Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies.

Journal article

PURPOSE: To develop, by specific genetic modification, a differentiated human retinal pigment epithelial (RPE) cell line with an extended life span that can be used for investigating their function in vitro and for in vivo transplantation studies. METHODS: Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-19. Factors reported to be important in RPE and photoreceptor cell function and survival in vivo were examined. RESULTS: Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology, expressed cytokeratins, and displayed junctional distribution of ZO-1, p100-p120 and beta-catenin. The cells expressed mRNA for RPE65 and cellular retinaldehyde-binding protein (CRALBP) and the trophic and growth factors brain-derived neurotropic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-alpha, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF, bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59. After activation with IFN-gamma both HLA-ABC and CD54 were upregulated, and the expression of HLA-DR was induced. Both cell lines failed to express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte reaction were unable to induce T-cell proliferation. Fas ligand (CD95L) was not detected in vitro by RT-PCR. Similar results were obtained with the ARPE-19 cell line. CONCLUSIONS: RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.

Authors: Kanuga, N; Winton, H; Beauchéne, L; Koman, A; Zerbib, A

• Publication status: Published
• Journal: Investigative ophthalmology and visual science
• Volume: 43
• Issue: 2
• Pages: 546-555
• Publication date: 2002-02-01
• EISSN: 1552-5783
• ISSN: 0146-0404
• Language: English
• Keywords: Female; Humans; Middle Aged; Eye Proteins; Lymphocyte Activation; Cell Line, Transformed; Cell Separation; Cell Transplantation; DNA Primers; Cell Survival; Transfection; Fluorescent Antibody Technique, Indirect; Pigment Epithelium of Eye; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; RNA, Messenger; Antigens, Polyomavirus Transforming