Stromal Niche Controls the Plasticity of Limbal and Corneal Epithelial Differentiation in a Rabbit Model of Recombined Tissue

PURPOSE. The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation. METHODS. Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4°C. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin Β1, and collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for 1 week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining. RESULTS. Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% ± 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin Β1, negative for K3, but weakly positive for Cx43, and still retained patches of collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks. CONCLUSIONS. These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring stemness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stemness is controlled.