Generation of iPS from marmosets for hematopoietic therapies
Priv.-Doz. Dr. P.A. Horn

The goal of this subproject is to develop induced pluripotent stem cells (iPS) in a clinically relevant nonhuman primate model, the marmoset (Callithrix jacchus). Marmoset iPS will be derived from skin fibroblasts or bone marrow mesenchymal cells by gammaretroviral or lentiviral transfer of a combination of genes that have been described for reprogramming of human and mouse iPS (combining either Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog and Lin28). In addition, we will assess the suitability of foamyviral vectors for reprogramming since those display improved safety features and different silencing kinetics. Marmoset iPS will be characterized in direct comparison to marmoset ESC. This includes expression of pluripotency genes, methylation status, embryoid body formation and targeted differentiation in vitro as well as teratoma formation in vivo. We will differentiate marmoset iPS to hematopoietic stem cells. These will be transplanted in a NOD/SCID xenotransplant model to assess their engrafting and in vivo differentiation capabilities. Finally, we will perform autologous transplantation of iPS-derived hematopoietic stem cells in marmosets in a pilot study.


Generation of iPS from rhesus monkeys
Prof. Dr. U. Martin

The recent generation of mouse and human induced pluripotent stem cells (iPS) represents a major breakthrough in stem cell research. Isolation of iPS from large animals including primates is now mandatory with respect to future clinical applications. In the case of embryonic stem cells (ESCs) it has been shown that rhesus monkey cells are indistinguishable from human ESCs on a phenotypical and functional level but behave entirely differently from murine ones in many aspects. Accordingly, we anticipate only minor differences between iPS of human and rhesus monkey origin. Rhesus monkey iPS (rhiPS) represent an excellent complementation to human iPS enabling not only detailed comparative in vitro characterization but also functional analyses in preclinical autologous cardiac and pulmonary transplantation models. The need for suitable large animal models in these areas is obvious since existing mouse models suffer from major animal-sizerelated limitations. Based on our experience in cultivation, lentiviral transgenesis and differentiation of rhesus monkey ESCs (RESCs), it is the goal of this subproject to generate iPS from rhesus monkeys. Our well established system for cultivation and mesendodermal / cardiac and pulmonary differentiation of ESCs will allow direct comparison of RESCs and rhiPS on a phenotypical and functional level.

iPS in a marmoset model for Parkinson’s disease
Dr. R. Behr/Dr. T. Müller

The long-term goal of this subproject is the development of a preclinical non-human primate model for cell replacement therapy in Parkinson’s disease using induced pluripotent cells (iPS). The use of retroviral vectors induces genomic alterations in the cells and hence have tumorigenic potential. Here, a method for the generation of iPS without inducing genomic alterations will be explored in its efficiency to reprogram marmoset fibroblasts. Primary ibroblasts will be transfected with episomal vectors encoding Oct4, Sox2, Klf4, and c-Myc (or alternative combinations) to obtain epiPS (episomal iPS), which will eventually be used for preclinical testings in non-human primates. After successful derivation and characterization of epiPS (in comparison with marmoset ESC), the epiPS will be retransplanted into the original cell donor and immunological tolerance by the recipient will be determined. It is the long term goal to differentiate the epiPS to dopaminergic neurons, which will then be transplanted into an established primate Parkinson’s disease model. If iPS derived using the viral approach appear to be superior to epiPS, the differentiation and transplantation experiments will be performed with “classical” iPS established by partner 2.