From the Stanford Cardiovascular Institute, CA (D.T.P., L.T., J.L., N.S., I.Y.C., J.-W.R., Y.K., R.C.W., J.W.B., S.M.W., K.R.-H., T.Q., J.C.W.).
Department of Medicine, Division of Cardiology, Stanford University School of Medicine, CA (D.T.P., L.T., J.L., N.S., J.-W.R., R.C.W., S.M.W., T.Q., J.C.W.).
Circ Res. 2018 Aug 3;123(4):443-450. doi: 10.1161/CIRCRESAHA.118.312913.
Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology.
Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity.
Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively.
Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.
人诱导多能干细胞衍生的内皮细胞（iPSC-ECs）作为心血管研究的有用工具已经兴起，提供了广泛的转化和临床应用。然而，目前可用的 iPSC-EC 分化方案效率低下，以及衍生的 iPSC-ECs 的异质性仍然是 iPSC-EC 技术的主要限制。
在此，我们对 iPSC-EC 分化后的人类 iPSCs 进行了基于液滴的单细胞 RNA 测序（scRNA-seq）。基于液滴的 scRNA-seq 能够平行分析数千个细胞，从而能够全面分析转录异质性。
通过 scRNA-seq 鉴定了真正的 iPSC-EC 簇，该簇表达高水平的内皮特异性基因。通过 CD144 抗体偶联磁分选对 iPSC-EC 进行分选，显示出标准的内皮形态和功能，包括管形成、对炎症信号的反应以及 NO 的产生。鉴定出分化方案产生的非内皮细胞群体，包括未成熟的心肌细胞、肝样细胞和血管平滑肌细胞。此外，对纯化的 iPSC-EC 进行 scRNA-seq 分析显示出转录异质性，有 4 个主要亚群，分别由 CLDN5、APLNR、GJA5 和 ESM1 基因的丰富表达标记。
大规模平行、基于液滴的 scRNA-seq 允许对数千个接受 iPSC-EC 分化的人类 iPSCs 进行细致的分析。结果表明分化技术效率低下，可以通过进一步研究基于识别抑制非内皮细胞类型扩增的分子特征来改进。还鉴定了真正的人类 iPSC-EC 的亚型，使我们能够对具有特定生物学功能和身份的 iPSC-EC 进行分选。
