Abstract
Purpose :
Retinal pigment epithelium (RPE) plays a crucial role for the integrity and function of the retina as well as its pathologies and has become a target for gene therapy approaches in recent years. For in vitro studies ARPE-19 cells are commonly used as an alternative to primary RPE, despite the disadvantage that these cells undergo epithelial-mesenchymal transition (EMT), losing many features of primary RPE such as pigmentation and expression of RPE signature markers. We aimed to determine whether combination of RPE-specific laminin (LN) coating and media supplementation with nicotinamide (NAM) could improve ARPE-19 expression of genes and proteins, along with morphological phenotype resembling mature RPE. Further we assessed if matured ARPE-19 cells can be used as a model for RPE specific gene therapy using adeno associated virus (AAV) as a delivery vector.
Methods :
ARPE-19 cells were cultured on tissue culture plastic, with or without NAM supplementation and/or coated with human recombinant LN-521. Cell morphology was evaluated by phase microscopy. RPE maturation was demonstrated by immunocytochemistry and gene expression assayed by qPCR. Viral transduction experiments with adeno associated virus AAV1 or AAV2, VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with low multiplicity of infection.
Results :
The combination of LN-521 coating with NAM supplementation promoted cytoskeletal and tight junction reorganization, and expression of differentiation markers such as VMD2, RPE65 and PDGFRB. In addition, the combination reduced the expression of the EMT markers, TNFα and TGF-β1; and NCAM1, an immature phenotype RPE marker. Both AAV1- and AAV2-VMD2-GFP expression was detected from 2-weeks and improved at 4-weeks post-plating. Further we could demonstrate that AAV1 exhibits superior in vitro tropism for differentiated ARPE-19 cells.
Conclusions :
We demonstrate that the combination of NAM with LN-521 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. Moreover ARPE-19 grown on standard tissue culture plastic, can be reliably and accurately transduced with low viral titer as demonstrated in this study, facilitating the investigation of RPE specific gene therapy approaches.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.