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非人类灵长类动物 iPSC 的生成、培养和在化学定义条件下的心脏分化。

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

机构信息

Research Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany.

DZHK (German Centre for Cardiovascular Research), Partner Site Göttingen, 37077 Göttingen, Germany.

出版信息

Cells. 2020 May 29;9(6):1349. doi: 10.3390/cells9061349.

Abstract

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 () 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 (). 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.

摘要

非人类灵长类动物(NHP)是用于晚期临床前开发先进治疗药物产品(ATMP)的重要替代模型,包括诱导多能干细胞(iPSC)为基础的治疗方法,也正在开发用于心力衰竭修复。为了通过再肌化进行有效的心脏修复,需要大量的心肌细胞,可以通过有效的 iPSC 分化获得。然而,NHP-iPSC 的生成和在无饲养细胞的条件下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()iPSC 系的可重复开发。使用非整合载体,在化学定义条件下对产后恒河猴皮肤成纤维细胞进行重编程。该协议的稳健性通过另一种 NHP 物种,即橄榄狒狒()得到了证实。无饲养细胞的 NHP-iPSC 维持基本上取决于 Wnt 激活的同时通过 GSK 抑制(Gi)和 Wnt 抑制(Wi)。使用组合生长因子/GiWi 方案成功地将生成的 NHP-iPSC 分化为心肌细胞。证明了 iPSC 衍生的心肌细胞能够自我组织成收缩性工程心肌(EHM)。总之,这项研究建立了一种可重复的 NHP-iPSC 生成和培养的稳健方案,该方案可用于 NHP 中细胞替代治疗策略的临床前测试。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/878c/7349583/572bce8cb60c/cells-09-01349-g001.jpg

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