技术资料

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

UNLABELLED: AC133 cells,a subpopulation of CD34+ hematopoietic stem cells,can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected,magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma,and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However,low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature,and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors. View Publication

The homeostatic adult bone marrow (BM) is a complex tissue wherein physical and biochemical interactions serve to maintain a balance between the hematopoietic and nonhematopoietic compartments. To focus on soluble factor interactions occurring between mesenchymal and hematopoietic cells,a serum-free adhesion-independent culture system was developed that allows manipulation of the growth of both mesenchymal and hematopoietic human BM-derived progenitors and the balance between these compartments. Factorial experiments demonstrated a role for stem cell factor (SCF) and interleukin 3 (IL-3) in the concomitant growth of hematopoietic (CD45+) and nonhematopoietic (CD45-) cells,as well as their derivatives. Kinetic tracking of IL-3alpha receptor (CD123) and SCF receptor (CD117) expression on a sorted CD45- cell population revealed the emergence of CD45-CD123+ cells capable of osteogenesis. Of the total fibroblast colony-forming units (CFU-Fs) and osteoblast colony-forming units (CFU-O),approximately 24% of CFU-Fs and about 22% of CFU-Os were recovered from this population. Cell-sorting experiments demonstrated that the CD45+ cell population secreted soluble factors that positively affect the survival and proliferation of CFU-Fs and CFU-Os generated from the CD45- cells. Together,our results provide insight into the intercellular cytokine network between hematopoietic and mesenchymal cells and provide a strategy to mutually culture both mesenchymal and hematopoietic cells in a defined scalable bioprocess. View Publication

Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors,which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here,we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA,synthesized siRNA and transcribed shRNA,reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction,however,was sufficient to increase transduction efficiency to two- to four-fold in cell cultures,and followed by a seven- to eight-fold increase in mice. The RNAi treated,lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together,both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus,RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy. View Publication

The coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein (CRD-BP/IMP1) is an RNA-binding protein specifically recognizing c-myc,leader 3' IGF-II and tau mRNAs,and the H19 RNA. CRD-BP/IMP1 is predominantly expressed in embryonal tissues but is de novo activated and/or overexpressed in various human neoplasias. To address the question of whether CRD-BP/IMP1 expression characterizes certain cell types displaying distinct proliferation and/or differentiation properties (i.e. stem cells),we isolated cell subpopulations from human bone marrow,mobilized peripheral blood,and cord blood,all sources known to contain stem cells,and monitored for its expression. CRD-BP/IMP1 was detected only in cord blood-derived CD34(+) stem cells and not in any other cell type of either adult or cord blood origin. Adult BM CD34(+) cells cultured in the presence of 5'-azacytidine expressed de novo CRD-BP/IMP1,suggesting that epigenetic modifications may be responsible for its silencing in adult non-expressing cells. Furthermore,by applying the short interfering RNA methodology in MCF-7 cells,we observed,subsequent to knocking down CRD-BP/IMP1,decreased c-myc expression,increased IGF-II mRNA levels,and reduced cell proliferation rates. These data 1) suggest a normal role for CRD-BP/IMP1 in pluripotent stem cells with high renewal capacity,like the CB CD34(+) cells,2) indicate that altered methylation may directly or indirectly affect its expression in adult cells,3) imply that its de novo activation in cancer cells may affect the expression of c-Myc and insulin-like growth factor II,and 4) indicate that the inhibition of CRD-BP/IMP1 expression might affect cancer cell proliferation. View Publication

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA,encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast,no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines,mice,dogs,nonhuman primates,and human subjects. Here,we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose,and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells,such hits were overrepresented in leukemic clones,pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases,indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings,we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery. View Publication

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. 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

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

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

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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