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J. T. Daniels, G. A. Secker, Q. Schwarz, R. R. Ali; Pax6 Heterozygosity Causes Retention of Putative Limbal Epithelial Stem Cells and Novel Niche Structures in the Central Cornea. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2046.
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© ARVO (1962-2015); The Authors (2016-present)
To identify putative limbal epithelial stem cells (LESC) and their associated niche structures in a mouse model of aniridia.
Wild type (WT) and Pax6 heterozygous (Het) mice were injected with bromodeoxyuridine (BrDU) at post-natal day 12. Following chase periods of up to 10 weeks the numbers of putative LESCs, identified as label retaining cells (LRCs), were counted. FITC-phallodin and DAPI staining and immunohistochemistry of whole-mounted corneas was used to assess epithelial cell size, nuclear:cytoplasmic ratio, β1 integrin brightness and putative niche structures by confocal microscopy. Proliferation in the limbal and corneal epithelia was determined with a 4 hour BrDU pulse-chase in 10 week old animals. Blood vessels were immunolocalised with anti-CD31 antibodies in whole-mounted corneas.
Initially the majority of corneal and limbal basal epithelium was labelled with BrDU. By 8 weeks, LRCs were only detectable in the limbus of WT individuals, while the Hets displayed LRCs in the central cornea for at least 8 weeks (p<0.05). ‘Corneal pits’ (CP) were found to be associated with clusters of LRCs in both groups, however these developed later in the WT mice and did not penetrate the stroma as they were found to do in the Hets. The CP contained small tightly packed cells of high nuclear:cytoplasmic ratio and were β1 integrin-bright. Proliferation was higher in the corneal than the limbal epithelium of the WT individuals. The opposite was true for the Hets. Neovascularisation of the Het corneas was evident by 5 weeks.
A putative LESC niche was identified in the normal and pathogenic mouse cornea. We propose that aniridia-associated ocular surface failure may be the result of insufficient stem cell differentiation in the corneal epithelium during development rather than LESC deficiency. These results may have implications for the design of future therapeutic strategies for aniridia-associated ocular surface failure and other diseases characterised clinically as being the result of stem cell deficiency.
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