Modulation of Keratin, Connexin, and Integrin Expression in Limbal Epithelium Expanded on Intact or Denuded Amniotic Membrane with and without a 3T3 Fibroblast Feeder Layer

Abstract

Abstract: : Purpose:The stem cell (SC)-containing limbal basal epithelium does not express keratin K3, connexin (Cx)43 and Cx50. In contrast, the transient amplifying cell-containing corneal basal epithelium expresses K3 and Cx43; the corneal suprabasal epithelium expresses K3 and Cx50. Amniotic membrane (AM) is an ideal substrate for ex vivo expansion of limbal epithelium. We would like to determine AM culturing conditions that may preferentially promote limbal SC expansion. Methods:Human limbal epithelium (HLE) was expanded by explant cultures on intact and epithelially denuded AM with or without a 3T3 fibroblast feeder layer. Sections of epithelial monolayers for each condition were immunostained for integrin α and ß subunits. Western blot analysis was used to analyze K3, Cx43, and Cx50 expression as well as those integrins showing differences by immunostaining. Xenotransplantation to NIH-nu-xid-bg-mice was performed to investigate the epithelial phenotype after stratification. Results:HLE on denuded AM with and without a 3T3 feeder layer showed a similar pericellular distribution of all integrin subunits. In contrast, expression of α3, α6, ß1 and ß4 integrins on intact AM was reduced at HLE basal membrane in contact with devitalized amniotic epithelial cells. Western blot analysis revealed that the levels of Cx43 and Cx50 proteins by HLE on intact AM were less than those on denuded AM. Addition of a 3T3 feeder layer to denuded AM increased the level of Cx43 and decreased that of Cx50. The protein level of α3 was decreased while that of α6 was increased by HLE on denuded AM with 3T3 fibroblasts as compared to that without 3T3 fibroblasts. After xenotransplantation the basal epithelium of HLE on intact AM did not express K3, Cx43 and Cx50, while that on denuded AM expressed all three markers. The addition of 3T3 fibroblasts resulted in positive staining of K3 and Cx43 but negative staining of Cx50 in the basal epithelium. Conclusion:HLE on intact AM retains a limbal basal epithelial phenotype, while HLE on denuded AM differentiates into a corneal phenotype. The addition of a 3T3 feeder layer slows but does not prevent differentiation of HLE on denuded AM. Future studies are needed to clarify how integrin mediated adhesion complexes may control epithelial stemness.

Keywords: 370 cornea: basic science • 339 cell adhesions/cell junctions • 607 transplantation