In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
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DOI：
10.21769/bioprotoc.3755
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发表时间：
2020-09-20
期刊：
影响因子：
0.8
通讯作者：
Frazer, Kelly A.
中科院分区：
文献类型：
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作者：
D'Antonio-Chronowska, Agnieszka;D'Antonio, Matteo;Frazer, Kelly A.
诱导多能干细胞来源的心血管前体细胞为研究心脏发育和疾病病因学的分子基础提供了一个前所未有的平台，但也具有在未来再生医学和药物基因组研究中发挥关键作用的巨大潜力。像iPSCORE这样的生物库(Stacey等人，2013年；Panopoulos等人，2017年)，其中包含从数百个遗传和种族多样化的个人产生的IPSCs，是进行这些研究的宝贵资源。在这里，我们提出了一种优化的、经济有效的和高度标准化的方案，用于使用小分子大规模衍生人IPSC-CVPC，并使用代谢选择进行纯化。我们已经成功地应用该方法从154个不同的iPSCORE iPSC系中分离出IPSC-CVPC，获得了大量高纯度的心肌细胞。我们协议的一个重要组成部分是细胞融合估计(CcEstimate)，这是一种自动方法，用于估计IPSC单层达到80%融合的时间，这是启动IPSC-CVPC派生的最佳选择，并使该协议能够方便地用于不同生长速度的IPSC系列。此外，我们发现IPSC-CVPC之间的细胞异质性是由于两种不同类型的心肌细胞所占比例的不同：心肌细胞(CMS)和心外膜来源细胞(EPDCs)，这两种细胞都被证明在心脏再生中具有关键功能。该协议消除了IPSC线对线优化的需要，并且可以很容易地适应和扩展到高通量研究或产生适合于再生医学应用的大量细胞。
Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE (Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications.