Department of Cardiology, Angiology, Hemostaseology and Medical Intensive Care, Medical Faculty Mannheim, University Medical Centre Mannheim (UMM), Heidelberg University, 68167 Mannheim, Germany.
Key Laboratory of Medical Electrophysiology of Ministry of Education, Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, China.
Int J Mol Sci. 2022 Jul 31;23(15):8507. doi: 10.3390/ijms23158507.
Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), I, I and I were similar in hiPSC-ECs and HCMECs. I was larger and I was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.
人诱导多能干细胞（hiPSC）衍生的内皮细胞（hiPSC-ECs）为血管再生和药物筛选的机制研究提供了新的机会。然而，hiPSC-ECs 的功能仍需要进行表征。本研究旨在比较 hiPSC-ECs 与原代人心脏微血管内皮细胞（HCMECs）的电生理和功能特性，主要集中在离子通道和膜受体信号转导以及特定的细胞功能上。hiPSC-ECs 是从三个独立的健康供体的人皮肤成纤维细胞中生成的 hiPS 细胞衍生而来的。通过流式细胞术、免疫荧光染色、实时定量聚合酶链反应（qPCR）、酶联免疫吸附试验（ELISA）、管形成、LDL 摄取、外泌体释放试验以及重要的膜片钳技术对 hiPSC-ECs 与 HCMECs 进行表型和功能比较。成功地从 hiPS 细胞中生成了 hiPSC-ECs，并通过内皮标志物进行了鉴定。hiPSC-ECs 中 KCNN2、KCNN4、KCNMA1、TRPV2 和 SLC8A1 的 mRNA 水平明显高于 HCMECs。hiPSC-ECs 中 AT1 受体 mRNA 水平高于 HCMECs。AT2 受体 mRNA 水平在所有受体中最高。hiPSC-ECs 中肾上腺素能受体 ADRA2 的表达低于 HCMECs，而 ADRA1、ADRB1、ADRB2 和 G 蛋白 GNA11 和 Gai 的表达在两种细胞类型中相似。hiPSC-ECs 中胆碱能和多巴胺能受体 CHRM3、DRD2、DRD3 和 DRD4 的表达水平明显低于 HCMECs。内皮细胞的功能特征，如管形成和 LDL 摄取试验，在 hiPSC-ECs 和 HCMECs 之间没有统计学差异。苯肾上腺素同样增加了 hiPSC-ECs 和 HCMECs 中血管收缩素内皮素-1（ET-1）的释放。乙酰胆碱也同样增加了 hiPSC-ECs 和 HCMECs 中一氧化氮的生成。hiPSC-ECs 和 HCMECs 的静息电位（RPs）、I、I 和 I 相似。hiPSC-ECs 中的 I 较大，I 较小。此外，我们还注意到 hiPSC-ECs 中高表达外泌体标志物 CD81，而 HCMECs 中高表达 CD9 和 CD63。然而，从这两种类型的细胞中提取的外泌体数量没有显著差异。本研究表明，hiPSC-ECs 在离子通道功能和膜受体偶联信号转导以及生理细胞功能方面与天然内皮细胞相似，尽管存在一些差异。这些信息可能有助于使用 hiPSC-ECs 进行研究。
