Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good 'cobblestone-like' morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.
Keywords: AA, antibiotic/antimycotic; ALCAM/CD166; CM, conditioned medium; DM, descement membrane; DMEM, Dulbecco's modified Eagle's medium; DSAEK, Descement's stripping automated endothelial keratoplasty; FBS, fetal bovine serum; FGF-2, fibroblast growth factor-2; FNC, fibronectin and collagen-based; FT, flowthrough; GPC-4, Glypican-4; HRP, horseradish peroxidase; ICC, immunocytochemistry; IP, immunoprecipitation; LEC, lens epithelial cells; MACS, magnetic affinity cell separations; MFI, mean fluorescence intensity; MPL, monophosphryl-lipid A; Na+K+ATPase, sodium potassium ATPase; Peroxiredoxin-6; Prdx-6, Peroxiredoxin-6; TDM, trehalose dichorynmycolate; ZO-1, zonula occludins-1; cell enrichment; characterization; hCENC, human corneal endothelial cells; hCSF, human corneal stromal fibroblasts; hPSC, human pluripotent stem cells; human corneal endothelial cells; mAbs, monoclonal antibodies; monoclonal antibodies; nMFI, normalized mean fluorescence intensity.