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.
