Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

中文翻译：

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在化学定义的条件下，非人类灵长类动物iPSC的产生，培养和心脏分化。

非人灵长类动物（NHP）是晚期治疗药物（ATMP）的临床前后期开发的重要替代模型，包括基于诱导多能干细胞（iPSC）的疗法，该疗法也正在开发中，用于心力衰竭修复。为了通过肌肉再生进行有效的心脏修复，需要大量心肌细胞，这可以通过有效分化iPSC来获得。但是，在无饲养细胞的条件下，NHP-iPSC的产生和在未分化状态下的长期培养被证明是有问题的。在这里，我们描述了恒河猴（猕猴）iPSC线路。使用非整合载体在化学定义的条件下对产后恒河猴皮肤成纤维细胞进行重新编程。使用另一个NHP物种橄榄狒狒（Papio anubis）。NHP-iPSC的无饲养者维持基本上取决于通过GSK抑制（Gi）和Wnt抑制（Wi）同时进行的Wnt激活。使用组合的生长因子/ GiWi协议成功地将生成的NHP-iPSCs分化为心肌细胞。证明了iPSC衍生的心肌细胞自组织为可收缩的工程心肌（EHM）的能力。总的来说，这项研究为NHP-iPSC的稳健产生和培养建立了可重复的方案，这对于NHP中细胞置换治疗策略的临床前测试很有用。