Haematopoietic stem cells (HSCs) are responsible for the maintenance of haematopoiesis under normal conditions and its adaptation to haematopoietic stress. 1 HSCs are localized in the bone marrow (BM) in a micro-anatomic space designated as the HSC niche2 comprising different cellular components. While osteoblasts predominantly contribute to the restoration of haematopoiesis after transplantation, endothelial and perivascular stromal cells form a perivascular niche, which promotes the self-renewal of HSCs and their multi-lineage differentiation, and hence the maintenance of haematopoiesis. 2 Cells of the perivascular niche not only provide the mechanical barrier between the BM microenvironment and circulation, but also regulate haematopoiesis, either through adhesive interactions with HSCs3 or by the release of paracrine factors. 4, 5 The adhesive interactions of HSCs with endothelial and perivascular cells mediate both the trafficking of HSCs into and out of the BM6 and their maintenance and proliferation potential. 3 Developmental endothelial locus-1 (Del-1) is a secreted glycoprotein that interacts with integrins. 7–9 We have recently shown that Del-1 is expressed by important cellular components (endothelial, mesenchymal stromal cells, and cells of the osteoblastic lineage) of the HSC niche, where it regulates haematopoietic progenitor function and their myeloid differentiation. 10 This ability of Del-1 to act as an HSC niche factor was attributed to its interaction with β3 integrin on haematopoietic progenitors. 10 Here, we set out to precisely define the role of Del-1 as an endothelial cell–derived perivascular niche factor. To this end, we studied steady-state and regeneration haematopoiesis using an independent in vivo approach, namely mice with endothelial cell–specific overexpression of Del-1 (EC-Del1 mice).

We engaged EC-Del1 transgenic mice, generated by utilizing a tie2 promoter/enhancer construct in the C57BL/6JOlaHsd background. 11, 12 EC-Del1 mice exhibited significantly elevated Del-1 gene (Edil3) expression in the BM (► Fig. 1A), as assessed by quantitative polymerase chain reaction (qPCR), which was performed as described. 10 Relative messenger ribonucleic acid (mRNA) expression levels were calculated according to the ΔΔCt method upon normalization to 18S. 10, 13 Enhanced Del-1 expression in the BM was owing to increased expression in endothelial cells (Lin–CD45–CD31þSca1þ;► Fig. 1B), isolated from the BM by fluorescence-activated cell sorting (FACS) as previously described. 10 On the contrary, no expression of Del-1 in haematopoietic progenitors was observed (data not shown). Endothelial overexpression of Del-1 resulted in an increase in total BM cellularity (data not shown). We then performed phenotypic analysis of haematopoietic cells in the BM by flow cytometry, 10 to address whether Del-1 overexpression can affect the numbers of haematopoietic progenitors in the BM. Flow cytometry analysis was performed by FACS Canto II flow cytometer using the FACSDiva 6.1. 3 software (BD, Heidelberg, Germany). FlowJo (TreeStar, Ashland, OR, USA) software was used for data analysis. Antibodies used for FACS analysis are described in► Supplementary Table 1. Consistent with a role for Del-1 as an HSC niche factor, EC-Del1 mice exhibited an expansion in the absolute cell numbers of haematopoietic progenitors (Lin–Sca1þcKitþ [LSK]), long-term HSCs (LT-HSCs; Lin–cKitþSca1þCD48–CD150þ) and short-term HSCs (ST-HSCs; Lin–cKitþSca1þ CD48–CD150–), while the cell numbers of multi-potent