Co–localization and High Expression Level of ß–1 integrins and K3 Keratins in Expansion of Human Corneolimbal Explants.

Abstract

Abstract: : Purpose: Transplantation of expanded human corneolimbal explants is a new treatment for ocular surface reconstruction. Limbal epithelial stem cells are characterized by a slow cell cycle and are thought to be devoid of K3 keratin, a basic cytokeratin specific to corneal epithelium. Our previous report (Yiu SC et al, IOVS 2001; 42: ARVO Abstract 5477) showed high expression of ß–1 integrins in the human corneolimbal stem cell compartment, which suggests that ß–1 integrin could be used as a marker for corneal stem cells. If this unique property remains unchanged in primary culture, it may be possible to isolate these cells individually and expand them to confluence to obtain a pure population of stem cells. The present study is to determine whether cultured corneolimbal epithelial cells continue to express high levels of ß1 integrin and continue to be K3 keratin negative. Methods: Human corneal donor rims were obtained from corneal transplant surgery. Explants of 2 mm x 2 mm were obtained from the corneolimbal area and placed on a sterile petri dish with growth media (EpiLife, Cascade Biologic). The cultured epithelial cells were allowed to grow to confluence and fixed with 4% paraformaldehyde. The cultured epithelial cells were then stained with ß1, α6, K3 keratin, and other antibodies. Confocal microscopy was used to detect and analyze staining patterns. Results: One cluster of cells was brightly stained with ß–1 integrins, while others were brightly stained with α–6 integrins. But not all cells stained brightly with either ß–1 integrins or α–6 integrins. In addition, cells that were brightly stained with ß–1 also had higher expression K3 keratin. This co–localization and high expression of ß–1 and K3 keratin differs from our findings in the whole tissue study (Yiu SC et al, IOVS 2001; 42: ARVO Abstract 5477). Conclusions: Our results demonstrate that the primary culture of corneolimbal explants retained characteristics similar to those of cells in the human corneolimbal stem compartment except for the high expression of K3 keratin. The bright ß–1 integrin staining of the one cluster of cells suggested that they could be putative corneal stem cells while the bright α–6 integrin staining of other cells suggested that they could be transient amplifying cells. However, the high expression of K3 keratin among cells that stained brightly with ß–1 suggested these cells may lose some of their "stemness" during the expansion of human corneolimbal explants. Additional work is required to answer this very important question.

Keywords: cornea: epithelium • cornea: basic science