Abstract
Abstract: :
Purpose: The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of K3 keratin (K3) and a weak expression of connexin 43 (Cx43). We would like to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation. Materials and Methods: We developed a technique for isolating an entire viable limbal (Le) and corneal (Ke) epithelial sheet from the pigmented limbus and the central cornea, respectively, of the dutch belted rabbits by 8.5 mm trephination and incubation for 18 h at 4 oC in SHEM medium containing 50 mg/ml of Dispase II and 100 mM sorbitol. A video of such a technique will be presented.. The resultant sheet was characterized by immunostaining with antibodies against K3 and Cx43. Isolated Le and Ke were recombined with Dispase-denuded limbal (Ls) or corneal (Ks) stroma, and then cultured in SHEM for 10 days before air-lifting for one week. The resultant epithelial phenotype was determined by histology and immunostaining to K3, Cx43 and BrdU after 24 hr labeling. Results: An intact Le and Ke obtained by this new technique exhibited a high cell viability of 92 % after being disintegrated into single cells and tested by ethidium and calcein staining. Le showed basal negativity to K3 and weak Cx43, a pattern resembling the in vivo limbus. After air-lifting, the resultant epithelium was stratified in all recombinants. K3 expression was positive in full thickness of Ke/Ks, negative in the basal layer of Le/Ls and Ke/Ls, and sporadically positive in the basal layer of Le/Ks. Cx43 expression was uniformly positive in the basal layer of Ke/Ks, but weak in that of Le/Ls, Ke/Ls and Le/Ks. Twenty-four hour BrdU labeling for rapid cycling cells showed positive nuclei in basal and suprabasal layers of Ke/Ks, Ke/Ls and Le/Ks, but exclusively in the basal layer of Le/Ls, which also contained cells with negative staining. Conclusion: Both corneal and limbal epithelial sheets can be easily and reproducibly isolated from rabbits and recombined on different stroma to test their differentiation. Reprogramming of adult corneal transient amplifying cells to a limbal phenotype can be induced when exposed to limbal stromal niche. Conversely, differentiation of limbal epithelial stem cells to corneal epithelial phenotype can be induced upon contact with corneal stroma. Further characterization of the differences in such stromal niches is warranted.
Keywords: cornea: epithelium • cornea: basic science • microscopy: light/fluorescence/immunohistochem