Abstract
INTRODUCTION: We aimed to evaluate biophysical compatibility of corneal endothelial cells in genetically-engineered (GE) pigs to identify the best candidate for in vivo nonhuman primate (NHP) study and to collect baseline data for future clinical trials from a biological function perspective. METHODS: Triple or quadruple knock-out (T[Q]KO) pigs with inactivation of α1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β1,4-N-acetylgalactosaminyltransferase (β4GalNT2), and/or isoglobotrihexosylceramide synthase (iGB3s) genes were used in combination with insertion of Human(h) CD55, CD39, CD46, and thrombomodulin (TBM) genes. Twenty-seven eyeballs obtained from 14 GE pigs were used to evaluate the physical parameters and functional properties as a suitable donor. Corneal endothelial cell density (ECD) was serially measured for 7 days in a preservative solution. Doubling time (DT) for EC proliferation and immunofluorescence staining of corneal endothelial pump channels and tight junctional proteins were evaluated. RESULTS: Mean age of GE pigs was 11.9 months old and mean central corneal thickness was 718.2 µm. Mean ECD loss/week was significantly higher in GE pigs younger than 6 months, which was 55.1%, while it was 8.8% in GE pigs older than 6 months (p < 0.001). T(Q)KO/hCD46/hTBM cornea showed early severe apoptotic or necrotic changes of endothelial cells, whereas other GE corneas did not. Although the mean DT increased in all GE pigs, it showed no difference between the younger and older groups. ZO-1, N-cadherin, ATPase, SLC4A1, and aquaporin 1 were well expressed on endothelial cells regardless of modified gene types. CONCLUSION: It suggests that no effect of the added gene was observed on the endothelial cell functional capacities in TKO(QKO) pig corneas, and GE pigs over 6 months old are suitable as corneal transplant donors.