Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.
Bottom Line: A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis.We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants.Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.
Affiliation: Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138.Show MeSH
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Mentions: To test whether adenosine signaling would have an effect on mammalian hematopoiesis, we assessed the effect of adenosine on the development of mouse HSPC populations. We first used an mESC hematopoietic differentiation system, an accessible in vitro model which recapitulates mouse embryonic hematopoiesis (Kim et al., 2013). In this model, mESCs are aggregated into embryoid bodies (EBs) and differentiated into primitive streak-like mesoderm on day 2, hemangioblasts on day 3.25, and hematopoietic precursors after day 5. A2b receptor is present between days 0 and 6 of EB differentiation (not depicted). Analysis of gene expression data in sorted populations from day 6 EBs (Kim et al., 2013) showed that A2b is high in the VE-cadherin+CD41− endothelial cells and low in the CD41+ populations (Fig. 4 A), indicating that A2b likely regulates VE-cadherin+ cell intermediates, a fraction from which HSCs emerge. Methylcellulose-based colony-forming assay was performed to measure both hematopoietic progenitor types and numbers. Addition of A2b selective agonist BAY 60-6583 to EBs between days 4 and 6 significantly increased the number of hematopoietic colonies, including definitive erythroid (CFU-E), granulocyte/monocyte (CFU-GM), and multipotent granulocyte/erythrocyte/monocyte/macrophage (CFU-E/GM/GEMM) colonies, at a concentration of 0.5–2.5 µM (Fig. 4 B). Consistently, qPCR analysis revealed that many hematopoietic genes, including scl, lmo2, runx1, gata1, bH1, and βmajor (adult globin), were up-regulated on day 6 after BAY 60-6583 treatment (Fig. 4 C). In contrast, BAY 60-6583 did not increase the expression of the mesoderm marker Cerberus, the expression of endothelial gene fli1 (Fig. 4 D). Interestingly, the expression of flk1 was decreased after BAY 60-6583 treatment (Fig. 4 D), consistent with the previous study showing the antagonist effect of runx1 on flk1 (Swiers et al., 2013). These data support a positive role of adenosine and its A2b receptor in regulating mESC hematopoietic differentiation.
Affiliation: Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138.