TY - JOUR
T1 - Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line
AU - Robertson, Danielle M
AU - Li, Li
AU - Fisher, Stephen
AU - Pearce, Virginia P.
AU - Shay, Jerry W
AU - Wright, Woodring E
AU - Cavanagh, Harrison D
AU - Jester, James V.
PY - 2005/2
Y1 - 2005/2
N2 - PURPOSE. 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 longterm, 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.
AB - PURPOSE. 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 longterm, 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.
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U2 - 10.1167/iovs.04-0528
DO - 10.1167/iovs.04-0528
M3 - Article
C2 - 15671271
AN - SCOPUS:13944283269
SN - 0146-0404
VL - 46
SP - 470
EP - 478
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 2
ER -