Identification of Distinct Pathways Involved in Regulation of Mesenchymoangioblasts and Hemangioblasts Development From Mesoderm

Abstract 1326

Using hESC differentiation system we recently identified a mesenchymoangioblast (MAB) as a novel precursor for mesenchymal stem cells (MSCs) and endothelial cells and demonstrated that mesenchymal and hematopoietic cells develop sequentially from mesodermal precursors with primary angiogenic potential – MAB and hemangioblasts (HB), respectively. In addition, we found that angiogenic mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by surface expression of apelin receptor (APLNR) and lack of expression of typical endothelial (CD31, CD144), hematopoietic (CD43, CD45) and mesenchymal (CD73, CD195) markers i.e. as ^EHMlin-APLNR+ cells. (Vodyanik et al. Cell Stem Cell 2010;7:718). In response to FGF2 APLNR+ cells form morphologically distinct compact mesenchymal or MAB colonies and disperse hematopoietic or blast (HB) colonies in serum-free clonogenic semisolid medium. When transferred to the adherent cultures in serum-free medium with FGF2, individual colonies gave rise to multipotential mesenchymal cell lines with typical phenotype (CD146+CD105+CD73+CD31-CD43-45-), differentiation (chondro-, osteo-, and adipogenesis) and robust proliferation (>80 doublings) potentials. In contrast, HB colonies consisted almost entirely of CD235a and CD41a expressing cells with morphology resembling erythroblasts. Replating of HB colonies in hematopoietic serum-free and serum-containing clonogenic medium demonstrated that they gave rise to erythroid, megakaryocytic and mixed colonies composed of erythroid, megakaryocytic cells and macrophages indicating that BL-CFCs probably reflected the first wave of embryonic hematopoiesis initiated in the yolk sac. To define pathways involved in MAB and HB development we tested the effect of different growth factors and cytokines on mesenchymal and blast colony formation. We confirmed that the development of MAB and HB depends on FGF2, by demonstrating complete suppression of blast and mesenchymal colony formation by elimination of FGF2 from clonogenic culture or by abrogation of FGF2 signaling using PD 173074 inhibitor of FGF2 receptor autophosphorylation. The formation of MAB and HB colonies was also completely abrogated by adding TGFb or activin A, and increased in the presence of SB431542 TGFb signaling inhibitor. PDGF-BB alone lacked colony-forming activity, but its addition to FGF2 significantly increased the frequency and size of mesenchymal but not blast colonies. In contrast, the addition of VEGF essentially abrogated mesenchymal colony formation. Although VEGF had little effect on BL-CFCs, the addition KI8751 KDR inhibitor significantly decreased the number of blast colonies, confirming that their development depends on VEGF signaling. The addition of individual hematopoietic cytokines to FGF2 had a relatively mild effect on the number of blast colonies. However, they increased the size of colonies, which was especially obvious with the addition of EPO. When IL3, IL6, EPO, TPO, and SCF were added to clonogenic cultures together with FGF2, we observed a significant increase in the number and size of blast colonies, some of which had grown into very large grape-like structures. Because we found that emerging CD144⁺CD235a⁺ cells generated hematopoietic colonies morphologically resembling blast colonies in the presence of FGF2 and hematopoietic cytokines, we concluded that hematopoietic cytokine-free clonogenic cultures would be more appropriate for detection of BL-CFCs and enhancing the specificity of this assay. The finding that mesenchymal and blast CFCs arise from cells expressing APLNR prompted us to test whether this receptor is involved in the regulation of MAB and HB development. We found that APLNR agonist aplein-12 inhibits mesenchymal colonies, while it significantly increases the formation of blast colonies in synergy with VEGF. Together, these studies demonstrated that the activation of multiple but different signaling pathways regulates the formation of mesenchymal and blast colonies.