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Method Article
Brandon Hadland
Fred Hutchinson Cancer Research Center
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.
Keywords
Haematopoietic stem cell (HSC), aorta-gonad-mesonephros (AGM), embryonic haematopoiesis, haematopoietic niche, endothelial cell
Figure 1
Figure 2
This is a list of supplementary files associated with this preprint. Click to download.
- AGMAktECMediaTables.pdf
Table 1. AGM Akt-EC culture media. This table contains detailed information regarding the quantities and concentrations of reagents needed to make AGM EC base media and AGM EC+ media.
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Associated Publications
Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros–derived hematopoietic stem cells
by Brandon K. Hadland, Barbara Varnum-Finney, Michael G. Poulos, +4
Journal of Clinical Investigation (12 April, 2015)
10.1016/j.stemcr.2017.04.007
Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture
by Brandon K. Hadland, Barbara Varnum-Finney, Cynthia Nourigat-Mckay, +2
Journal of Visualized Experiments (08 May, 2018)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Brandon Hadland
Fred Hutchinson Cancer Research Center
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 02 Sep, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.
Figure 1
Figure 2
This is a list of supplementary files associated with this preprint. Click to download.
- AGMAktECMediaTables.pdf
Table 1. AGM Akt-EC culture media. This table contains detailed information regarding the quantities and concentrations of reagents needed to make AGM EC base media and AGM EC+ media.
Loading...
Associated Publications
Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros–derived hematopoietic stem cells
by Brandon K. Hadland, Barbara Varnum-Finney, Michael G. Poulos, +4
Journal of Clinical Investigation (12 April, 2015)
10.1016/j.stemcr.2017.04.007
Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture
by Brandon K. Hadland, Barbara Varnum-Finney, Cynthia Nourigat-Mckay, +2
Journal of Visualized Experiments (08 May, 2018)