Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.
OBJECTIVE
To develop and characterize a telomerase-immortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms involved in regulating human corneal epithelial cell differentiation.


METHODS
Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air-lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti-Ki-67, anti-p16), stratification (phalloidin and anti-ZO-1), and differentiation (anti-K3, anti-BCL-2 and TUNEL labeling).


RESULTS
hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 10. Air-lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO-1-stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5/K14) with K3-corneal keratin marker expression in long-term, air-lifted culture. Anti-BCL-2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL-2 expression in TUNEL-labeled surface epithelial cells.


CONCLUSIONS
hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecular mechanisms regulating corneal epithelial cell differentiation and desquamation.
Robertson
Danielle M
DM
Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Li
Li
L
Fisher
Stephen
S
Pearce
Virginia P
VP
Shay
Jerry W
JW
Wright
Woodring E
WE
Cavanagh
H Dwight
HD
Jester
James V
JV
D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D020287:In Situ Nick-End Labeling; D007621:Karyotyping; D007633:Keratins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D019394:Ki-67 Antigen; D008565:Membrane Proteins; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D007633:Keratins; D019098:Telomerase