Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

Abstract

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and to identify the niche cells and factors for HSC expansion. To understand the spatial distribution and interactions between HSCs and niche cells in fetal liver, we introduced 2 spatial transcriptomic methods, Slide-seq (Stickels et al. Nature Biotechnology, 2021), and 10x Genomics Visium Spatial Gene Expression, in our study on E14.5 and E16.5 mouse fetal liver. By integrating with a 10x Genomics' Chromium™ Single Cell 3' Solution, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that PMCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs and endothelial cells, indicating a supportive role of N-cadherin-expressing PMCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGFs, TGFβs, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding is consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. In addition, endothelium cells have enriched expression of several niche factors, including BMPs, KITL, IGFs and TGFβs. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Interestingly, conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, flowcytometry analysis and scRNA-seq on conditional knockout of Cxcxl12 in N-cadherin-expressing cells showed a myeloid-biased differentiation. Subsequential slide-seq showed that fetal liver HSCs were located close to N-cadherin-expressing PMCs and endothelial cells. Intriguingly, fetal liver HSCs migrated toward and resided in close proximity to megakaryocytes after conditional knockout of Cxcxl12 in N-cadherin-expressing cells. In summary, we postulate that N-cadherin-expressing cells, especially PMCs, maintain fetal liver HSCs. The knockout of Cxcl12 in N-cadherin-expressing cells leads to the migration of HSCs towards megakaryocytes, which may induce HSC expansion and biased differentiation. In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 and E16.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches to verify the distribution changes of HSCs in N-cadCreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs.