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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Research Article Hematology

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 2

Coculture on AGM AKT-ECs generates long-term HSCs from E9.5–E10 P-Sp/AGM VE-cadherin+ precursors.

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Coculture on AGM AKT-ECs generates long-term HSCs from E9.5–E10 P-Sp/AGM...
(A) Schematic of experimental design. Dotted box indicates approximate region of the P-Sp/AGM. (B) Formation of hematopoietic colonies (magnification ×100) and (C) CD45+ cells from sorted P-Sp/AGM VE-cadherin+ cells during coculture with AGM AKT-ECs or without ECs. Also shown are AGM AKT-ECs cultured with hematopoietic cyto­kines but without P-Sp/AGM cells (AGM AKT-EC only). (D) Total CD45+, myeloid (Gr1+ and/or F4/80+), and LSK cells generated per embryo equivalent (ee) of starting cells. Shown is mean ± SD (n = 3), from representative experiment (n = 3). (E) CFU progenitors per ee of starting cells. Shown is mean ± SD (n = 3), from representative experiment (n = 2). (F) Engraftment of VE-cadherin+ cells cultured on AGM AKT-ECs or control (no EC). Shown at each time point is mean ± SD of PB engraftment (n = 4 experiments, 23 total mice), transplanted at 0.5–2 ee. (G) Donor-derived PB engraftment at ≥16 weeks from n = 4 primary recipients transplanted to each of 2 secondary recipients. (H) Engraftment in PB at ≥16 weeks after transplant from E9.5–E10 VE-cadherin+ cells transplanted directly after sort (uncultured) with 2 ee, following coculture on OP9, or on multiple independent AGM AKT-ECs (#1–4) transplanted with 1–2 ee of cells. ‡Transplant from cocultured cells from E9 P-Sp (13–20 sp). Control AGM AKT-ECs cultured with hematopoietic cytokines but without P-Sp/AGM cells were also tested for engraftment (AGM AKT-EC only). Numbers above indicate fraction of mice with multilineage engraftment, designated by data points in red. *P < 0.05, **P < 0.01 AGM, AKT-EC coculture vs. no EC; unpaired Student’s t test.