Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

Abstract

Purpose Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (i) to document the lack of NeuGc expression on corneas and aortas, and cultured endothelial cells (aortic [AECs]; corneal [CECs]) of GTKO/NeuGcKO pigs, and (ii) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. Methods Wild-type (WT), GTKO, and GTKO/NeuGcKO pig were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and to determine human IgM and IgG binding to tissues. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. Results Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither human nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared to binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. Conclusions The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.

Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs

“…4A) and corneas [Fig. 4B, detailed in [38]] expressed Gal and NeuGc as did corresponding cultured pAECs and pCECs (Fig. 4C,D).…”

“…Flow cytometric analysis of expression of Gal, NeuGc, and hCD46 on RBCs, PBMCs, AECs, CECs, and cultured pig islets Surface expression of Gal, NeuGc, and hCD46 was investigated by flow cytometry, as previously described [33,34,38]. Cultured porcine islet cells were dissociated into single-cell suspensions by gentle agitation in 0.25% trypsin-EDTA (Invitrogen) for flow cytometric analysis, as previously described [36].…”

“…Flow cytometric analysis for human IgM/IgG binding to RBCs, PBMCs, AECs, CECs, and cultured pig islets IgM and IgG binding assays using human sera (n = 6, including all ABO blood types) were carried out as previously described [33,34,38,39]. FITC-conjugated goat-derived anti-human IgM (l chain-specific) or IgG (c chain-specific) polyclonal antibodies (concentration 1 : 100; Invitrogen) were used as secondary antibodies.…”

Human antibody recognition of xenogeneic antigens (NeuGc and Gal) on porcine heart valves: could genetically modified pig heart valves reduce structural valve deterioration?

“…However, to our knowledge, there is no report regarding the role of another carbohydrate antigen, N ‐glycolylneuraminic acid (NeuGc), against which humans also have natural antibodies and which may be just as important as the Gal antigen in the human‐porcine/bovine xenograft combination. NeuGc epitopes are strongly and broadly expressed on the vascular endothelium throughout the pig, such as on the vascular endothelium of the kidney, liver, heart, and pancreas , and also on cell clusters, for example, islets , as well as on all layers of the cornea . There are also reports of expression of NeuGc on pig aortic and pulmonary valves, using mass spectrometry , but no report has been published using immunofluorescence staining.…”

Xenotransplantation

“…Lee et al studied the impact of the expression of N-glycolylneuraminic acid on pig corneas, concluding that the absence of N-glycolylneuraminic acid expression on GTKO pig corneas may not prove an advantage over GTKO pig corneas [52,53]. Dong et al recently published their initial results of GTKO.hCD46 pig full-thickness corneal xenografts in NHPs, which were comparable to the survival of wild-type pig corneas [54].…”