Abstract
Abstract: :
Purpose: Limbal epithelial stem cells (LSC) are the progenitors of corneal epithelium. LSC cultured on human amniotic membrane has been successfully used to reconstruct corneal epithelium in limbal deficient patients, suggesting that bioengineering of corneal epithelium by LSC is fessible. The phenotypic characteristics of the expanded epithelium from limbal explant on amniotic membrane need to be established if the fabrication of corneal epithelium were to become a reality. Methods: To characterize the phenotypic characteristics of the limbal epithelial outgrowth, we employ the immunofluorescent staining and confocal microscopy to examine the expression patterns of p63, Ki-67, keratin 3, connexin 43, and integrin ß4 subunit in freshly prepared corneal and limbal tissue, and in limbal explant and outgrowth cultured on amniotic membrane. Results: P63 is a putative limbal epithelial stem cell marker, Ki-67 is a proliferating cell nuclear marker, keratin 3 is a corneal epithelial marker, connexin 43 is a gap junction protein expressed in corneal epithelial cells and integrin ß4 subunit is a laminin receptor expressed throughout the epithelia of the cornea and limbus. Our results showed that corneal epithelium is negative with p63 and Ki-67, weakly positive with connexin 43, and strongly positive with keratin 3. In addition, integrin ß4 subunit is positive in suprabasal and basal cells, and in the basal cells it is mostly localized to the lateral membranes. In limbus, the nuclei of basal and supra basal cells are positive with p63 and Ki-67. Keratin 3 and connexin 43 positive cells are confined to stratified superficial layers. All limbal epithelial cells are positive with integrin ß4 subunit, and in the basal layer, it is localized to the bottom and top membranes. In limbal explant and its epithelial outgrowth on amniotic membrane, both p63 and Ki-67 positive cells are present only in the basal layer, and keratin 3 positive cells are confined to the stratified superficial layers. Interestingly, the fluorescent signal of integrin ß4 subunit is evenly distributed in cells of the limbal epithelial explant and its outgrowth. Conclusions: Our results suggested that the epithelial outgrowth of limbal explant on amniotic membrane exhibits a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.
Keywords: cornea: epithelium