Abstract
Abstract: :
Purpose: Purification of stem cells from the corneoscleral limbus is hampered by the presence of contaminating cells, coupled with a shortage of unambiguous stem cell markers. The nuclear transcription factor p63 and the cell surface drug resistance marker ABCG2 have been proposed as potential markers for stem cells of the corneoscleral limbus. In this study, we sought to co–localize these proteins in cells grown from primary human corneoscleral rims in order to design a stem cell purification strategy. Methods: Human corneoscleral rims were obtained from Upstate New York Transplant Services. Tissues were plated as explant cultures within 24 hours of death or cryopreserved for tissue sectioning. Cell cultures were maintained in DMEM for up to four weeks and fixed in 4% paraformaldehyde for immunocytochemistry or harvested for western immunoblot analysis. Cryosections were cut and mounted onto slides for immunohistochemistry. Results: Immunoreactivity to the nuclear marker p63 and cell surface marker ABCG2 was seen clearly in the basal epithelial cell layer of the corneoscleral limbus, but did not appear in the central cornea. Co–localization of p63 and ABCG2 was seen in subpopulations of corneoscleral explant cultures, but appeared to decrease over time. Conclusions: The cell surface marker ABCG2 may be useful for purification of stem cells from the corneoscleral limbus at early timepoints. Co–localization of ABCG2 and p63 may help identify the slowly cycling stem cells from other contaminating cell types.
Keywords: cornea: epithelium • immunohistochemistry • cornea: basic science