Comparison of the histology, gene expression profile, and phenotype of cultured human limbal epithelial cells from different limbal regions

¹ Center for Clinical Research, Ulleval University Hospital, Oslo, Norway; Department of Ophthalmology, Ulleval University Hospital, Oslo, Norway
² Department of Clinical Chemistry, Ulleval University Hospital, Oslo, Norway
³ Department of Ophthalmology, Ulleval University Hospital, Oslo, Norway
⁴ Institut Universitari Barraquer/Universitat Autonoma de Barcelona, Barcelona, Spain
⁵ Laboratory of Metabolism, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland, United States
⁶ Center for Clinical Research, Ulleval University Hospital, Oslo, Norway
⁷ Dental Faculty, University of Oslo, Norway, Institute for Oral Biology, Oslo, Norway
⁸ Department of Pathology, Ulleval University Hospital, Oslo, Norway

^* To whom correspondence should be addressed. E-mail: sten.rader{at}medisin.uio.no.

	Abstract

PURPOSE. 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 using Affymetrix GeneChip Human 1.0 ST Array. The microarray results were validated using RT-PCR. Epithelial morphology was studied using 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 compared with cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed when comparing superior region 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+, Np63+, ABCG2+, K19+, vimentin+, Integrin 1+, Nestin-, K3-, K5+, 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.

Key Words: corneal transplantation, corneal epithelium, gene expression, tissue engineering, limbus, immunohistochemistry