Comparison of proliferative capacity of genetically-engineered pig and human corneal endothelial cells.

PURPOSE: The possibility of providing cultured corneal endothelial cells (CECs) for clinical transplantation has gained much attention. However, the worldwide need for human (h) donor corneas far exceeds supply. The pig (p) might provide an alternative source. The aim of this study was to compare the proliferative capacity of CECs from wild-type (WT) pigs, genetically-engineered (GE) pigs, and humans. METHODS: The following CECs were cultured: hCECs from donors (i) ≤36 years (young), (ii) ≥49 years (old), and WT pCECs from (iii) neonatal (<5 days), (iv) young (<2 months), and (v) old (>20 months) pigs, and CECs from young (vi) GE pigs (GTKO/CD46 and GTKO/CD46/CD55). Proliferative capacity of CECs was assessed by direct cell counting over 15 days of culture and by BrdU assay. Cell viability during culture was assessed by annexin V staining. The MTT assay assessed cell metabolic activity. RESULTS: There was significantly lower proliferative capacity of old CECs than of young CECs (p < 0.01) in both pigs and humans. There was no significant difference in proliferative capacity/metabolic activity between young pCECs and young hCECs. However, there was a significantly higher percentage of cell death in hCECs compared to pCECs during culture (p < 0.01). Young GE pCECs showed similar proliferative capacity/cell viability/metabolic activity to young WT pCECs. CONCLUSIONS: Because of the greater availability of young pigs and the excellent proliferative capacity of cultured GE pCECs, GE pigs could provide a source of CECs for clinical transplantation.

Distribution of non-gal antigens in pig cornea: relevance to corneal xenotransplantation.

PURPOSE: The aim of this study was to investigate the distribution of antigens other than galactose-α-1,3-galactose (Gal) (non-Gal) recognized by human and rhesus monkey serum antibodies in the α-1,3-galactosyltransferase gene-knockout (GTKO) pig cornea. METHODS: The distribution of non-Gal, specifically N-glycolylneuraminic acid (NeuGc), in the corneas from wild-type (WT) and GTKO pigs was identified. Corneal sections from WT and GTKO pigs were incubated with human or rhesus monkey serum to determine immunoglobulin (Ig)M and IgG binding to corneal tissue by means of fluorescent microscopy. RESULTS: Strong expression of NeuGc was found in all layers of both WT and GTKO pig corneas. In both humans and monkeys, antibody binding (IgG > IgM) to GTKO was found to be weaker than that to entire WT pig corneas, but in both, most antibody binding, especially IgG, was to the epithelium. There was weak diffuse antibody binding, especially of IgG, to the corneal stroma, suggesting binding to antigens expressed on collagen. There was no or minimal binding of IgM/IgG to the corneal endothelium. CONCLUSIONS: Although the cornea is avascular, antibodies in primate serum can bind to pig antigens, especially on epithelial cells and stromal collagen. Although the binding to entire GTKO corneas was weaker than that to WT corneas, deletion of the expression of NeuGc and expression of human complement-regulatory proteins in the pig cornea will be important if prolonged clinical corneal xenograft survival is to be achieved.

Characterization of porcine corneal endothelium for xenotransplantation.

PURPOSE: Endothelial keratoplasty (EKP) has become increasingly popular in the treatment of corneal disease. However, the global shortage of human donor corneas limits clinical corneal transplantation. Genetically engineered (GE) pigs may provide an alternative source of corneas for EKP. The aim of this study was to evaluate corneal endothelial cells (CECs) from wild-type (WT) and GE pigs. METHODS: Density, size of CECs, and the percentage of hexagonal cells (as a measure of heterogeneity) were measured by ex vivo confocal microscopy in corneas from WT and GE pigs of different ages - neonatal (4-5 days), young (5-15 weeks), adult (5-15 months), and old (20-42 months). α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for the human complement-regulatory protein(s), CD46 (GTKO/CD46) +/- CD55 (GTKO/CD46/CD55) were used as sources of GE corneas. RESULTS: Mean CEC densities (cells/mm²) were neonatal (5968), young (3789), adult (2589), and old (2070). As with human corneas, there was an age-dependent decrease in pig CEC density and increase in pig CEC size. However, unlike human corneas, there was no correlation between the percentage of hexagonal cells (approximately 50% in all pig corneas) and age, suggesting that heterogeneity is intrinsic for pig corneas. Genetic modification did not affect CEC density, size, or morphology compared to WT pigs. CONCLUSION: Because of the availability of young pigs and their greater CEC density (and the protection afforded against the human immune response), GE pigs could provide an unlimited source of corneas for clinical EKP.