Work package 2

In WP 2, somatic cell nuclear transfer (SCNT) will be used to produce porcine fetuses with different sets of genetic modifications as selected in WP 1. We will terminate the pregnancies after 60 days to isolate organspecific porcine endothelial cells (pEC) carrying the selected genetic modifications. These pEC will undergo a second round of in vitro testing to assess the effects of specific sets of genetic modifications on humoral and cellular innate immune activation, activation of the coagulation system, and direct activation of T cells. Emphasis will be laid on assessing specific functions of EC, which cannot be reliably tested with pKC. Porcine EC will also be tested by the Bern group with whole, non-anticoagulated human blood. These assays will allow the selection of sets of advanced genetic modifications which optimally inhibit activation of the human coagulation cascade. In addition, other EC-specific functions, like the preservation of a pro-fibrinolytic phenotype of the EC, will be assessed. The possibility to grow the pEC under physiological, organ-specific flow conditions in the microfluidic EC culture model will help us to decide whether the same set of genetic modifications can be used for flow- and shear stress conditions of all organs or whether organ-specific sets of genetic modifications should be used. The aim of WP 2 is to define a set of advanced genetic modifications which shall be used to produce piglets for testing in limb perfusion assays and finally, outside of this project, in pig-to-NHP xenotransplantation.