Characterization of Corneal Pannus Removed from Patients with Total Limbal Stem Cell Deficiency

Abstract

Abstract: : Purpose: To determine the origin of epithelial lineage in cells of and derived from entire corneal pannus tissue removed from patients with total limbal stem cell (SC) deficiency. Methods: Entire corneal pannus, removed during ocular surface reconstruction from 8 patients with total limbal SC deficiency – aniridia (2), chemical burns (3), idiopathic (2) and Stevens Johnson syndrome (1), was subjected to immunostaining with anti K3 keratin and anti K19 keratin monoclonal antibodies. A small pannus segment (1 x 1 mm) obtained from 5 of these patients was cultured in SHEM until confluency. Cells extracts were subjected to western blotting and RT–PCR analyses. Results: Hematoxylin staining showed variable degrees of epithelial stratification with goblet cells present in all patients. The stroma of all specimens showed blood vessels and inflammatory cell infiltrates. Immunostaining showed full thickness expression of K19 keratin in the entire pannus of all patients. Immunostaining to K3 keratin was negative in 7 patients, and sporadic suprabasally in a patient with Stevens–Johnson syndrome. After culturing, all 5 pannus specimens showed compact small epithelial cells in outgrowth, and except in one reached confluency after 2 to 3 weeks. Cultured cell extracts from all five cultured specimens and the control of limbal epithelial outgrowth showed a positive 60 kDa band of ΔNp63, but were negative to K3/K12 keratin pair. Expression of K3 keratin mRNA was observed in 3 but expression of K12 keratin mRNA was negative to all five outgrowth specimens. Conclusions: The resultant epithelial phenotype was not corneal as evidenced by the negative staining to cornea–specific keratin K3, and only minimal expression of K3 keratin but not of K12 keratin protein and mRNA, but was conjunctival as evidenced by the presence of goblet cells and K19 keratin in the entirely removed pannus tissue. The abundant expression of ΔNp63 in such conjunctiva–derived epithelium in eyes with total limbal SC deficiency raises doubts in its validity of being a limbal SC marker.

Keywords: cornea: epithelium • conjunctiva • cornea: basic science