Abstract
Abstract: :
Purpose: p63, a critical nuclearly localized developmental morphogen, has been proposed to be a marker for stem cells in the limbus and skin epithelia (Pellegrini et al, 2001). This view has been challenged by studies showing widespread p63 staining within the limbus and even the stem cell–free corneal domain. We have now reexamined the p63/stem cell relationship under the hypothesis that the level of p63, rather than its presence, indicates cell stemness. Methods: Human and rabbit limbo–corneal cryosections and limbal epithelial cells seeded in clonogenic conditions (2,000 cells/cm2 over a 3T3 feeder layer) were stained for p63 by indirect immunofluorescence using the Y4A3 and 4A4 anti–p63 clones. Growing clones were fixed and stained daily for the first 7 days of culture. The intensity of nuclear staining (rel. measure of p63 content) was measured and plotted as a function of colony size by image analysis. Results: In the tissue sections approx. 5–7% of the cells possessed distinct high stain intensities, 40% had intermediate values (30–60% of the maximum value) and the rest had staining intensities ≤10% of the maximum value. Replication rates for the isolated limbal cells were markedly different. For example, on Day 5, colonies contained between 256 (eq. ≤16 hr/cycle) to 2 (eq. 96 hr/cycle) cells. These later cells exhibiting slow–cycling (or delayed proliferative start) possessed several features consistent with a stem cell phenotype, including, small size, low intracellular complexity and high ability to efflux Hoechst 33342 (Goodell et al, J Exp Med. 183:1797, 1996). All colony cells were p63+. The intensity stain in the slow cycling cells though, was 3–4 times higher than in the rapidly proliferating congeners. Nuclear fluorescence intensity did not substantially decrease with the increase in cell doublings, suggesting that the difference between slow and fast cycling cells could not be attributed to this factor. Conclusions: The expression of p63, per se, cannot be construed to indicate cell stemness. Rather, the present results indicate that the level of p63 expression seems to correlate with the degree of cell stemness.
Keywords: cornea: epithelium • receptors