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T. Utheim, S. Raeder, O. K. Olstad, O. Utheim, M. de la Paz, W. B. Medin, B. Roald, T. Lyberg; Identification of Predilection Target Sites for Tissue Harvesting in Corneal Epithelial Tissue Engineering. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5746. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The present study aimed at comparing gene expression, histology, and phenotypes of cultured human limbal epithelial cells (HLEC) for transplantation derived from four target limbal sites along the limbal circumference.
Cadaveric human corneas with research consent were obtained from the Centro de Oftalmologia Barraquer (Spain). HLEC were cultured on intact amniotic membranes for 21 days from human limbal explants of 1-clock-hour width from the superior, nasal, inferior, and temporal regions. Following RNA Qiagen extraction and purification from 5 mm trephinated disks of cultured epithelium, gene expression in cultured HLEC was determined using Affymetrix GeneChip Human 1.0 ST Array. Cultured HLEC were characterised by histological and immunophenotypical analysis.
The mean RNA yield was highest in extracts from the superior regions (287.8 ng/µl) and lowest from temporal regions (86.3 ng/µl) which provided sufficient RNA for microarray analysis in only two out of four temporal biological replicates, but the differences were not statistically significant. Of the 28.869 genes tested in HLEC cultures, six genes (KRT12, KRT16, MFSD4, ZNF667, RHOU, and SILV) were differentially overexpressed (more than twofold, p<0.05, unpaired t test)) in HLEC cultures from superior limbus compared with nasal limbus and only one gene (HEPHL1) compared with inferior limbus. Three genes (PLD5, SNORD57, and BEX1) were differentially overexpressed in HLEC cultures from inferior limbus compared with cultures from nasal limbus. HLEC cultures derived from the superior region, demonstrated the highest success rate (83%, 5/6) in terms of formation of a confluent multilayered epithelium of up to seven layers thickness in sharp contrast to cultures from the temporal region that were mostly monolayered or devoid of cells. Independent of regional limbal localization, cultured HLEC demonstrated an undifferentiated nature (p63+/ΔNp63α+/ABCG2+/K19+/Vimentin+/Integrin β1+/K3-/K5+/E-Cadherin+)
These data suggest that HLEC cultures from superior limbal biopsies most frequently succeed in the formation of multilayered epithelia. However, the differences in gene expression in HLEC cultures derived from different regions were minimal.
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