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Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by tack 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.
The hierarchy of human hematolymphopoietic cells is defined by functional assays. HSCs with extensive self-renewal capacity are assayed in vivo for their capacity to xenograft nonobese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice and sheep fetuses (1-3). These models are surrogates for a syngeneic transplantation assay. Primitive HPCs with limited self-renewal potential are identified in vitro as high-proliferative potential colony-forming cells (HPP-CFCs) (4). Lineagecommitted HPCs with no self-renewal activity are also defined in vitro by clonogenic assays as colony-forming units (CFUs) or burst-forming units (BFUs) (1,5). Dexter-type LTC, consisting of a liquid phase on irradiated BM stroma, identifies LTC-initiating cells (LTC-ICs, generating HPCs assayed in secondary culture) (6) and cobblestone area-forming cells (CAFCs, generating hematopoietic colonies recognized as "cobblestone areas" in LTC stroma) (7). Depending on the LTC duration, LTC-ICs represent primitive HPCs (5- to 8-week LTC) (8) or highly quiescent putative HSCs resistant to retroviral gene transfer (12-week LTC) (9) (see below).
Although HSC identification is still elusive, recent observations have suggested a role for VEGFR2 (Flk1 in mice) in marine embryonic hematoangiogenesis. Targeted gene disruption studies indicate that Flk1 is required for initiation of hematolymphopoiesis and vasculogenesis (10), implying that Flk1 may be required for generation of hemoangioblasts, that is, the hypothetical stem cells for both hematolymphopoietic and endothelial lineages (11). Other studies also suggested the existence of embryonic Flk1+...