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Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

Jing L, Tamplin OJ, Chen MJ, Deng Q, Patterson S, Kim PG, Durand EM, McNeil A, Green JM, Matsuura S, Ablain J, Brandt MK, Schlaeger TM, Huttenlocher A, Daley GQ, Ravid K, Zon LI - J. Exp. Med. (2015)

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.

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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.

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Treatment with an A2b receptor agonist promotes hematopoietic development in mESC culture and AGM explants. (A) Microarray gene expression analysis of A2b, scl, and runx1 in sorted VE-cadherin+CD41−, VE-cadherin+CD41+, and VE-cadherin−CD41+ cells from day 6 EBs. (B) CFUs were measured 7 d after culture in M3434 of day 6 whole EBs treated with DMSO or BAY 60-6583 (0.5 µM and 2.5 µM; Student’s t test: *, P < 0.05; **, P < 0.01; n = 3–5 per group). (C) qPCR analysis of hematopoietic genes (scl, lmo2, gata1, bH1, bMajor, runx1, and cmyb) measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (D) qPCR analysis of fli1, cerberus, and flk1 measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (E) Microarray gene expression analysis of selected genes in nonhemogenic endothelial cells (EC), HE, and hematopoietic progenitor cells (HPC) from Affymetrix microarrays under GEO accession no. GSE52075 (Swiers et al., 2013; n = 3). (F) Expression of A2b and GADPH genes by RT-PCR in VE-cadherin+ cells purified from the E10.5 AGM. (G) CFU-C assay from E10.5 AGM explants. Numbers are per embryo (Student’s t test: *, P < 0.05; **, P < 0.01; n = 4 per group). The results are presented as mean ± SE.
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fig4: Treatment with an A2b receptor agonist promotes hematopoietic development in mESC culture and AGM explants. (A) Microarray gene expression analysis of A2b, scl, and runx1 in sorted VE-cadherin+CD41−, VE-cadherin+CD41+, and VE-cadherin−CD41+ cells from day 6 EBs. (B) CFUs were measured 7 d after culture in M3434 of day 6 whole EBs treated with DMSO or BAY 60-6583 (0.5 µM and 2.5 µM; Student’s t test: *, P < 0.05; **, P < 0.01; n = 3–5 per group). (C) qPCR analysis of hematopoietic genes (scl, lmo2, gata1, bH1, bMajor, runx1, and cmyb) measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (D) qPCR analysis of fli1, cerberus, and flk1 measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (E) Microarray gene expression analysis of selected genes in nonhemogenic endothelial cells (EC), HE, and hematopoietic progenitor cells (HPC) from Affymetrix microarrays under GEO accession no. GSE52075 (Swiers et al., 2013; n = 3). (F) Expression of A2b and GADPH genes by RT-PCR in VE-cadherin+ cells purified from the E10.5 AGM. (G) CFU-C assay from E10.5 AGM explants. Numbers are per embryo (Student’s t test: *, P < 0.05; **, P < 0.01; n = 4 per group). The results are presented as mean ± SE.

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.


Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

Jing L, Tamplin OJ, Chen MJ, Deng Q, Patterson S, Kim PG, Durand EM, McNeil A, Green JM, Matsuura S, Ablain J, Brandt MK, Schlaeger TM, Huttenlocher A, Daley GQ, Ravid K, Zon LI - J. Exp. Med. (2015)

Treatment with an A2b receptor agonist promotes hematopoietic development in mESC culture and AGM explants. (A) Microarray gene expression analysis of A2b, scl, and runx1 in sorted VE-cadherin+CD41−, VE-cadherin+CD41+, and VE-cadherin−CD41+ cells from day 6 EBs. (B) CFUs were measured 7 d after culture in M3434 of day 6 whole EBs treated with DMSO or BAY 60-6583 (0.5 µM and 2.5 µM; Student’s t test: *, P < 0.05; **, P < 0.01; n = 3–5 per group). (C) qPCR analysis of hematopoietic genes (scl, lmo2, gata1, bH1, bMajor, runx1, and cmyb) measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (D) qPCR analysis of fli1, cerberus, and flk1 measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (E) Microarray gene expression analysis of selected genes in nonhemogenic endothelial cells (EC), HE, and hematopoietic progenitor cells (HPC) from Affymetrix microarrays under GEO accession no. GSE52075 (Swiers et al., 2013; n = 3). (F) Expression of A2b and GADPH genes by RT-PCR in VE-cadherin+ cells purified from the E10.5 AGM. (G) CFU-C assay from E10.5 AGM explants. Numbers are per embryo (Student’s t test: *, P < 0.05; **, P < 0.01; n = 4 per group). The results are presented as mean ± SE.
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fig4: Treatment with an A2b receptor agonist promotes hematopoietic development in mESC culture and AGM explants. (A) Microarray gene expression analysis of A2b, scl, and runx1 in sorted VE-cadherin+CD41−, VE-cadherin+CD41+, and VE-cadherin−CD41+ cells from day 6 EBs. (B) CFUs were measured 7 d after culture in M3434 of day 6 whole EBs treated with DMSO or BAY 60-6583 (0.5 µM and 2.5 µM; Student’s t test: *, P < 0.05; **, P < 0.01; n = 3–5 per group). (C) qPCR analysis of hematopoietic genes (scl, lmo2, gata1, bH1, bMajor, runx1, and cmyb) measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (D) qPCR analysis of fli1, cerberus, and flk1 measured in day 6 whole EBs treated with DMSO or 0.5 µM BAY 60-6583 (Student’s t test: *, P < 0.05; n = 3 per group). (E) Microarray gene expression analysis of selected genes in nonhemogenic endothelial cells (EC), HE, and hematopoietic progenitor cells (HPC) from Affymetrix microarrays under GEO accession no. GSE52075 (Swiers et al., 2013; n = 3). (F) Expression of A2b and GADPH genes by RT-PCR in VE-cadherin+ cells purified from the E10.5 AGM. (G) CFU-C assay from E10.5 AGM explants. Numbers are per embryo (Student’s t test: *, P < 0.05; **, P < 0.01; n = 4 per group). The results are presented as mean ± SE.
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.

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.

View Article: PubMed Central - HTML - PubMed

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.

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Related in: MedlinePlus