IntroductionDuring megakaryocytic (Mk) differentiation, Mk precursors switch from a mitotic to an endomitotic process characterized by DNA duplication without cytokinesis. This still poorly understood process leads to the formation of large polyploid cells with polylobulated nuclei that, in turn, give rise to platelets by cytoplasm fragmentation. 1,2 The major regulator of Mk development, Mpl ligand/ thrombopoietin (TPO), acts at all stages of megakaryocytopoiesis: commitment and proliferation of hematopoietic progenitor cells (HPCs), polyploidization of Mk precursors, and final maturation, including the formation of membrane demarcations and platelet production (reviewed in Kaushansky, 1 ZuckerFranklin and Kaushansky, 2 Zimmet and Ravid, 3 Cramer et al 4 ). However, despite these properties, TPO fails to induce in vitro a level of Mk polyploidization comparable to that observed in vivo. 5-7 Addition of either single or combined cytokines (ie, kit ligand, interleukin-3, interleukin-6) to TPO-containing cultures, although improving Mk proliferation, negatively affects cytoplasmic maturation and polyploidization. 5,6 Similarly, although erythropoietin (Epo) is considered the main growth factor stimulating erythropoiesis, additional cytokines are required at early and late erythroid (E) stages. 8 Vascular endothelial growth factor (VEGF) is a key factor for proliferation and survival of endothelial cells. [9][10][11] The VEGF family, including VEGF/VEGF-A, -B, -C, -D, and -E, 10-12 as well as the placenta growth factor (PlGF), 13 mediates angiogenic signals to endothelial cells through the binding with tyrosine kinase receptors designated VEGFR-1/Flt1, VEGFR-2/KDR/Flk1, and VEGFR-3/Flt-4. 14 VEGF is the ligand of both Flt1 and kinase domain receptor (KDR) and consists of several isoforms generated by alternative splicing of a single mRNA precursor (VEGF121, 145, 165, 189, or 206), which differ in their molecular mass and their biologic properties, such as the ability to bind heparin or heparinlike molecules on cell surface. 10,15 VEGF expression is enhanced spatially and temporally and is associated with physiologic events leading to angiogenesis in vivo, and its production is potentiated by hypoxia. 16 Studies on gene knockout mice demonstrated the physiologic role of VEGF and its receptors, as central regulators of the development of vascular and hemopoietic tissues. Flt1 knockout causes a selective defect in the assembly and organization of vasculature. 17 Lack of either VEGF or KDR gene causes major defects in both vasculogenesis and blood island formation, 18-21 suggesting the existence in embryonic life of a bipotent stem cell (SC) for hematopoietic and endothelial lineages, the hemangioblast.In postnatal life, both Flt1 and KDR are expressed at low levels on CD34 ϩ HPCs. [22][23][24][25][26][27] More important, the small fraction of CD34 ϩ Materials and methods Hematopoietic growth factors (HGFs) and culture mediaRecombinant human interleukin 3 (rhIL-3), granulomonocytic colony-stimulating factor (rhGM-C...
Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a developmentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, gamma----beta) globin, but little is known about the embryonic----fetal (that is, zeta----alpha and epsilon----gamma) switches, as well as the transition from 'primitive' yolk sac to 'definitive' liver erythropoiesis. Here we have studied the embryonic----fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified 'primitive' and 'definitive' erythroblasts. Primitive erythroblasts synthesize essentially zeta and epsilon chains at 5 weeks and alpha- and epsilon-globin with a minor aliquot of zeta and gamma chains at 6-7 weeks, whereas definitive erythroblasts produce alpha and epsilon + gamma + beta-globin at 6 weeks but only alpha and gamma + beta chains from 8 weeks onward. In both lineages the zeta----alpha and the epsilon----gamma switches are asynchronous, the former preceding the latter. Furthermore, zeta- and beta-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny.
The alpha chemokine receptor CXCR4 has been shown to be expressed on human hematopoietic progenitor cells and during the megakaryocytic differentiation pathway. Stromal cell-derived factor 1 (SDF-1) is the ligand for CXCR4. In this study, the role of SDF-1␣ in megakaryocytopoiesis was investigated. CD34 ؉ progenitors purified from peripheral blood were grown in serum-free liquid suspension culture supplemented with thrombopoietin to obtain a virtually pure megakaryocytic progeny. In this condition, the addition of SDF-1␣ gives rise to megakaryocytes (
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