In vitro differentiation capacity of telomerase immortalized human RPE cells.
OBJECTIVE
To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells.


METHODS
Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation.


RESULTS
Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation.


CONCLUSIONS
Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.
Rambhatla
Lakshmi
L
Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025, USA.
Chiu
Choy-Pik
CP
Glickman
Randolph D
RD
Rowe-Rendleman
Cheryl
C
D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D004261:DNA Replication; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D007633:Keratins / Q000378:metabolism; D008543:Melanins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000201:enzymology*; D019098:Telomerase / Q000235:genetics*; D014162:Transfection; D014746:Vimentin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D008543:Melanins; D014746:Vimentin; D007633:Keratins; D019098:Telomerase; D001616:beta-Galactosidase