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It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12,previously called stromal cell-derived factor 1,are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However,this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and,strikingly,is absent in circulating mature B cells,which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK),a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation,whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover,sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways. View Publication

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report,we show that marrow cells from WAS protein (WASP)-deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells,including hematopoietic progenitors and stem cells (HSCs),have decreased homing capacities that were associated with a defect in adhesion to collagen. During development,HSCs migrate from the liver to the marrow and the spleen,prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that,in contrast to marrow progenitor cells,fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients,a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore,the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs. View Publication

In a previous study of prevalidation,a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed,to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up,by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper,we describe an international blind trial designed to apply this model to the clinical neutropenia,by testing 20 drugs,including 14 antineoplastics (Cytosar-U,5-Fluorouracil,Myleran,Thioguanine,Fludarabine,Bleomycin,Methotrexate,Gemcitabine,Carmustine,Etoposide,Teniposide,Cytoxan,Taxol,Adriamycin); two antivirals (Retrovir,Zovirax,); three drugs for other therapeutic indications (Cyclosporin,Thorazine,Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin,Bleomycin,Etoposide,Fludarabine,5-Fluorouracil,Myleran,Taxol,Teniposide,Thioguanine,and Thorazine),IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine,Cyclosporin,Cytosar-U,Cytoxan,Gemcitabine,Indocin,Lindane,Methotrexate,Retrovir,and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD� View Publication

Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development,however,must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor,EphB4,known to be spatially restricted in expression and critical for organized vessel formation,modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast,blood cell,cardiomyocyte,and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated,but not neuroectoderm-associated,genes. Therefore,EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone,increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the side population" (SP) of marrow stem cells� View Publication

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs,their identification and enumeration depends on functional long-term,multilineage,in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens,such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication

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Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34(+) cells,which are enriched in HSPCs,roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34(+) cells do not roll in these vessels,in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be alpha1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLe(x)). We found that inadequate alpha1-3 fucosylation of CB CD34(+) cells,particularly CD34(+)CD38(-/low) cells that are highly enriched in HSPCs,caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34(+) cells with guanosine diphosphate (GDP) fucose and exogenous alpha1-3 fucosyltransferase VI increased cell-surface sLe(x) determinants,augmented binding to fluid-phase P- and E-selectin,and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that alpha1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants. View Publication

Hematopoietic progenitor/stem cell homing to the bone marrow requires the concerted action of several adhesion molecules. Endothelial P- and E-selectins play an important role in this process,but their ligands on a large subset of neonate-derived human CD34+ cells are absent,leading to a reduced ability to interact with the bone marrow (BM) microvasculature. We report here that this deficiency results from reduced alpha1,3-fucosyltransferase (FucT) expression and activity in these CD34+ cells. Incubation of CD34+ cells with recombinant human FucTVI rapidly corrected the deficiency in nonbinding CD34+ cells and further increased the density of ligands for both P- and E-selectins on all cord blood-derived CD34+ cells. Intravital microscopy studies revealed that these FucTVI-treated CD34+ cells displayed a marked enhancement in their initial interactions with the BM microvasculature,but unexpectedly,homing into the BM was not improved by FucTVI treatment. These data indicate that,although exogenous FucT enzyme activity can rapidly modulate selectin binding avidity of cord blood CD34+ cells,further studies are needed to understand how to translate a positive effect on progenitor cell adhesion in bone marrow microvessels into one that significantly influences migration and lodgement into the parenchyma. View Publication

Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here,we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene,and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes,including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust,erythroid-specific production of therapeutically relevant levels of beta-globin protein. However,the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications. View Publication