Isolation of tissue-resident vascular endothelial stem cells from mouse liver
Isolation of tissue-resident vascular endothelial stem cells from mouse liver
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DOI：
10.1038/s41596-019-0276-x
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发表时间：
2020-01-31
期刊：
影响因子：
14.8
通讯作者：
Takakura, Nobuyuki
中科院分区：
文献类型：
--
作者：
Naito, Hisamichi;Wakabayashi, Taku;Takakura, Nobuyuki
内皮细胞（EC）是构成血管系统的血管的基本组成部分;促进血液流动;并调节渗透性、血管生成、炎症反应和稳态组织维持。越来越多的证据表明，在体内存在EC异质性。然而，从成年小鼠分离新鲜EC以进一步研究这一点是具有挑战性的。在这里，我们描述了一个简单的和可重复的协议，分离不同类型的EC和CD 157（+）血管驻留内皮干细胞（VESC）的机械酶组织消化，然后荧光激活细胞分选。该方法建立在肝组织上，但可以用于以最小的修改从其他器官分离EC。单细胞悬液的制备可在2.5 h内完成。我们还描述了EC克隆和网络形成的测定，以及分离的EC的转录组学分析。通过机械酶组织消化和荧光激活细胞分选，从小鼠中分离出各种类型的内皮细胞和CD 157（+）血管内皮干细胞。
Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157(+) vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.Various types of endothelial cells and CD157(+) vascular-resident endothelial stem cells are isolated from mice by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting.