Abstract
Purpose: :
Because of the lack of consensus biomarker(s) to identify limbal stem cells (SCs), it remains a great challenge to pinpoint their exact location in the limbal basal layer known to express p63. Pax 6, a well known master homeobox gene that directs eye morphogenesis, is expressed by the postnatal ocular surface epithelium. Because downregulation of Pax 6 is an early step during transdifferentiation of adult rabbit corneal transient amplifying cells to epidermal cells when engrafted in mouse embryonic epidermis as well as a hallmark during physiological and pathological squmamous metaplasia of the ocular surface epithelium, we speculate that there exist p63(+)/Pax 6(-) progenitor cells in the limbal basal epithelium.
Methods: :
Double immunostaining to p63 and Pax 6 were performed in cross-sections, flat mount preparation of human limbal epithlelia, cytospin of dispase-isolated and trypsin/EDTA-treated single cells, and clones generated by single limbal epithelial progenitor cells enriched by rapid adhesion to collagen I-coated dishes cocultured with feeder layers made of murine 3T3 fibroblasts or human amniotic epithelial cells.
Results: :
Double immunostaining to Pax 6 and p63 revealed clusters of limbal basal epithelial cells that were positive to nuclear staining of p63 but negative to nuclear staining of Pax 6 in both cross-sections and flat mount preparations. The same double immunostaining also confirmed that these p63(+)/Pax 6(-) cells were derived from the limbal epithelium, but not from the limbal stroma, by isolating the intact human limbal epithelial sheets by dispase, dissociating them into single cells, and submitting them to the cytospin preparation. When these single limbal epithelial cells were enriched by seeding on collagen I-coated dishes for 15 min and cocultured with 3T3 fibroblasts, all 134 resultant clones were p63(+)/Pax 6(+). In contrast, 24 clones generated on the feeder layer made of human amniotic epithelial cells consisted of p63 (+)/Pax 6(-) cells (n=9), p63 (+)/Pax 6(-/+) cells (n=6), and p63 (+)/Pax 6(+) cells (n=9).
Conclusions: :
Collectively, these results indicate that there exist p63 (+)/Pax 6(-) cells in the limbal basal epithelium. These progenitor cells can be maintained by human amniotic epithelial feeder layers but not by murine 3T3 fibroblast feeder layers, raising a question about the efficacy of using 3T3 fibroblasts to preserve/expand limbal epithelial progenitors. The SC status and plasticity of this p63 (+)/Pax 6(-) progenitors awaits further characterization.
Keywords: cornea: epithelium • gene/expression