Abstract
Purpose :
Retinal pigment epithelium (RPE) modulates the exchange of metabolite products to and from the retina, maintains the photoreceptor function by phagocytosis and processing of photoreceptor outer segments allowing their renewal. The human RPE cell-line ARPE-19 is commercially available and widely used as an alternative to primary RPE. However, these cells undergo epithelial-mesenchymal transition losing many features of primary RPE such as polarity, pigmentation, and expression of RPE signature markers. We aimed to determine whether a combination of laminin coating and media supplementation with nicotinamide could improve ARPE-19 expression of genes and proteins, along with morphological phenotype resembling mature RPE.
Methods :
ARPE-19 cells were cultured for up to 3 months with or without 10mM nicotinamide supplementation and/or coated with human recombinant laminin 521. Gross cell morphology was evaluated by phase microscopy. Assessment of maturation was performed by immunocytochemistry and gene expression by qPCR.
Results :
Laminin 521, in combination with nicotinamide supplementation, promoted cytoskeletal reorganization and expression of differentiation markers such as VMD2, RPE65 and PDGFRB compared to non-supplemented culture condition. Moreover, the combination of nicotinamide media supplementation and laminin 521 coating reduced the expression of the of EMT markers, TNFα and TGF-β1; and NCAM1, an immature phenotype RPE marker.
Conclusions :
This study demonstrates that the combination of nicotinamide and laminin 521 coating accelerated the kinetics of expression of mature RPE signature genes, reduced expression of EMT genes, accompanied with improved epithelial cell morphology and cytoskeletal organization, bringing the dedifferentiated ARPE-19 closer to their in vivo phenotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.