Isolation of Murine Embryonic Hemogenic Endothelial Cells

被引:7
作者
Fang, Jennifer S. [1 ,2 ,3 ]
Gritz, Emily C. [4 ]
Marcelo, Kathrina L. [5 ]
Hirschi, Karen K. [1 ,2 ,3 ]
机构
[1] Yale Univ, Vasc Biol & Therapeut Program, Yale Cardiovasc Res Ctr, Yale Stem Cell Ctr,Sch Med,Dept Med, New Haven, CT 06520 USA
[2] Yale Univ, Vasc Biol & Therapeut Program, Yale Cardiovasc Res Ctr, Yale Stem Cell Ctr,Sch Med,Dept Genet, New Haven, CT 06520 USA
[3] Yale Univ, Vasc Biol & Therapeut Program, Yale Cardiovasc Res Ctr, Yale Stem Cell Ctr,Sch Med,Dept Biomed Engn, New Haven, CT 06520 USA
[4] Yale Univ, Sch Med, Dept Pediat, Sect Neonatal Perinatal Med, New Haven, CT 06520 USA
[5] Baylor Coll Med, Dept Mol & Cellular Biol, Houston, TX 77030 USA
来源
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2016年 / 112期
关键词
Developmental Biology; Issue; 112; hemogenic endothelium; definitive hematopoiesis; vascular development; mouse embryos; FACS; hematopoietic stem and progenitor cells; HEMATOPOIETIC STEM-CELLS; AORTIC ENDOTHELIUM; PROGENITORS; MARKS;
D O I
10.3791/54150
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
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页数:10
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