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Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell–derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2+(VEGFR2+) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for α-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2+cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2+mesodermal cells.

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+ E-cadherin- proximal lateral mesoderm and the second being CD45- VE-cadherin+endothelial cells. To further dissect the CD45-VE-cadherin+ cells, we have examined distribution of ?4-integrin on this cell population, because ?4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+ E-cadherin- cells, CD45- VE-cadherin+?4-integrin- cells differentiate first, followed by ?4-integrin+ cells appearing in both CD45- VE-cadherin+ and CD45-VE-cadherin- cell populations. In the CD45-VE-cadherin+ cell population, ?4-integrin+ subset but not ?4-integrin- subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45-VE-cadherin- ?4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the ?4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45-VE-cadherin+ ?4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of ?4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM−cells, but not B220−cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM−cells to B220+, lgM+cells. B220+, lgM+cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1α inhibited IL-7-induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1α enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1α may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1α may play an important role in differentiation of myeloid and lymphoid lineages.

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3- G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kitwith its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

The receptor encoded by the W (c-kit)locus (Wreceptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+ / +) mice, whereas its ligand encoded by the SIlocus (SIligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of Wreceptors and SI ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/ Wgenotype did not express the extracellular domain of Wreceptors, and attachment of W/ WCMC to + / + fibroblasts was significantly impaired. Fibroblasts derived from em- bryos of the SI/SI genotype did not express SI ligands, and the attachment of +/+ CMC to SI/SI fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/W, W/W or W/W42 CMC to +/ + fibroblasts was comparable with that of +/ + CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/ + CMC to + / + fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.© 1992 by The American Society of Hematology. 0006–4971 / 92/7903-0007$3.00/0

The proto-oncogene c-kitencodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kitmolecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kitmolecule (approximately 5%) were B220(B)−, TER119(erythroid)−, Thy1negative-low, and WGA+. A small number of Mac-1(macro-phage)+or Gr-1(granulocyte)+cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)−c-kit+cells were rhodamine-123lowand this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin−WGA+c-kit+cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin−c-kit+cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 μg ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kitmolecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.