Abstract
Purpose :
The most successful treatment of limbal stem cell deficiency is the transplantation of limbal epithelial stem cells (LESCs) from the healthy eye or from donor. Clinical results have shown that cultured limbal epithelial cell sheets containing higher percentage of LESCs led to successful corneal epithelial regeneration. Unfortunately, the number of LESCs decreases throughout the culture out of their niche. We cultured LESCs in medium supplemented with interleukin-13 (IL-13) to determine the effect of IL-13 on the stemness and differentiation of the cell culture.
Methods :
Human limbal explants were used for culture of limbal epithelial cells up to the second passage (P0-P2) and the effect of IL-13 was determined by qPCR (the expression of p63α, keratin (K)3, K7, K12, K14 and K17). Cells were characterized by immunofluorescence staining for p63α and Ki-67. Clonogenic ability was determined by colony-forming efficiency (CFE) assay and their proliferation was measured by Wst-1 assay.
Results :
The use of IL-13 has no effect on the start, reach of the confluence or viability of the P0 and P1 cell cultures. The capability of reaching the confluence in P2 culture was lower when IL-13 was absent. The expression of putative stem cell markers (p63α, K14 and K17) was significantly higher in all P0-P2 IL-13+ cultures compared to IL-13-. IL-13 had no effect on the differentiation into corneal epithelial cells (K3 and K12 expressions), nevertheless the expression of conjunctival marker K7 was increased. The immunofluorescence analysis showed a comparable positivity for p63α in all cell cultures, but a higher positivity for proliferation marker Ki-67 in cultures with IL-13. Results from CFE assay showed significantly increased clonogenic ability when LECSs were cultured with IL-13 in P1 and P2 cultures. The proliferation activity was comparable in P1 but increased in P2 IL-13+ cell cultures compared to IL-13- cultures.
Conclusions :
We showed that IL-13 significantly increased the stemness of the LESCs cultures by increasing their clonal ability and stem cells gene expression. Cells cultured with IL-13 had higher proliferative activity. IL-13 had no effect on the differentiation of cells into corneal epithelial phenotype, nevertheless it increased the differentiation into conjunctival cells.
This is a 2020 ARVO Annual Meeting abstract.