Hyperoxia impairs iPSC derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype. Hyperoxia impairs iPSC derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

Induced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. We examined iPSC-ECs at late passage (>2 weeks) under hyperoxic (21%)conditions with single cell RNA sequencing Overall design: Y6 pluripotent stem cells were directed to endothelial identity by treatment with CHIR99021, a GSK3 inhibitor, as well as BMP4 for 3 days, followed by VEGFa and forskolin treatment for 2 days according to the protocol described by Cowen et al 2014, Nature Cell Biology DOI 10.1038/ncb3205. Then, cells were purified using CD144 magnetic beads, and were subsequently cultured for two weeks at atmospheric oxygen level (21%) in a conventional cell culture incubator before harvest and analysis by scRNAseq

诱导多能干细胞（induced pluripotent stem cells，iPSCs）定向分化为内皮细胞表型（iPSC-ECs）后，在标准体外培养条件下会丢失关键身份标记物的表达，这限制了其临床应用。我们针对传代晚期（培养时长超过2周）的iPSC-ECs，在高氧（21%氧浓度）条件下开展了单细胞RNA测序（single cell RNA sequencing，scRNAseq）分析。

实验整体设计如下：参照Cowen等人2014年发表于《Nature Cell Biology》（DOI: 10.1038/ncb3205）的实验方案，首先使用GSK3抑制剂CHIR99021与骨形态发生蛋白4（BMP4）处理Y6多能干细胞3天，将其定向诱导为内皮细胞表型；随后采用血管内皮生长因子A（VEGFa）与福司可林（forskolin）继续处理2天。之后通过CD144磁珠对诱导得到的细胞进行纯化，并在常规细胞培养箱中以常压氧浓度（21%）继续培养两周，最后收集细胞并通过scRNAseq完成检测分析。