Continuous Migration of Limbal Epithelial Progenitor Cells From the Limbal Explants to Intact Amniotic Membrane During ex vivo Expansion

Abstract

Abstract: : Purpose: Ex vivo expansion of limbal epithelial progenitor cells by amniotic membrane (AM) from a limbal biopsy is a new surgical approach for eyes with limbal stem cell deficiency. It remains unclear whether limbal progenitor cells migrate from the biopsy explant to AM during ex vivo expansion. Methods: Human limbal explants were cultured on intact AM under a submerge manner in SHEM medium for 2 weeks to generate epithelial outgrowth (reaching size of ? mm diameter). The explant was removed, transferred to another intact AM, and cultured for another 2 weeks. Such a maneuver of transfer and culturing was carried out until no epithelial outgrowth was detected. The resultant outgrowth surface area of each passage was digitized. Both epithelial cells from the explant and the outgrowth on AM were harvested by dispase treatment, and seeded at the density of 50 cells/cm² on 3T3 fibroblast feeder layers to assay the clonal growth. Cryosections of limbal explant and epithelial outgrowth were stained with H&E, and immunostained to p63, and pancytokeratins. Results: The surface area of epithelial outgrowth continuously decreased from passage 1 to 3, and no outgrowth was detected after passage 3. At each passage, colony–forming efficiency was higher for epithelial cells harvested from the outgrowth as compared to those harvested from the explant. H&E staining showed that the number of epithelial cell layers on the surface of the explant decreased to one to two after passage 1 or 2, and to only sporadic epithelial cells after passage 3. Interestingly, p63–positive cells were continuously noted in the epithelial outgrowth from passage 1 to 3, but only in the surface epithelium of the explant at passage 1. Besides migrating onto AM to generate epithelial outgrowth, groups of epithelial cells expressing pancytokeratins were also found in the stroma of the limbal explant at each passage, but p63–positive cells were only found in the limbal stroma at passage 1, confirming the existence of intrastromal invasion. Conclusions: Continuous migration of limbal progenitor cells onto intact AM takes place during ex vivo expansion. However, some progenitor cells also invade into the explant stroma. Whether invaded cells undergo epithelial–mesenchymal transition into fibroblasts at late passages will be further determined.

Keywords: cornea: epithelium • cornea: basic science