Hematopoietic stem cells (HSCs) are crucial for blood production, and understanding their development is vital for therapeutic applications like engineering HSCs *ex vivo*. HSCs emerge from hemogenic endothelium (HE) within embryonic arterial vessels, particularly the aorta-gonad-mesonephros (AGM) region. The process is complex, involving sequential waves of lineage-restricted progenitors before HSC emergence. HE cells downregulate endothelial genes and activate hematopoietic genes to produce HSCs. Functional HSCs exhibit self-renewal, homing, and multilineage hematopoiesis. The precise combination of cell-intrinsic and extrinsic signals needed to support these HSC properties during development remains unclear, hindering the goal of engineering HSC development *ex vivo*. The AGM's arterial environment suggests both cell-intrinsic arterial HE properties and cell-extrinsic arterial niche signals contribute to HSC fate. Studies have highlighted the importance of arterial programs, such as Notch pathway regulation, in definitive hematopoiesis and HSC formation. However, the exact signaling interactions between arterialized HE and the vascular niche remain poorly defined. The rarity of HSCs relative to other progenitors complicates their study. HSC activity is first consistently detected around embryonic day 11 (E11) in the AGM, with HSC precursors detectable earlier (E9) and increasing in number between E10 and E11. These precursors have been characterized phenotypically by surface markers (VE-Cadherin, CD41, CD45), progressing from VE-Cadherin+CD41+CD45− pro-HSCs/pre-HSCs I to VE-Cadherin+CD45+ pre-HSCs II, and finally to VE-Cadherin−/+CD45+ HSCs. While helpful, this phenotypic characterization doesn't fully explain the molecular properties of developing HSCs, given their asynchronous emergence and low numbers relative to other progenitors. Single-cell RNA sequencing (scRNA-seq) offers a powerful tool to overcome these challenges, enabling transcriptome-wide analysis of rare cell populations and developmental transitions. Previous scRNA-seq studies have provided insights into HSC precursors and supported the idea of HSCs arising from HE with arterial endothelial properties. This study aims to apply scRNA-seq to understand the interactions between the arterial vascular niche and HE in regulating HSC specification and self-renewal, leveraging a previously established model using AGM-derived endothelial cell stroma (AGM-EC) that supports HSC generation from embryonic precursors.

Several studies have investigated the development of hematopoietic stem cells (HSCs), focusing on the role of the aorta-gonad-mesonephros (AGM) region and the hemogenic endothelium (HE). Research has established the sequential waves of hematopoietic progenitors preceding HSC emergence, with HE playing a crucial role in this transition. Phenotypic characterization using surface markers has provided insights into the developmental stages of HSCs. However, the precise molecular mechanisms driving HSC development remain largely undefined. The importance of the arterial vascular niche and signaling pathways like Notch has been highlighted, but the exact interactions are not fully understood. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable tool to study rare cell populations and developmental transitions, offering a high-resolution view of gene expression dynamics during HSC development. Previous studies have applied scRNA-seq to investigate HSC emergence, revealing insights into transcriptional properties of HSC precursors and their relationship to arterial HE. This current study builds on these findings to further investigate the complex interactions between the arterial vascular niche and HE cells during HSC specification and self-renewal.

This study employed a multi-faceted approach combining *in vivo* and *in vitro* analyses to investigate the development of hematopoietic stem cells (HSCs) from hemogenic endothelium (HE). First, the researchers utilized an *ex vivo* vascular niche model mimicking the embryonic aorta-gonad-mesonephros (AGM) region, where HSCs originate. They identified a surface marker combination (VE-Cadherin+, CD61+, EPCR+) to enrich for HSC precursors at various developmental stages from HE to HSCs. This enriched population (V+61+E+) was then subjected to single-cell RNA sequencing (scRNA-seq) from embryonic days E10 and E11 to capture transcriptional profiles during HSC emergence. In parallel, they performed scRNA-seq on AGM-derived endothelial cells (AGM-EC) to identify transcriptional signatures supporting HSC development. AGM-EC, which can support HSC formation from early embryonic precursors, were classified as either HSC-supportive or non-supportive based on their ability to generate engrafting HSCs in co-culture experiments. Differentially expressed genes were analyzed using gene ontology analysis to understand their functional roles. To further study HSC development, the researchers used a previously established method involving single-cell index sorting, AGM-EC co-culture, and transplantation assays to assess HSC potential. They correlated clonal HSC potential with surface expression of various markers to pinpoint HSC precursors at different maturation stages. The *in vivo* scRNA-seq data from the V+61+E+ population were analyzed using pseudotemporal ordering to reconstruct the developmental trajectory from HE to HSCs. Gene expression changes over pseudotime were examined to identify genes and pathways involved in the transition. Published gene sets defining arterial endothelial cells, HSC-primed HE, pre-HSCs, and mature HSCs were used to further refine the identification of HSC precursors within the scRNA-seq data through in silico analysis, focusing on the identification of distinct cell types and their transcriptional properties at different stages of development. To study HSC self-renewal and differentiation *in vitro*, scRNA-seq was performed on the progeny of single V+61+E+ cells following co-culture with AGM-EC. This allowed characterization of the transcriptional signatures of self-renewing HSCs and their differentiation into hematopoietic progenitor cells (HPCs). Finally, the researchers integrated the scRNA-seq data from AGM-EC and developing HSCs to identify ligand-receptor interactions potentially regulating HSC development. A curated ligand-receptor database was used to infer functionally relevant interactions between niche endothelial cells and HSC precursors. This was employed to guide the design of a stroma-independent engineered niche, composed of immobilized ligands and cytokines, aiming to recapitulate the function of AGM-EC in supporting HSC generation from embryonic precursors. The efficacy of this engineered niche was assessed by culturing E11 and earlier stage AGM-derived V+61+E+ cells and assessing the generation of long-term engrafting HSCs through transplantation assays. Finally, scRNA-seq was used to compare hematopoiesis in the engineered niche with that in the AGM-EC niche.

This study identified a surface marker combination (VE-Cadherin+, CD61+, EPCR+) that enriches for HSC precursors spanning the developmental spectrum from HE to HSCs. Single-cell RNA sequencing (scRNA-seq) revealed the transcriptional landscape of these precursors during their transition, highlighting dynamic changes in gene expression consistent with their maturation. The study identified a group of genes associated with arterial endothelial cells and HSCs co-expressed during this critical transition. Pseudotemporal ordering of the scRNA-seq data revealed a developmental trajectory, identifying key genes and pathways (including Notch signaling) regulating HSC emergence from HE. In silico analysis further refined the identification of HSC precursors within the scRNA-seq data, by combining in silico analyses of publicly available gene signatures for arterial EC, HE, pre-HSC, and HSC throughout development, revealing that pre-HSC emergence from HE is characterized by overlapping arterial endothelial and HSC-specific transcriptional programs. Analysis of AGM-derived endothelial cells (AGM-EC) that vary in their ability to support HSC generation identified genes specifically expressed in HSC-supportive AGM-EC, focusing on cell adhesion molecules and integrins, that were essential for supporting HSC generation. An engineered niche mimicking the AGM vascular niche was successfully created by utilizing scRNA-seq data to identify ligand-receptor interactions crucial for HSC development. This engineered niche, which combined immobilized Notch ligands, cytokines, and an integrin ligand (fibronectin), supported the generation of long-term engrafting HSCs from embryonic precursors *in vitro*. The addition of CXCL12 to the engineered niche further enhanced HSC generation, particularly from earlier developmental stages (E10 and even E9). Long-term scRNA-seq revealed the generation of HSCs and other blood lineages in both the AGM-EC and engineered niches, indicating that the engineered niche successfully recapitulated, at least in part, the functional properties of the endogenous vascular niche in supporting HSC development.

This study significantly advances our understanding of HSC development by integrating single-cell transcriptomics with functional assays. The identification of a surface marker combination (V+61+E+) that specifically enriches for HSC precursors represents a major advance, enabling more precise study of this rare and heterogeneous population. The detailed transcriptional map generated provides a valuable resource for future studies and helps to identify specific genes and signaling pathways crucial for HSC development. The success in engineering a stroma-independent niche that can generate functional HSCs *in vitro* opens up new avenues for research. The identification of CXCL12 as a critical factor for HSC generation from earlier precursors highlights the importance of chemokines in regulating HSC fate. The observation that this engineered niche successfully recapitulates some aspects of the *in vivo* niche, but not entirely, indicates that further optimization is needed. Additional signals from the *in vivo* niche, perhaps from non-endothelial cells, may be necessary to fully replicate HSC development. Future studies using spatial transcriptomics could further enhance our understanding of the spatial organization and interactions within the AGM niche that support HSC development. Ultimately, the knowledge gained from this research will be crucial for developing strategies to engineer HSCs from pluripotent stem cells for therapeutic applications.

This study provides a comprehensive transcriptional map of hematopoietic stem cell (HSC) development from hemogenic endothelium (HE), identifying key signaling interactions within the AGM vascular niche. A novel surface marker combination (V+61+E+) effectively enriches HSC precursors, facilitating detailed single-cell RNA sequencing analysis. The findings led to the successful engineering of a stroma-independent niche capable of generating functional HSCs *in vitro*, highlighting the critical roles of Notch signaling, integrin interactions, and CXCL12. Further research should focus on incorporating additional niche factors and optimizing signal pathway modulation to enhance HSC generation from even earlier developmental stages. Spatial transcriptomic approaches are needed to provide more precise details on the spatial organization of HSC development *in vivo*. The overall implications of this work are significant, offering advancements toward disease modeling and developing cellular therapies from human pluripotent stem cells.

While the V+61+E+ immunophenotype effectively enriched for cells with HSC potential, this population remains functionally heterogeneous at the single-cell level. In silico analysis was necessary to further identify cells with HSC potential in the scRNA-seq data. The current methods do not allow for precise spatial localization of pre-HSC/HSCs *in vivo*. Future technologies like spatially resolved single-cell multi-omics will be crucial for determining the precise spatial organization of HSCs and their interactions within the AGM. The engineered niche, although successful, does not fully replicate the *in vivo* niche, suggesting additional factors may play a role in optimizing HSC generation.