Long-term survival of allogeneic donor cell-derived corneal epithelium in limbal deficient rabbits

Purpose. To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model. Methods. Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicatedin vitro without a supporting layer. The cell (3 × 10⁵) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis. Results. A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium. Conclusions. Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epitheliumin vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.