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C. J. Connon, B. Chen, S. Mi, B. Wright; Limitations of K14/K5 as a Stem Cell Marker In Bovine Corneal Epithelia. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1977.
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To investigate claims that K14/K5 is a limbal stem cell marker.
Expression of K14 was compared by immunohistochemistry, Western blotting (semi-quantitative) and Real time PCR (quantitative) between bovine epithelial cells taken from the limbus and central cornea (n=6). K14 antibody (Progen, Germany) was used at concentrations of 1:100 and 1:2000 for immunhistochemistry and Western blotting respectively. Secondary antibodies were incubated overnight at concentrations of 1:100 (donkey anti guinea pig) and 1:8000 (horse radish peroxidise conjugated to guinea pig) for immunohistochemistry and Western blotting respectively. A custom-made PerfectProbe assay (PrimerDesign, U.K.) was used to quantify Keratin 5 gene expression by real time PCR. A functional study was also included to compare K5/14 expression in limbal epithelial cells expanded upon amniotic membrane after airlifting - a known differentiation stimulant.
K14 expression (or its partner K5) was detected in epithelial cells from both the limbal area and central cornea by immunohistochemisrty, Western blotting and real time PCR. K14 was localised predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Semi quantitative Western blotting revealed K14 expression in both limbus and central cornea (higher level in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus (at a similar level). Following airlifting the limbal epithelial cells expanded upon amniotic membrane revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in K3 expression (a known marker for differentiation).
K14 and its partner K5 are found not just in the limbus but also the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific. Furthermore K14/K5 expression levels were not found to be reduced in an experimental culture system in which the limbal epithelial cells were forced to differentiate, suggesting K14/K5 is not a reliable maker for undifferentiated cells.
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