Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

Postnatal CD34 ؉ cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34 ؉ KDR ؉ cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34 ؉ KDR ؉ cells generate large hematoendothelial (Hem-End) colonies (5% of seeded cells), whereas CD34 ؉ KDR ؊ cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription-polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34 ؉ KDR ؉ cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds-that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic HemEnd colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34 ؉ KDR ؉ cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors. (Blood. 2002;100:3203-3208)

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...