1. Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy.
- Author
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Bernd J, Plastino F, Karayannis JJ, Kvanta A, Locri F, and André H
- Subjects
- Humans, Cell Line, Cell Differentiation, Laminin metabolism, Laminin genetics, Epithelial-Mesenchymal Transition, Bestrophins genetics, Bestrophins metabolism, Retinal Pigment Epithelium metabolism, Retinal Pigment Epithelium cytology, Genetic Therapy methods, Dependovirus genetics
- Abstract
The human retinal pigment epithelium (RPE) cell line ARPE-19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE-specific laminin (LN) and nicotinamide (NAM) could improve ARPE-19 redifferentiation to resemble mature RPE and improve the assessment of RPE-specific gene therapy strategies. ARPE-19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521-coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno-associated virus (AAV)1 or AAV2, carrying a VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin-1 and RPE 65 was promoted concomitantly with a reduction of several epithelial-mesenchymal transition markers, such as TNF-α, TGF-β, CDH2, and vimentin. Redifferentiated ARPE-19 transduced at low multiplicity of infection of both AAV1- and AAV2-VMD2-GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post-plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE-19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE-19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral-mediated RPE-specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE-centered diseases., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)
- Published
- 2024
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