Non-human primates (NHP), and in particular Old World monkeys including macaques and baboons, are key animal models for the late preclinical testing of novel stem cell-based therapies and other advanced therapy medical products (ATMP) for the treatment of degenerative diseases. These pathologies are characterized by the loss of functional cells in an organ, as in Parkinson’s disease, age-related macular degeneration, or after myocardial infarction. For preclinically relevant testing of induced pluripotent stem cell (iPSC)-based therapies, robust, and standardized protocols for the generation, characterization, and differentiation of NHP-iPSCs are required. Since the discovery of iPSCs by Takahashi and Yamanaka in 2006, human reprogramming protocols have been continuously refined. However, the generation of integration-free NHP-iPSC lines and a stable feeder- and serum-free long-term culture turned out to be difficult or even impossible with the current protocols established for human iPSCs. Here, we provide a robust protocol for the generation of transgene-free Old World monkey (and human) iPSCs and long-term cultivation under chemically defined conditions. This protocol was successfully applied to generate human, baboon (Papio anubis), rhesus (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis) iPSCs from skin fibroblasts. The resulting NHP-iPSCs provide a valuable resource for the preclinical testing of regenerative therapies in NHP.

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