Abstract
Abstract: :
Purpose: The developmental morphogen p63 has been proposed to be a marker for stem cells in the limbal/corneal epithelium (PNAS, 98:3156), a view undermined by studies showing p63 staining within the stem cell–free corneal domain (DNA Cell Biol. 21:443). Here, we examine our hypothesis (Epstein, ARVO 2004 #3770) that the level of p63, rather than its mere presence, reflects the degree of cell stemness. Methods: Flow cytometry was used to isolate Hoechst 33342 side population (SP) cells and extremely low side light scattering (LSSC; see Results) cells from enzymatically dissociated rabbit conjunctival and limbal epithelial cells, respectively. Two animals were infused with BrdU prior to sorting to assess cell cycling frequency according to BrdU labeling index. SP cells and control non–SP cells, representing the majority of the cell population, were cytospun onto glass slides and fixed in formalin. These cells and human and rabbit limbo–corneal cryosections were stained for p63 by indirect immunofluorescence using two anti–p63 clones. The intensity of nuclear staining, used as a measure of p63 concentration, was measured by image analysis. Results: SP and LSSC cells accounted for less than 1% of the total limbal or conjunctival epithelia cell populations. In all experiments, at least 50% of the SP cells displayed the LSSC phenotype and, conversely, more than 50% of the LSSC cells were found to be SP cells. Low levels of BrdU incorporation after 2–week nucleotide analog infusion demonstrated the slow cycling nature of the SP and LSSC cells. The average intensity of p63 staining for the conjunctival SP and LSSC cells was 2.5–fold higher than their respective non–SP or non–LSSC control populations. Limbal LSSC cells, in turn, were 3 times higher than their controls. In the tissue sections all basal limbal cells were p63+. Yet 5–7% of them expressed p63 at clearly unique high levels. Conclusions: Our results are consistent with a relationship in which the level of p63 nuclear concentration is maximal in stem cells and decrease gradually as cells become more differentiated.
Keywords: cornea: epithelium • conjunctiva • wound healing