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.

Figure 7. Model for vascular niche induction and NOTCH-mediated expansion of HSCs.

(A) The AGM AKT-EC vascular niche promotes (i) induction of HSCs from embryonic HE, which is dependent on NOTCH activation mediated by one or more NOTCH ligands expressed on ECs to promote the endothelial-to-hematopoietic transition, as well as other EC-derived niche factors (*) to promote the generation of engrafting HSCs. (ii) Subsequent to HSC specification, further amplification of AGM-derived HSC numbers continues to involve both ligand-mediated NOTCH activation and additional EC-niche factors (**), which contribute to HSC maturation and self-renewal. (B) NOTCH activation by immobilized NOTCH ligand Delta1^ext-IgG, combined with hematopoietic growth factors (SCF, TPO, IL6, FLT3L) and small molecule inhibition of the TGF-β pathway (SB+4GF), is sufficient to amplify AGM-derived HSC numbers in vitro in the absence of AGM AKT-EC stroma.

Figure 6. Delta1^ext-IgG increases multilineage progenitors and long-term HSCs from E11 AGM-derived CD45⁺VE-cadherin⁺ cells.

(A) Schematic of experimental design. Dotted box indicates approximate region of the AGM. (B) Phenotyping of cells cultured on Delta1^ext-IgG or control (hIgG). (C) Total, LSK (SCA1⁺c-KIT⁺Gr1^–F4/80^–), and myeloid (Gr1⁺ and/or F4/80⁺) cells generated on Delta1^ext-IgG or hIgG per embryo equivalent (ee) starting cells. (*P < 0.001, Delta^ext-IgG vs. hIgG, unpaired Student’s t test). Shown is mean ± SD (n = 3), from representative experiment (n > 3). (D) CFU progenitors from cells cultured on Delta1^ext-IgG or hIgG. Shown is mean ± SD (n = 3), from representative experiment (n = 3). (*P < 0.0001 for total, **P < 0.01 for CFU-Mix, Delta^ext-IgG vs. hIgG or uncultured, unpaired Student’s t test) (E) T cell precursor phenotyping following extended culture (8 days) on Delta1^ext-IgG or hIgG. (F) Generation of B cells (CD19⁺B220⁺) and T cells (CD4⁺CD8⁺) after culture on Delta1^ext-IgG or hIgG followed by secondary culture on OP9/OP9-DL1. (G) Engraftment of cells cultured on Delta1^ext-IgG or hIgG. Shown at each time point is mean ± SD of donor PB engraftment (n = 4 experiments, 37 total mice). (H) PB engraftment at ≥16 weeks from n = 5 primary recipients transplanted to each of 2 secondary recipients. (I) Engraftment at ≥24 weeks in mice transplanted with freshly sorted cells (uncultured), or cells cultured on Delta1^ext-IgG or hIgG, transplanted at 1 ee. Shown above is a fraction of mice with multilineage engraftment, designated by data points in red. (*P < 0.0001, Delta1^ext-IgG vs. uncultured, 2-sided Fisher’s exact test). (J) Limit-dilution analysis of HSC repopulating units (HSC/RU) of cells transplanted prior to culture (uncultured) or following Delta1^ext-IgG culture. Dotted lines represent 95% confidence interval (P = 0.00005, ELDA) (60).

Figure 5. NOTCH pathway inhibition reduces generation of phenotypic HSCs during AGM AKT-EC coculture.

(A) Surface expression by staining with antibodies specific for NOTCH1 and NOTCH2 on E11 VE-cadherin⁺CD45⁺ and E9.5 VE-cadherin⁺c-KIT⁺ hematopoietic progenitor cells from dissected P-Sp/AGM. (B) Total live cells, myeloid cells (Gr1⁺ and/or F4/80⁺), LSK (SCA1⁺c-KIT⁺Gr1^–F4/80^–) cells, and LSK-SLAM (SCA1⁺c-KIT⁺Gr1^–F4/80^–CD150⁺CD48^–) cells generated from sorted E11.5 AGM-derived VE-cadherin⁺CD45⁺ cells following 5 days cultures on AGM AKT-ECs in the presence of gamma-secretase inhibitor DAPT or control (DMSO). Shown is mean ± SD of n = 3 replicate samples, from a representative experiment (n = 2).

Figure 4. Coculture on AGM AKT-ECs increases long-term HSCs from E11 AGM CD45⁺VE-cadherin⁺ cells.

(A) Schematic of coculture experiments. Dotted box indicates approximate region of the AGM. (B) Surface phenotyping for LSK and LSK-SLAM subset (CD48^–CD150⁺), designated by red boxes, of cells cultured on AGM AKT-ECs or control (no EC). (C) Total CD45⁺, myeloid (Gr1⁺ and/or F4/80⁺), LSK, and LSK-SLAM cells generated per embryo equivalent (ee) of starting cells. (*P < 0.001, **P < 0.01, AGM AKT-EC vs. no EC, unpaired Student’s t test ). Shown is mean ± SD from replicate samples (n = 3), from representative experiment (n = 3). (D) Engraftment following transplantation of E11 CD45⁺VE-cadherin⁺ cells cultured on AGM AKT-ECs or control (no EC). Shown at each time point is mean ± SD of donor PB engraftment (n = 3 experiments, 12 total mice), transplanted at 1 ee. (E) Donor-derived PB engraftment at ≥16 weeks from n = 7 primary recipients (transplanted with AGM AKT-cultured cells) transplanted to each of 2 secondary recipients. (F) Engraftment in PB at ≥16 weeks after transplant from control (uncultured) cells transplanted at 1 ee, OP9-cultured cells transplanted at 0.1 ee, or cells cultured on multiple independent AGM AKT-ECs (#1-3) transplanted with dilutions of cultured cells expressed per ee of starting population. Numbers above indicate fraction of mice with multilineage engraftment, designated by data points in red. Limit-dilution analysis of HSC/RU (repopulating units) generated following culture on AGM AKT-ECs (#3) by ELDA analysis (60). Dotted lines represent 95% confidence interval (216-2268 HSC/RU per ee).

Figure 3. Coculture on AGM AKT-ECs generates long-term HSCs from both hematopoietic progenitor cells and HE.

(A) Engraftment in PB at ≥16 weeks after transplant from VE-cadherin⁺c-KIT⁺ HPCs or HE sorted as indicated (see also Supplemental Figure 4 for sorting windows and after-sort analysis), following coculture on AGM AKT-ECs, each transplanted with 1–2 embryo equivalent (ee) of cells. Numbers above indicate fraction of mice with multilineage engraftment, designated by data points in red. (B) Donor-derived PB engraftment at ≥16 weeks from n = 5 primary recipients (transplanted with AGM AKT-cultured HE cells) transplanted to each of 2 secondary recipients. (C) CD45⁺, myeloid (Gr1⁺ and/or F4/80⁺), and LSK cells generated from P-Sp/AGM VE-cadherin⁺CD41^–CD45^– HE following coculture with AGM AKT-ECs or without AGM AKT-ECs (no EC). (D) CFU progenitors per ee of HE cells, freshly sorted (uncultured HE), following AGM AKT-EC culture, or cultured without ECs (no EC). Shown is mean ± SD of CFU (n = 3), from representative experiment (n = 2). (E) Total CD45⁺ hematopoietic cells generated from HE following AGM AKT-EC coculture in the presence of DMSO (carrier control) or gamma-secretase inhibitor DAPT. Shown is mean ± SD from replicate samples (n = 3), from representative experiment (n = 3). (*P < 0.001, DMSO vs. DAPT, unpaired Student’s t test ).

Figure 2. Coculture on AGM AKT-ECs generates long-term HSCs from E9.5–E10 P-Sp/AGM VE-cadherin⁺ precursors.

(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.

Figure 1. Constitutive AKT expression permits culture of AGM AKT-EC –expressing NOTCH ligands.

(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.