Production of cloned pigs expressing human thrombomodulin in endothelial cells

Abstract

Yazaki S, Iwamoto M, Onishi A, Miwa Y, Hashimoto M, Oishi T, Suzuki S, Fuchimoto D‐I, Sembon S, Furusawa T, Liu DG, Nagasaka T, Kuzuya T, Ogawa H, Yamamoto K, Iwasaki K, Haneda M, Maruyama S, Kobayashi T. Production of cloned pigs expressing human thrombomodulin in endothelial cells. Xenotransplantation 2012; 19: 82–91. © 2012 John Wiley & Sons A/S. Abstract: For long‐term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti‐inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK‐puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM‐PAEC. Although no bleeding tendency was observed in hTM‐cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen‐antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.

Role of anti‐CD40 antibody‐mediated costimulation blockade on non‐Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig‐to‐baboon model

“…Variable graft survival results with these genetic modifications are reported . Some recent modifications to GTKO pigs include expression of human thromboregulatory proteins such as CD39 and thrombomodulin but the graft survival data for organs from these pigs are not yet available. Besides genetic modifications of the donor, the immunosuppressive regimen is continuously being modified with more emphasis on reducing the lethal side effects by replacing generalized immunosuppression with target‐specific immunosuppression or immunomodulation.…”

Expression of human thrombomodulin on porcine endothelial cells can reduce platelet aggregation but did not reduce activation of complement or endothelium – an experimental study

“…Therefore, it is difficult to rule out the effect of hTBM alone in this setting. There are only few studies focusing on the effect of hTBM alone . Two major effects of hTBM in this context have been described: first, regulation of the procoagulant activity by prevention of thrombin generation and second, anti‐inflammatory and complement regulatory properties .…”

Recent advances in understanding xenotransplantation: implications for the clinic

“…This problem is being resolved by the development of pigs that express one or more human coagulation-regulatory proteins, e.g., thrombomodulin (TBM), endothelial protein C-receptor, CD39, or tissue factor pathway inhibitor, thus balancing procoagulation and anticoagulation in the graft [46,47]. …”