Single-cell transcriptomics identifies CD44 as a new marker and regulator of haematopoietic stem cells development

Abstract

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening we identified CD44 as a new marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta gonad mesonephros (AGM) region. This allowed us to provide a very detailed phenotypical and transcriptional profile for haemogenic endothelial cells, characterising them with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists (Smad6, Smad7 and Bmper) and a downregulation of genes related to glycolysis and the TCA cycle. Moreover, we demonstrated that by inhibiting the interaction between CD44 and its ligand hyaluronan we could block EHT, identifying a new regulator of HSPC development.

De novo generation of macrophage from placenta-derived hemogenic endothelium

“…To compare the molecular features of HE cells in placenta and AGM, we first verified that CD44 can also be used to enrich HE cells in the AGM region (Figures S3A and B), consistent with a recent report (Oatley et al, 2020). Then we enriched HE cells and non-HE cells (CD44 -ECs) from AGM and placenta at E11.0 and performed modified Smart-seq2 due to the rarity of HE cells (Figure S3C, Table S2).…”

“…During mouse embryogenesis, HE cells can be identified in different anatomical locations including yolk sac and AGM (Gao et al, 2018). In this study, for the first time, we identified HE cells in the mouse placenta by using a surface marker CD44 that can purify ECs with hemogenic potential as reported in mouse and human AGM region recently (Oatley et al, 2020, Zeng et al, 2019. In addition, we also verified the conserved expression of CD44 in human placental ECs.…”

Single cell transcriptome analysis reveals markers of naïve and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells

“…Noteworthy, RUNX3 expression was specifically associated with the monocyte 323 subcluster ( Figure 6I progenitors, we observed high levels of CD44 mRNA expression, a protein reported to be 347 expressed in normal and leukemic hematopoietic cells (Zöller, 2015). Recently, a single 348 cell transcriptome analysis reported that CD44 marks hematopoietic stem and progenitor 349 cells in the mouse AGM, and functions in endothelium to hematopoietic transition, EHT 350 (Oatley et al, 2018). We confirmed CD44 expression on the aortic endothelium of the 351 AGM region and, showed expression on some of the vessels in the yolk sac together with 352 the hematopoietic clusters associated with these.…”

Endothelial-to-haematopoietic transition: an update on the process of making blood

“…Other studies have focused on one or two developmental time points and compared the different cell populations from non-HEC to HSPCs with the aim of identifying, and ordering along a differentiation trajectory, the intermediate cellular states with the accompanying transcriptional stages. The identification of additional cell surface markers [69,71] and the use of further fluorescent reporter mouse lines [66,68,70] have made these isolation strategies ever more sophisticated.…”

“…The advent of single-cell RNA-Seq has allowed a more detailed dissection of the differentiation pathway from endothelial cell to haematopoietic cell [66,69,71]. Additional intermediate cellular states have been uncovered that can be ordered along a differentiation trajectory using pseudotime algorithms, which is most prominently marked by a gradual down-regulation of endothelial-specific genes and an up-regulation of haematopoietic genes.…”