Comparison of the histology, gene expression profile, and phenotype of cultured human limbal epithelial cells from different limbal regions.
OBJECTIVE
To investigate whether human limbal epithelial cells (HLECs) derived from various regions of the limbus exhibit differences in gene expression and epithelial characteristics.


METHODS
HLECs were derived from explants taken from the superior, nasal, inferior, and temporal limbus and cultured for 21 days. Whole genome transcript profiling was performed with a gene microarray. The microarray results were validated by using RT-PCR. Epithelial morphology was studied with light microscopy and transmission electron microscopy, and phenotype was evaluated by immunohistochemistry.


RESULTS
Epithelial outgrowth was present in most cultures of superior origin (88%) in contrast to cultures of temporal origin (38%). The epithelial thickness and number of cell layers were significantly greater in cultures of superior origin than in cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed in the superior region, compared with the other regions. Proposed limbal stem cell, progenitor, and differentiation markers were not differentially expressed. The uniform gene expression of ocular surface markers correlated with homogeneous immunostaining of corresponding protein markers in HLEC cultures from all regions, demonstrating an undifferentiated phenotype (p63(+), DeltaNp63alpha(+), ABCG2(+), K19(+), vimentin(+), integrin beta1(+), nestin(-), K3(-), K5(+), and E-cadherin(+)).


CONCLUSIONS
No major transcriptional or phenotypic differences were observed in cultured HLECs derived from different regions of the limbus. However, explants of superior origin demonstrated the highest outgrowth success rate and generated epithelia with greater epithelial thickness and number of cell layers, which may prove useful for transplantation purposes.
Utheim
Tor P
TP
Center for Clinical Research, Oslo University Hospital, Ulleval, University of Oslo, Oslo, Norway.
Raeder
Sten
S
Olstad
Ole K
OK
Utheim
Øygunn A
ØA
de La Paz
Maria
M
Cheng
Robert
R
Huynh
Trang T
TT
Messelt
Edward
E
Roald
Borghild
B
Lyberg
Torstein
T
D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D002452:Cell Count; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D019573:Epithelium, Corneal / Q000166:cytology; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007124:Immunoenzyme Techniques; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000235:genetics; D044767:Ubiquitin-Protein Ligases; D000070584:Tripartite Motif Proteins
D015415:Biomarkers; D002352:Carrier Proteins; D014157:Transcription Factors; D020741:rho GTP-Binding Proteins; C486680:OSR2 protein, human; C488674:TRIM36 protein, human; D044767:Ubiquitin-Protein Ligases; D000070584:Tripartite Motif Proteins; C432668:RHOU protein, human