Accelerated maturation of ARPE‐19 cells for the translational assessment of gene therapy

Abstract

AbstractPurpose: Retinal pigment epithelium (RPE) modulates the exchange of metabolite products to and from the retina, and are paramount to maintain retinal function. 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 (EMT) losing many features of primary RPE such as polarity, pigmentation, and expression of RPE signature markers. We aimed to determine whether a 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 to improve assessment of RPE‐specific gene therapy strategies.Methods: ARPE‐19 cells were propagated 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 performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno associated virus (AAV)1 or AAV2, VMD2‐driven GFP, were assessed at 2‐ and 4‐weeks post‐plating in the different culturing condition with low multiplicity of infection.Results: The combination of LN‐521 coating with NAM supplementation promoted cytoskeletal and tight junction proteins 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.Conclusions: Our study demonstrates 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, viral‐mediated RPE‐specific gene expression demonstrates the amenability of the combinatorial cultures for translational assessment of RPE gene therapies.