Rodríguez-Polo Ignacio, Stauske Michael, Behr Rüdiger
Research Platform Degenerative Diseases, German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany.
German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Göttingen, Germany.
Methods Mol Biol. 2022;2454:697-716. doi: 10.1007/7651_2021_380.
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.
非人灵长类动物（NHP），尤其是包括猕猴和狒狒在内的旧世界猴，是用于新型干细胞疗法及其他先进治疗医药产品（ATMP）治疗退行性疾病的临床前后期测试的关键动物模型。这些病症的特征是器官中功能细胞的丧失，如帕金森病、年龄相关性黄斑变性或心肌梗死后的情况。对于基于诱导多能干细胞（iPSC）疗法的临床前相关测试，需要用于NHP-iPSC生成、表征和分化的强大且标准化的方案。自2006年高桥和山中发现iPSC以来，人类重编程方案不断完善。然而，使用目前为人类iPSC建立的方案，生成无整合的NHP-iPSC系以及稳定的无饲养层和无血清长期培养变得困难甚至不可能。在此，我们提供了一种用于生成无转基因旧世界猴（和人类）iPSC并在化学限定条件下进行长期培养的强大方案。该方案已成功应用于从皮肤成纤维细胞生成人类、狒狒（埃及狒狒）、恒河猴和食蟹猴的iPSC。所得的NHP-iPSC为NHP中再生疗法的临床前测试提供了宝贵资源。
