Isolation of Murine Embryonic Hemogenic Endothelial Cells.
Isolation of Murine Embryonic Hemogenic Endothelial Cells.
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DOI：
10.3791/54150
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发表时间：
2016-06-17
期刊：
影响因子：
--
通讯作者：
Hirschi KK
中科院分区：
文献类型：
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作者：
Fang JS;Gritz EC;Marcelo KL;Hirschi KK
胚胎血管内皮的生血内皮细胞的特化发生在不同组织内的短暂发育期，并且对于从小鼠胚胎外卵黄囊、胎盘、脐血管和胚胎性腺-中肾（AGM）区域出现永久性HSPC是必需的。该细胞群的瞬时性质和小尺寸使得其用于仔细定量和实验应用的可再现分离在技术上困难。我们已经建立了一种基于荧光激活细胞分选（FACS）的方案，用于在卵黄囊和AGM中的生血内皮细胞和HSPC的峰值生成时间期间同时分离它们。我们演示了从小鼠胚胎中解剖卵黄囊和AGM组织的方法，并且我们提出了优化的组织消化和抗体偶联条件，以便在通过FACS进行鉴定和检索之前获得最大的细胞存活率。显示了代表性的FACS分析图，其鉴定了生血内皮细胞和HSPC表型，并描述了用于在克隆水平上评价其血液形成潜力的基于甲基纤维素的测定。
The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.