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E.C. Figueira, M.T. Coroneo, D. Wakefield; ‘Stem Cell Pathway’ Related Gene Expression Profile of the Human Fetal Limbus and Primary Cultured Human Limbal Epithelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3022.
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To characterize the ‘Stem cell pathway’ related gene expression profile of human fetal limbus and human limbal explant epithelial cells (primarily cultured in serum free media) .
Total RNA was extracted from an 18 weeks old human fetal cornea. Central and limbal corneal epithelium RNA was amplified into antisense RNA (T7 RNA amplification) , reverse transcribed and hybridized onto a 256 gene ‘Stem cell pathway’ cDNA microarray membrane and the resulting differential expression (after subtracting signal from background and normalizing to house keeping gene) were compared. Likewise, total RNA extracted from human cadaveric limbal explant epithelial cells in primary culture and passaged (5 times) epithelium were reverse transcribed and then hybridized on the same type of cDNA array. The differential expression was compared between the two samples. The differential expression of 4 selected limbal upregulated genes was studied at the mRNA level using RT–PCR and protein expression level using immunohistochemistry.
Analyses of the differential expression of the microarray genes ( significant difference considered >2 fold) in the human fetal limbus compared with central cornea and primary limbal explant epithelial cells versus 5th trypsin passaged cells identified 33 genes significantly differentially expressed more in the human fetal limbus and in the primary limbal explant epithelial cells (13 genes were exclusively expressed by the fetal limbus and 22 genes exclusively by the primary limbal explant epithelial cells). The microarray differential expressions of 4 selected limbal up regulated genes were confirmed by RT PCR and immunohistochemistry. Limbal expression of these four genes was confirmed on immunohistochemistry to the limbal basal epithelium within the limbal palisades in the fetal and adult human cornea.
A ‘stem cell pathway’ specific related gene expression profile of the basal limbal epithelium, that includes limbal epithelial stem cells, has been identified and four proteins have been localized to the limbal basal epithelium.
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