Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros–derived hematopoietic stem cells
Brandon K. Hadland, … , Shahin Rafii, Irwin D. Bernstein
Brandon K. Hadland, … , Shahin Rafii, Irwin D. Bernstein
Published April 13, 2015
Citation Information: J Clin Invest. 2015;125(5):2032-2045. https://doi.org/10.1172/JCI80137.
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Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros–derived hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin–expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherin+CD45+ AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.

Authors

Brandon K. Hadland, Barbara Varnum-Finney, Michael G. Poulos, Randall T. Moon, Jason M. Butler, Shahin Rafii, Irwin D. Bernstein

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Figure 1

Constitutive AKT expression permits culture of AGM AKT-EC –expressing NOTCH ligands.

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Constitutive AKT expression permits culture of AGM AKT-EC –expressing NO...
(A) Schematic of method for generation of AGM AKT-ECs. Image of cultured AGM AKT-ECs, magnification ×100. Dotted box indicates approximate region of the AGM. (B) Surface expression of endothelial markers VE-cadherin and CD31 on primary EC colonies cultured from AGM region and in AGM AKT-ECs following MyrAKT lentiviral transduction and expansion. Surface expression of FLK1, SCA-1, CD34, and CD45 in AGM AKT-ECs. Sub-plots show EC staining with isotype control antibodies. (C) Surface expression of NOTCH ligands JAG1, JAG2, DLL1, and DLL4, and corresponding isotype controls (shown in gray) on freshly sorted AGM endothelium (gated as VE-cadherin+CD45–CD41–) and AGM AKT-ECs.