Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies.
OBJECTIVE
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.
Kanuga
Naheed
N
Division of Cell Biology, Institute of Ophthalmology, University College London, London, United Kingdom.
Winton
Helen L
HL
Beauchéne
Laurence
L
Koman
Ahmet
A
Zerbib
Anne
A
Halford
Stephanie
S
Couraud
Pierre-Olivier
PO
Keegan
David
D
Coffey
Pete
P
Lund
Raymond D
RD
Adamson
Peter
P
Greenwood
John
J
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002461:Cell Line, Transformed; D002469:Cell Separation; D002470:Cell Survival; D017690:Cell Transplantation; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008213:Lymphocyte Activation; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000502:physiology; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D005136:Eye Proteins; D012333:RNA, Messenger