pauly Diana
Experimental Ophthalmology, Philipps-Universität Marburg, Marburg, Germany.
Institute of Molecular Physiology, Johannes Gutenberg University, Mainz, Germany.
Invest Ophthalmol Vis Sci. 2025 Jul 1;66(9):67. doi: 10.1167/iovs.66.9.67.
Epithelial maturation is essential for the specificity and functionality of retinal pigment epithelial (RPE) cell models. This study investigates how different maturation conditions shape RPE characteristics and cellular complement biology in two commonly used in vitro models: ARPE-19 and induced pluripotent stem cell-derived RPE (iPSC-RPE).
ARPE-19 and iPSC-RPE cells were cultured under low maturation (LM) or high maturation (HM) conditions. Phenotype, RPE marker expression, and functional properties were assessed, and expression of local complement components was analyzed at the transcript, protein, and secretion levels. Intracellular complement C3 processing was characterized using epitope-specific antibodies.
HM conditions enhanced epithelial features in both models, with HM iPSC-RPE displaying enhanced apical localization of RPE markers, polarity, reduced cilia length, and higher transepithelial resistance. Expression and secretion of complement components C3, FB, FH/FHL-1, and FI, as well as FH staining patterns, varied with maturation condition. HM iPSC-RPE secreted increased levels of C3a, C3(H2O), and iC3b, whereas LM cells retained C3 fragments intracellularly. Western blotting and immunostaining revealed maturation-dependent C3 fragment profiles, with apical localization of C3 and intracellular presence of the intact C3 β chain in HM iPSC-RPE, while C3 fragments were present in LM and ARPE-19 cells.
HM iPSC-RPE cells most closely mimic native RPE features and provide a robust model for in vitro studies. RPE cells exhibit cell-autonomous complement production and maturation-dependent C3 activation profiles, providing a foundation for future studies on C3 functionality in RPE homeostasis and retinal degeneration.
上皮细胞成熟对于视网膜色素上皮（RPE）细胞模型的特异性和功能至关重要。本研究调查了在两种常用的体外模型：ARPE-19和诱导多能干细胞衍生的RPE（iPSC-RPE）中，不同的成熟条件如何塑造RPE特征和细胞补体生物学特性。
将ARPE-19和iPSC-RPE细胞在低成熟（LM）或高成熟（HM）条件下培养。评估细胞表型、RPE标志物表达和功能特性，并在转录、蛋白质和分泌水平分析局部补体成分的表达。使用表位特异性抗体对细胞内补体C3加工进行表征。
HM条件增强了两种模型中的上皮特征，HM iPSC-RPE表现出RPE标志物的顶端定位增强、极性增加、纤毛长度缩短以及跨上皮电阻升高。补体成分C3、FB、FH/FHL-1和FI的表达和分泌，以及FH染色模式，随成熟条件而变化。HM iPSC-RPE分泌的C3a、C3(H2O)和iC3b水平增加，而LM细胞在细胞内保留C3片段。蛋白质印迹和免疫染色揭示了成熟依赖性的C3片段谱，HM iPSC-RPE中C3顶端定位且细胞内存在完整的C3β链，而LM和ARPE-19细胞中存在C3片段。
HM iPSC-RPE细胞最接近天然RPE特征，为体外研究提供了一个强大的模型。RPE细胞表现出细胞自主补体产生和成熟依赖性C3激活谱，为未来研究RPE稳态和视网膜变性中C3的功能奠定了基础。
