Abstract
Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven techniques for treating limbal stem cell deficiency (LSCD). However, the precise regions that are most suitable for preparing explants for transplantation have not been identified conclusively. Accordingly, this in vitro study aimed at determining ideal sites to be selected for tissue harvest for limbal stem cell culture and transplantation. We evaluated cell outgrowth potential and the expression of stem cell markers in cultures from 48 limbal explants from five cadaveric donors. The limbal explants were generated from the three specific sites: Lcor (located innermost and adjacent to the cornea), Lm (middle limbus), and Lconj (located outermost adjacent to the conjunctiva). We found that explants from the Lconj and Lm sites exhibited higher growth potential than those from the Lcor site. Transcript encoding the stem cell marker and p63 isoform, ΔNp63, was detected in cells from Lm and Lconj explants; expression levels were slightly, though significantly (p-value < 0.05), higher in Lm than in Lconj, although expression of ΔNp63α protein was similar in cells from all explants. Differential expression of ATP-Binding Cassette Subfamily G Member 2 (ABCG2) did not reach statistical significance. Immunohistochemistry by indirect immunofluorescence analysis of limbus tissue revealed that the basal layer in explant tissue from Lconj and Lm contained markedly more stem cells than found in Lcor explant tissue; these findings correlate with a higher capacity for growth. Collectively, our findings suggest that explants from the Lconj and Lm sites should be selected for limbal cell expansion for both CLET and SLET procedures. These new insights may guide surgeons toward specific limbal sites that are most suitable for stem cell culture and transplantation and may ultimately improve treatment outcomes in the patients with LSCD.
Highlights
Limbal epithelial stem cells reside in the limbus, which is the area of the eye located directly between the cornea and conjunctiva
simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are currently used as treatments for Limbal stem cell deficiency (LSCD)
We examined the capacity for cell proliferation and expression of stem cell markers using tissue explants from three distinct regions of the limbus, including Lcor, Lm, and limbus adjacent to the conjunctiva (Lconj)
Summary
Limbal epithelial stem cells reside in the limbus, which is the area of the eye located directly between the cornea and conjunctiva. These stem cells have the capacity for self-renewal, are capable of proliferation and migration, and are indispensable for continuous regeneration of the corneal epithelium [1,2,3,4]. Limbal stem cell deficiency (LSCD) is a clinical condition resulting from limbal epithelial stem cell damage. Current treatments for LSCD that aim to restore the defective corneal stem cell population include cultivated limbal epithelial transplantation (CLET) [7, 8], cultivated oral mucosal epithelial transplantation [9, 10], and simple limbal epithelial transplantation (SLET) [11, 12]. Additional information about stemness and proliferation potential of various sites within the limbus would serve to improve harvesting techniques
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