Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells

- Venus labeled LESCs can proliferate and maintain the LESC features in vitro.
- Venus-labeled corneal epithelial sheet possessed characteristics of normal corneal epithelium.
- Many donor cells P63+ were unevenly distributed among basal and suprabasal epithelial layers after surgery 3 months.
- The location and survival of donor cells varied according to the corneal epithelial region.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63+ cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.