Abstract
BackgroundIdentification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.Methodology/Principal FindingsK14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.Conclusions/SignificanceK14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.
Highlights
The cornea is the transparent tissue at the front of the eye; corneal epithelial cells, which are its external cell layers, are supported by corneal epithelial stem cells, located in the limbus, an area where the central cornea and conjunctiva meets
We report here that K14 expression was observed in both bovine limbus and central cornea and that K14 expression was found to increase in limbal epithelial cells, after air-lifting treatment, a cell culture technique well known to increase cell differentiation and promote cell-cell barrier function [18,27,28,29]
Epithelial cells stained with K3 were detected in limbal superior layers, but absent in the basal cell layers (Figure 1C); K3 positive cells were highly expressed in the central cornea, and present throughout all the epithelial cell layers (Figure 1D)
Summary
The cornea is the transparent tissue at the front of the eye; corneal epithelial cells, which are its external cell layers, are supported by corneal epithelial stem cells, located in the limbus, an area where the central cornea and conjunctiva meets. Identification of the epithelial stem cells from a corneal cell population by specific molecular markers has been intensively investigated for over the last 10 years. P63 [8], ABCG2 [9] and K14 [10] have been reported to be positively expressed in mammalian corneal epithelial stem cells, and K3 [11,12], K12 [13], and Cx43 [14] have been shown to be only positively expressed in terminally differentiated corneal epithelial cells. Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. We report on the limitations of K14/K5 as a limbal stem cell marker
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