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Comparison of the Histology, Gene Expression Profile, and Phenotype of Cultured Human Limbal Epithelial Cells from Different Limbal Regions

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

Corresponding author: Sten Raeder, Center for Clinical Research, University of Oslo, Oslo University Hospital, Ulleval, Kirkeveien 166, 0407 Oslo, Norway; sten.rader{at}medisin.uio.no.

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 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⁺, Np63⁺, ABCG2⁺, K19⁺, vimentin⁺, integrin β1⁺, 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.