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Isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy

Item Type:Article
Title:Isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy
Creators Name:Bascuas, T. and Kropp, M. and Harmening, N. and Wong, B.M. and Johnen, S. and Izsvák, Z. and Thumann, G.
Abstract:Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.
Keywords:Cell Separation, Cell Survival, Cultured Cells, Electroporation, Eye Proteins, Genetic Engineering, Genetic Therapy, Granulocyte-Macrophage Colony-Stimulating Factor, Mammals, Nerve Growth Factors, Oxidative Stress, Reporter Genes, Retinal Pigment Epithelium, Serpins, Transfection, Animals, Cattle, Mice, Rats, Swine
Source:Journal of Visualized Experiments
Page Range:e62145
Date:26 February 2021
Official Publication:https://doi.org/10.3791/62145
PubMed:View item in PubMed

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