Complement C3 Activation in the Human Retinal Pigment Epithelium

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.