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P. Douvaras, J. R. Dorin, R. E. Hill, J. D. West; Are Limbal Stem Cells Depleted in Older Mice and Pax6+/- Heterozygotes?. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1770.
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Previous studies of corneal epithelial stripe patterns in chimeras and X-inactivation mosaics provided indirect evidence that limbal stem cell (LSC) function is depleted in older mice and in Pax6+/- heterozygotes. This could reflect fewer LSCs or more quiescent LSCs. The purpose of this study was to use two approaches to evaluate these predictions.
The first approach involved using a low-frequency lineage marker to compare the frequency of clones of cells produced by LSCs. K5-LacZ mice express LacZ from the keratin 5 promoter and produce rare stripes of β-galactosidase-positive cells extending from the limbus into the corneal epithelium. As the stripes are rare, each is likely to be a clone of cells produced by a single active stem cell and the frequency of marked clones is likely to be proportional to the number of active LSCs. The second approach involved comparing the numbers of BrdU label-retaining cells (mostly slow-cycling putative stem cells). This provides an index that should be proportional to the number of active stem cells. Mice were exposed to BrdU for 1 week and chased for 10 weeks. Putative stem cells divide less frequently than other cells and so retain the BrdU during the chase period while faster cycling cells dilute it by cell division.
Comparison of K5-LacZ stripe frequencies supported the original predictions since wild-type mice produced significantly fewer stripes at 30 weeks than 15 weeks and Pax6+/- heterozygotes produced fewer stripes than age-matched wild-types. In contrast, the second approach failed to show the predicted reduction of label-retaining cells in older mice or in Pax6+/- mice.
It is unclear why the label-retaining cell experiment failed to support the mosaic studies but differences in cell-turnover between groups might affect label-retaining cell numbers. Further work is needed to investigate variables that affect the number of label-retaining cells and the discrepancy between the two methods.
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