Spatial Transcriptomics Characterize Dynamic and Polarized Niche for Embryonic Hematopoiesis at Single-Cell Level

The demand for high quantities of donor hematopoietic stem cells (HSCs) remains a significant hurdle for life-saving HSC transplantation in clinical settings. Directed differentiation of pluripotent stem cells (PSCs) to HSCs offers a promising strategy that theoretically could provide unlimited autologous HSCs for both research and clinical application. An in-depth understanding of embryonic HSC development would facilitate the PSC to HSC induction. The first transplantable HSC emerges in the dorsal aorta of aorta-gonad-mesonephros (AGM) region through stagewise development of hemogenic endothelial cell (HEC) specification and the endothelial-to-hematopoietic transition (EHT). These processes dynamically occur at the ventral, but not dorsal, side of the aorta within a narrow time window (mouse embryonic day [E] 9.5-12.5), indicating an essential role of the unique AGM microenvironment in HSC development. However, a comprehensive profiling of niche cells and their emanated signals in the AGM region at spatial-resolved and single-cell levels remains elusive. To address this question, we conducted scRNA-seq and sequencing-based spatial transcriptomics (Curio-seq) on mouse embryonic AGM-containing trunk sections at E10.5, E11.5 and E12.5, capturing key timepoints of HSC development. While the dorsal side of the aorta is predominantly occupied by a Nfgr+ cell cluster expressing BMP signals, the ventral side is composed of a complex cellular architecture and signals (e.g., WNT, NOTCH and retinoic acid). Specifically, a cell population co-expressing Cdh2 (encodes N-cadherin) and Pdgfra (named N-cad+ MSCs) forms a single cell layer sandwiched between aortic endothelial cells (AECs) and Wt1+ mesonephric cells. Importantly, all pre-HSCs identified in our study are in direct contact with N-cad+ MSCs. We next utilized CellChat and StringDB to predict ligand-receptor interactions and signaling crosstalk between niche cells and pre-HSCs. This reveals previously unreported interactions between the N-cad+ MSCs and pre-HSCs via multiple signal axes. Strikingly, these interactions change dynamically during E10.5-E12.5, with the interactions via Dlk1-Notch1,Tgfb1-Tgfbr1 and Jag1-Notch1 reaching the peak at E10.5, E11.5 and E12.5, respectively. Overall, the Curio-seq data profiled the dynamic and asymmetric distribution of cell identities and signals in the AGM region at a resolution of 10 µm. We next conducted image-based spatial transcriptomics MERFISH on E11.5 trunk sections by customizing RNA probes for 140 genes selected from the Curio-seq analysis, which included cell markers and signaling molecules. The MERFISH data effectively validated the spatially resolved niche cells and their associated signals in the AGM region at the single-cell level, as revealed by Curio-seq assay. For instance, it confirmed that the N-cad+ MSCs were enriched at the ventral side of the aorta, in direct contact with pre-HSCs, and served as the primary source of Jag1 and Cxcl12 around the aorta. We hypothesized that N-cad+ MSCs play a crucial role in HSC development due to their unique position and intricate interactions with pre-HSCs. To investigate this, we generated NcadCreER; Jag1flox/flox mouse model to conditional knockout Jag1 from N-cadherin-expressing cell at E9.5 and collected E11.5 AGM cells for transplantation assays. The recipient mice in the Jag1 knockout group exhibited higher peripheral blood CD45.2 chimerism at 16 weeks compared to the mice in the wild-type group (24.2% Vs 0.14%), indicating that N-cad+ MSC derived Jag1 serves as a negative regulator of HSC development. To further understand the underlying mechanisms, we are performing scMultiome-seq to co-profile the transcriptional and epigenetic landscapes of the HECs and pre-HSCs from wild-type and mutant embryos. In summary, we employed a combination of sequencing-based and imaging-based spatial transcriptomics technologies to comprehensively characterize the dynamic, spatial-resolved profiles of niche cells and their associated signals in the AGM region at the single-cell level. Functionally, we revealed an inhibitory role of N-cad+ MSC derived Jag1 in HSC development, thus validating the results obtained from the spatial transcriptomics analyses. This study enhances our understanding of the niche of embryonic hematopoiesis and provides insight into developing novel methods for PSC to HSC induction.