Final Report Summary - INTEREXT (Interspecies endotheliarization for organ xenotransplantation) Organ transplantation is a very effective approach to treat several deadly diseases. Over 1 million people worldwide have received an organ transplant, which in some cases have allowed the patient to survive for more than 25 years. Nevertheless, the number of organs available for transplantation do not cover the actual demand. Moreover, with the population becoming older, this demand is increasing, and so are the transplantation waiting lists, consequently raising the medical support requirement of the patients, as well as the number of people dying yearly without having received the transplantation needed (at this moment more than 5000/year) (http://www.unos.org/ and http://www.edqm.eu/). Besides the efforts to increase organ donation, new medical strategies are required. The performed project seeks to address a new strategy to solve the problem of organ demand for clinical practice. Transplantation of swine’s organs in humans is a visionary-old idea, which combines the advantage of whole organ replacement, with the potential broad availability of donors. However, despite the big efforts made in the last decades to generate genetically modified pigs and new immunosuppressive treatments, organ xenotransplantation is not clinically applicable due the strong immune reaction against donor organs and the molecular incompatibilities between species which trigger spontaneous coagulation. In recent years, the possibility to generate inter-specie chimera animals has been demonstrated, which opens doors for new strategies to create chimera organs for clinical applications. The knowledge on the mouse genome has made possible to identify specific genes responsible for the development of a given tissue-organ. Mouse models in which one of these genes has been deleted are available. It has been shown that injecting rat pluripotent cells in mice embryos lacking the two copies of the gene responsible for the formation of the pancreas is possible to generate a living mouse-rat chimera with a pancreas of rat origin. This phenomenon is called interspecific embryo complementation. This study provides a breakthrough proof of principle, demonstrating that is possible to generate interspecies chimera, and that is possible to direct exogenous complementation to a desired tissue, through genetic targeting of TFs determinant of one particular cell type. The main objective of the performed project was to generate mouse-rat chimera presenting the vascular system exclusively of rat origin, thanks to the interspecific complementation of vascular-deficient embryos. As pluripotent cells microinjected in preimplantational embryo can contribute to most if not all organs, we decided to generate a strategy to confine the chimerism to the desired tissue i.e. vascular system. Finally we pretended to test the degree of protection against rejection upon xenotransplantation that this revascularization produces. The work of the last two years financed by the Marie Curie action produced the following results: Generation of rat pluripotent stem cells: we have isolated 11 rat embryonic stem (ES) cell lines from two different background rat: Sprague-Dawley (SD) and Wistar-Kyoto. Additionally we have generated 3 iPS cell lines from SD rats using a doxycycline inducible lentiviral system and overexpression of 3 of Yamanaka factors (Oct3/4, Sox2 and Klf4 and 2 iPS cell lines using a retroviral system with constitutive promoter. The different ES and iPS cell lines show naïve-like cell morphology, can grow indefinitely and express markers of pluripotency, as detected both by PCR and by immunofluorescence analysis. Additionally, they express alkaline phosphatase activity and they are able to differentiate form embryoid bodies in vitro and to form teratoma in vivo in Rag2 mice. The primers for real-time PCR were designed in regions with 100% homology with the mouse genome in order to directly compare the similarity of our cells with a standard mouse ES cell line (R1). Our rat cell lines express pluripotency markers at very similar level to R1 mouse ES cell line suggesting the high quality of our cells. This cell lines are being used to generate inter-specific chimerae.Generation of inter-specific chimerae: we have set up for the first time in our group all the tools for mouse embryo handling, microinjection and transfer to foster mothers. Mostly, we collected the manipulated embryos at the gestational age E14.5. This time point has been chosen because it is posterior to the formation of the vascular system. From rat into-mouse microinjection experiments we have obtained a total of 19 embryos and 1 newborn pup, from which 8 were mouse-rat interspecific chimerae. Rat pluripotent cells were able to strongly contribute to different organs of the embryos, differentiating to the corresponding cell types. Importantly, rat cells can form vascular networks in the mouse chimerae. So far we have not obtained any mouse in which the vascular system is exclusively formed by rat cells. For this reason, we have not performed heart xenotransplantation studies. When we will obtain vascular-deficient complemented chimerae that can reach adulthood we will perform these experiments. In conclusion, during the two years period financed by the Marie Curie fellowship we have generated all the tools and optimized the sophisticated technology to generate the mouse-rat interspecies chimerae, which indeed have been obtained during this time frame. We think that our results can notably contribute to understand the potential of blastocyst complementation as a reliable technique for inter-species organ creation. Until now, no European group has published yet about the potential of blastocyst complementation for inter-species organ development, a research field that is leaded by Japan and USA.We have now all the tools and capacity to generate the chimerae with the rat vascular system needed to test the proposed proof of principle: that substitution of the vascular system of the donor species with the cells of the recipient species will drastically reduce the rejection upon organ xenotransplantation. The potential socio-economical impact of these studies is great, as it may constitute the base for further studies on big animals to lead finally to the generation of humanized organs in pig, as a broad source of patient-tailored organs for clinical transplantation.
