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Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells
Nature Protocols ( IF 14.8 ) Pub Date : 2021-03-26 , DOI: 10.1038/s41596-021-00497-2
Giulia Campostrini 1 , Viviana Meraviglia 1 , Elisa Giacomelli 1, 2 , Ruben W J van Helden 1 , Loukia Yiangou 1 , Richard P Davis 1 , Milena Bellin 1, 3, 4 , Valeria V Orlova 1 , Christine L Mummery 1, 5
Affiliation  

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.



中文翻译:

人多能干细胞同基因三维自聚集心脏微组织的生成、功能分析及应用

来自包含多种细胞类型的人类多能干细胞的组织样结构正在改变我们模拟和理解人类发育和疾病的能力。在这里,我们描述了从人类诱导的多能干细胞 (hiPSC) 生成心肌细胞 (CMs)、心脏成纤维细胞 (CFs) 和心脏内皮细胞 (ECs) 的方案,这三种主要细胞类型来自人类诱导的多能干细胞 (hiPSC),并将它们组合成三维(3D) 心脏微组织 (MT)。我们详细介绍了如何区分、分离、冷冻保存和解冻组成细胞,以及如何构建和分析 MT。该协议支持 hiPSC-CM 成熟,并允许用带有疾病突变的等基因变异体替换 MT 中三种心脏细胞类型中的一种或多种。每种细胞类型的分化需要约 30 天,而 MT 的形成和成熟还需要 20 d。不需要专业设备,而且该方法成本低廉,每 MT 仅需要 5,000 个细胞。

更新日期:2021-03-26
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