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...

Astrocyte-enriched cultures were established upon passaging of primary cultures from the myelencephalon and mesencephalon of 7-9-week-old human embryos. Immunocytochemical analysis showed that third-fourth passage cultures were composed of a highly enriched population of proliferating, epithelioid cells, up to 90% of which expressed glial fibrillary acidic protein (GFAP); no macrophages and very few fibroblasts (less than 2%) were present. GFAP expression and proliferation declined upon further culturing in serum-containing medium but could be transiently reinduced by growing the cells in a serum-free chemically defined medium. Large numbers of GFAP+ astrocytes were obtained from each embryo and could be stored frozen and recultured. Using flow cytometric analysis, human astrocyte cultures were examined for basal and cytokine [interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha)]-induced expression of molecules that may be involved in astrocyte-T-lymphocyte interactions. Cultured human astrocytes spontaneously expressed major histocompatibility complex (MHC) class I antigens and variable levels of MHC class II; MHC class I levels were increased upon IFN-gamma and TNF-alpha treatment, whereas MHC class II antigens were induced on most of the astrocytes by IFN-gamma. Among the molecules involved in antigen-independent interactions between T lymphocytes and target cells, lymphocyte function-associated molecule-3 (LFA-3) was spontaneously expressed by most cultured human astrocytes, whereas intercellular adhesion molecule-1 (ICAM-1) was present at variable levels in non-stimulated astrocytes and was greatly induced by IFN-gamma, TNF-alpha, and IL-1 beta. In this study we also show that the above cytokines upregulate astroglial expression of adhesion molecules of the integrin family (VLA-1, VLA-2, and VLA-6) that may be involved in astrocyte-extracellular matrix interaction and play a role in the astrocyte reactive changes occurring at sites of brain injury and inflammation. The human astrocyte cultures developed here represent a useful in vitro model to further investigate mechanisms involved in bidirectional communication between central glia and cells of the immune system.

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