Abstract
SummaryApelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance.
Highlights
Definitive hematopoietic stem and progenitor cells (HSPCs) generate all cells of the blood and immune system, with the most potent of these, the hematopoietic stem cell (HSC), being capable of repopulating the entire blood system upon transplantation
We showed that the expression of genes encoding APLNR and one of its ligands, APELIN, correlated with the increased production of HSPCs when the transcription factor, HOXB4 was activated in differentiating mouse and human pluripotent stem cells (PSCs) (Jackson et al, 2012, 2016)
Aplnr-tdTomato Reporter Marks Mesodermal Cells in Differentiating ESCs To define the phenotype of cells expressing the APLNR receptor during hematopoietic differentiation of ESCs we generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line using CRISPRCas9-mediated genome editing (Figure 1A). mESCs were transfected with the Aplnr targeting vector, Cas9 plasmid, and specific Guide RNAs (gRNA)
Summary
Definitive hematopoietic stem and progenitor cells (HSPCs) generate all cells of the blood and immune system, with the most potent of these, the hematopoietic stem cell (HSC), being capable of repopulating the entire blood system upon transplantation. In the mouse embryo the first wave of hematopoietic development originates in the yolk sac around embryonic day 7.25 (E7.25) and gives rise to primitive embryonic erythrocytes, megakaryocytes, and macrophages (Palis, 2014). The second wave originates from the yolk sac from E8.25 and gives rise to erythromyeloid progenitors that are defined by their ability to generate granulocytes and natural killer cells (McGrath et al, 2015; Dege et al, 2020). Research on the molecular mechanisms associated with the development of HSPCs in the embryo has instructed the design of culture protocols to model hematopoiesis in vitro from pluripotent stem cells (PSCs) (Ivanovs et al, 2017).
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