Trypsin was then removed, and the AGM cells in Dulbecco modified Eagle medium containing 15% heat-inactivated fetal calf serum were passed through a micropipette to make a single-cell suspension. 22-24 Taken together, these studies establish that VEGF plays a key role in the development of hematopoietic and endothelial cells.ĪGM tissues removed from embryos were washed once with 0.25% trypsin in HEPES-buffered saline (Gibco-BRL, Gaithersburg, MD) and incubated at 37☌ for 9 minutes in fresh trypsin solution. 19-21 In addition, recent studies on the in vitro differentiation of embryonic stem cells also indicate that hematopoietic cells and endothelial cells are derived from the same origin in the presence of VEGF. 16 Flk-1 knockout mice exhibited severe defects in hematopoiesis and vasculogenesis in the yolk sac, 17, 18 and a similar phenotype was found by gene disruption of the SCL/Tal-1 transcription factor. 15 Similarly, mouse cells expressing Flk-1 (mouse counterpart of VEGF-R2) and vascular endothelial cadherin (VE-cadherin) in the yolk sac and the pSp region at E9.5 mouse embryos gave rise to lymphohematopoietic cells in vitro. In the splanchnopleural mesoderm, cells expressing VEGF receptor 2 (VEGF-R2) were shown to form hematopoietic and endothelial colonies. Hematogenic activity is regulated by cytokines such as vascular endothelial growth factor (VEGF). These results suggest that M-CSF modulates the development of hematopoiesis by stimulating the differentiation of PCLP-1 +CD45 − cells to endothelial cells in the AGM region. The M-CSF receptor was expressed in PCLP1 +CD45 − cells, the precursors of endothelial cells, and M-CSF up-regulated the expression of more mature endothelial cell markers-VCAM-1, PECAM-1, and E-selectin-in PCLP1 +CD45 − cells. On the other hand, the number of cells expressing endothelial markers, vascular endothelial-cadherin, intercellular adhesion molecule 2, and Flk-1 was reduced in op/op AGM culture. The addition of M-CSF to op/op AGM culture decreased colony-forming unit (CFU)-GEMM, granulocyte macrophage–CFU, and erythroid–CFU, but it increased CFU-M. The number of hematopoietic progenitors including multipotential cells was significantly increased in the AGM culture of op/opembryos. Here we describe a role for macrophage–colony-stimulating factor (M-CSF) in the development of hematopoietic and endothelial cells in AGM culture. Previously, we showed that multipotential hematopoietic progenitors and endothelial cells were massively produced in primary culture of the AGM region in the presence of oncostatin M. Definitive hematopoietic stem cells arise in the aorta–gonad–mesonephros (AGM) region from hemangioblasts, common precursors for hematopoietic and endothelial cells.

Pclp scholar