Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

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.

p63: a crucial player in epithelial stemness regulation

“…Heterozygous mutation in the TP63 gene is also responsible for LESCs failure and results in drastic degeneration of corneal epithelial tissues [ 96 ]. ΔNp63α is the predominantly expressed isoform in LESC and in limbal basal cells [ 97 – 99 ], but not in cells that cover the central corneal surface [ 96 ] (Fig. 3C ).…”

Genetic analysis of allogenic donor cells after successful allo-limbal epithelial transplantation in simple and cultivated limbal epithelial transplantation procedures

“…Moreover, a murine study showed that a small population of LSCs regulated the immune reaction by inhibiting lymphocyte proliferation and altering cytokine production and highly expressed genes for various anti-apoptotic proteins 26 . This explanation might apply especially to patients with partial LSCD when the donor graft helps support the patient's remaining stem cell to repopulate the corneal epithelium, which gradually grows and eventually replaces the decaying donor cells, and maintain a normal ocular surface 27 .…”

The immunology of corneal limbal stem cells: the up-to-date approach to stem cell transplantation

“…The study of Ekpo et al was based on five human cadaveric donor corneoscleral tissues. The culture details were described by Prabhasawat et al [30,31]. After preparation, the explant culture was washed in phosphate-buffered saline (PBS), incubated in dispase for 20 minutes at 37°C, washed in PBS again and then placed on a culture plate.…”