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New transgenic pigs for xenotransplantation, part 2: Strategies to overcome cellular rejection
Author(s) -
Keßler B.,
Kurome M.,
Klymiuk N.,
Wünsch A.,
Seissler J.,
Wolf E.
Publication year - 2011
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/j.1399-3089.2010.00607_7.x
Subject(s) - xenotransplantation , thrombomodulin , transplantation , biology , transgene , immunology , endothelial protein c receptor , transfection , microbiology and biotechnology , thrombin , cell culture , medicine , platelet , gene , genetics
The successful generation of α‐1,3 galactosyltransferase knockout (αGal‐KO) pigs and pigs expressing human complement regulators was a crucial step forward in xenotransplantation research. These genetic modifications helped to control hyperacute rejection, but vascular and cellular mechanisms still represent a major hurdle for pig‐to‐primate xenotransplantation. Coagulation incompatibilities between human blood and porcine endothelium lead to thrombotic microangiopathy, which could be observed as a key rejection mechanism in pig‐to‐primate transplantation using αGal‐KO pigs as organ donors. Circulating human thrombin is bound by porcine thrombomodulin, but the resulting heterodimer fails to activate protein C, which inhibits the coagulation cascade. We established a vector for endothelial‐specific expression of human thrombomodulin (hTM) in αGal‐KOxCD46 pigs. The hTM construct was combined with a floxed blasticidin resistance cassette and transfected mixed cell populations were used as donors for somatic cell nuclear transfer (SCNT). Two hundred and ninety‐eight reconstructed embryos were transferred to four recipients, resulting in one pregnancy, which lead to the birth of three living piglets. All of them showed strong endothelial hTM expression. In contrast to vascularised organs, pancreatic islets are not subject to hyperacute rejection, but to cellular rejection, mainly based on T‐cell stimulation as islets of mature pigs express αGal‐epitopes only at a very low level and are vascularized by recipient capillaries after transplantation. Human CTLA4‐Ig is a potent inhibitor of T‐cell activation, excelled by its derivate LEA29Y which has higher binding affinity to the costimulatory B7 receptors. We have established the coding sequence of LEA29Y under the transcriptional control of a regulatory fragment of the porcine insulin gene. The construct also contained a floxed neomycin resistance cassette. Porcine fetal fibroblasts were transfected, followed by selection under geneticin for 7 days. Stably transfected cell clones were pooled and used as donor cells for SCNT. Transfer of 216 reconstructed embryos to three recipients resulted in two pregnancies with nine born piglets, including two stillbirths. Seven piglets were shown to be transgenic. Immunohistochemistry of the pancreata of two transgenic founders showed high expression levels of LEA29Y at an age of 3 months.