D'Antonio-Chronowska Agnieszka, D'Antonio Matteo, Frazer Kelly A
Department of Pediatrics, University of California, San Diego, La Jolla, USA.
Institue for Genomic Medicine, University of California, San Diego, La Jolla, USA.
Bio Protoc. 2020 Sep 20;10(18):e3755. doi: 10.21769/BioProtoc.3755.
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 , 2013 ; Panopoulos , 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.
诱导多能干细胞衍生的心血管祖细胞（iPSC-CVPCs）为研究心脏发育的分子基础和疾病病因提供了一个前所未有的平台，同时在再生医学和药物基因组学研究的未来也具有发挥关键作用的巨大潜力。像iPSCORE（Stacey，2013；Panopoulos，2017）这样的生物样本库包含了从数百名遗传和种族多样化个体中产生的诱导多能干细胞，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、具有成本效益且高度标准化的方案，用于使用小分子大规模诱导人iPSC-CVPCs的产生，并通过代谢筛选进行纯化。我们已成功应用该方案从154种不同的iPSCORE诱导多能干细胞系中诱导出iPSC-CVPCs，获得了大量高度纯化的心脏细胞。我们方案的一个重要组成部分是细胞汇合度估计（ccEstimate），这是一种自动方法，用于估计诱导多能干细胞单层达到80%汇合度的时间，这是启动iPSC-CVPC诱导的最佳时间，并使该方案能够轻松应用于不同生长速率的诱导多能干细胞系。此外，我们表明iPSC-CVPCs之间的细胞异质性是由于两种不同心脏细胞类型比例的变化：心肌细胞（CMs）和心外膜衍生细胞（EPDCs），这两种细胞在心脏再生中都已被证明具有关键功能。该方案无需对诱导多能干细胞系进行逐系优化，并且可以轻松调整和扩展以适用于高通量研究或生成大量适合再生医学应用的细胞。
