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J Vis Exp. 2016 Jun 17;(112). doi: 10.3791/54150.

Isolation of Murine Embryonic Hemogenic Endothelial Cells.

Author information

1
Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine.
2
Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine.
3
Department of Molecular and Cellular Biology, Baylor College of Medicine.
4
Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine; karen.hirschi@yale.edu.

Abstract

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.

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