The Aorta-Gonad-Mesonephros niche shapes the functions of yolk sac-derived macrophages involved in hematopoietic stem and progenitor cell generation ex vivo

Technical Summary: The Aorta-Gonad-Mesonephros niche shapes the functions of yolk sac-derived macrophages involved in hematopoietic stem and progenitor cell generation ex vivo

Problem Statement
Hematopoietic stem cell (HSC) transplantation is a curative therapy for blood disorders, yet its application is limited by donor scarcity and graft-versus-host disease. Generating patient-specific HSCs from induced pluripotent stem cells (iPSCs) offers a solution, but current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, HSCs emerge in the aorta-gonad-mesonephros (AGM) region via endothelial-to-hematopoietic transition (EHT). While yolk sac (YS)-derived macrophages are known to populate the AGM at the time of HSC emergence, the specific mechanisms by which they support HSPC generation ex vivo remain poorly defined. Furthermore, it is unclear whether macrophage functions are intrinsic to their YS origin or shaped by the AGM niche.

Methodology
The study employed a combination of in vivo mouse models and in vitro functional assays:

• Mouse Models: Wild-type C57BL/6, MacGreen (Csf1r-EGFP), F4/80 knockout (KO), and lineage-tracing (Cdh5-CreERT2; Rosa26LSL-tdTomato) mice were used. Embryos were staged at E10.5.
• Cell Sorting and Characterization: AGM and YS tissues were dissected and digested. Flow cytometry was used to distinguish myeloid populations based on CD45, CD11b, F4/80, cKit, and CD206. A refined gating strategy was developed to separate CD206⁺ mature macrophages from CD206⁻ myeloid progenitors.
• Functional Assays:
  • Colony-Forming Unit (CFU-C) assays assessed progenitor potential of sorted cells on OP9 stromal cells.
  • Co-culture systems tested the ability of CD206⁺ macrophages to enhance the progenitor potential of hemogenic endothelial cells (ECs) from both AGM and YS sources, including reciprocal co-cultures (AGM macrophages with YS ECs and vice versa).
  • Lineage Tracing: 4-OHT pulse-chase experiments at E7.5 and E8.5 tracked the origin of AGM macrophages.
• Transcriptomics: Bulk RNA sequencing (RNA-seq) was performed on sorted CD206⁺ and CD206⁻ macrophages from AGM and YS. Differential expression analysis (DESeq2) and NicheNet ligand-receptor interaction analysis were used to identify niche-specific genes and predicted interactions with endothelial and cluster cells.
• Validation: RT-qPCR and immunostaining validated RNA-seq findings and protein expression levels (e.g., F4/80, Ccr2, Tnfr).

Key Contributions and Results

1. Definition of Macrophage Maturity in the AGM:
   The study establishes that CD206 expression defines terminally differentiated macrophages in the mouse AGM at E10.5. CD206⁺ macrophages lack in vitro progenitor potential, whereas CD206⁻ myeloid cells (even those expressing F4/80) retain multipotent progenitor capacity and can generate hematopoietic colonies.

2. Niche-Specific Acquisition of Function:
   Lineage tracing confirmed that both CD206⁺ and CD206⁻ AGM macrophages originate from early YS-derived endothelial precursors, with no evidence of de novo macrophage generation within the AGM. Despite this shared origin, AGM macrophages acquire niche-specific properties:

   • Transcriptomic Shift: AGM CD206⁺ macrophages upregulate pro-inflammatory cytokines (e.g., Tnf, Il18) and adhesion molecules (Adgre1/F4/80) compared to their YS counterparts.
   • Functional Divergence: AGM CD206⁺ macrophages enhance the progenitor potential of AGM endothelial cells in co-culture, whereas YS CD206⁺ macrophages do not enhance YS endothelial cells. Reciprocal co-cultures demonstrated that AGM macrophages cannot confer this benefit to YS endothelium, indicating the function is dependent on crosstalk with the AGM microenvironment rather than being an intrinsic, ontogeny-determined property.

3. Identification of AGM-Restricted Genes:
   RNA-seq identified a small set of genes preferentially upregulated in AGM CD206⁺ macrophages compared to all other populations: Mmp2, Nrep, Ccl2, and Cxcl16 (validated by RT-qPCR). NicheNet analysis predicted that these macrophages interact with both endothelial cells (ECs) and cluster cells (CCs) via ligands such as Ccl2 (binding Ccr2) and Tnfα (binding Tnfr), with receptor expression being higher on CCs.

4. A Novel Regulatory Role for F4/80:
   Using F4/80 KO embryos, the study identified a previously unreported, niche-specific role for F4/80 in the AGM:

   • AGM Effects: In the absence of F4/80, the frequency of endothelial cells decreased, while the frequency of cKit⁺ hematopoietic progenitors and colony-forming activity increased. This suggests F4/80 normally restrains HSPC frequency while supporting endothelial maintenance in the AGM.
   • YS Specificity: These effects were absent in the YS of F4/80 KO embryos, where endothelial and progenitor frequencies remained unchanged compared to controls.

Significance and Claims
The paper claims that AGM macrophages, despite sharing a YS origin with other myeloid cells, undergo a transcriptional and functional reprogramming upon entering the AGM niche. This reprogramming equips them with specific factors (e.g., Mmp2, Ccl2, Cxcl16) and a regulatory role for F4/80 that are critical for shaping HSPC output in the AGM.

The authors posit that these findings provide a mechanistic basis for refining iPSC-based HSC differentiation protocols. Specifically, they suggest that the targeted, temporally controlled addition of macrophage-derived signals (such as Ccl2, Tnfα, and Cxcl16) and the modulation of niche interactions could improve the generation of functional HSCs in vitro. The study modestly notes that while these insights are derived from mouse models, validation in human cellular models is essential to determine translational relevance, and that the in vivo role of F4/80 in HSC generation requires further investigation via transplantation assays.