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Hematopoietic Stem Cells (hematopoietic + stem_cell)
Terms modified by Hematopoietic Stem Cells Selected AbstractsAdult bone marrow,derived stem cells for organ regeneration and repairDEVELOPMENTAL DYNAMICS, Issue 12 2007Florian Tögel Abstract Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow,derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine. Developmental Dynamics 236:3321,3331, 2007. © 2007 Wiley-Liss, Inc. [source] Dermal benzene and trichloroethylene induce aneuploidy in immature hematopoietic subpopulations in vivoENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2001Cynthia R. Giver Abstract Accumulation of genetic damage in long-lived cell populations with proliferative capacity is implicated in tumorigenesis. Hematopoietic stem cells (hsc) maintain lifetime hematopoiesis, and recent studies demonstrate that hsc in leukemic patients are cytogenetically aberrant. We postulated that exposure to agents associated with increased leukemia risk would induce genomic changes in cells in the hsc compartment. Aneusomy involving chromosomes 2 and 11 in sorted hsc (Lin,c-kit+Sca-1+) and maturing lymphoid and myeloid cells from mice that received topical doses of benzene (bz) or trichloroethylene (TCE) was quantified using fluorescence in situ hybridization. Six days after bz or TCE exposure, aneuploid cells in the hsc compartment increase four- to eightfold in a dose- and schedule-independent manner. Aneuploid lymphoid and myeloid cells from bz- and TCE-treated mice approximate controls, except after repeated benzene exposures. Aneuploid cells are more frequent in the hsc compartment than in mature hematopoietic subpopulations. Hematotoxicity was also quantified in bz- and TCE-exposed hematopoietic subpopulations using two colony-forming assays: CFU-GM (colony-forming units/granulocyte-macrophage progenitors) and CAFC (cobblestone area,forming cells). Data indicate that bz is transiently cytotoxic (,1 week) to hsc subpopulations, and induces more persistent toxicity (>2 weeks) in maturing, committed progenitor subpopulations. TCE is not hematotoxic at the doses applied. In conclusion, we provide direct evidence for induction of aneuploidy in cells in the hsc compartment by topical exposure to bz and TCE. Disruption of genomic integrity and/or toxicity in hsc subpopulations may be one step in leukemic progression. Environ. Mol. Mutagen. 37:185,194, 2001. © 2001 Wiley-Liss, Inc. [source] Stemness, fusion and renewal of hematopoietic and embryonic stem cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2003S. Constantinescu Abstract Development of replacement cell therapies awaits the identification of factors that regulate nuclear reprogramming and the mechanisms that control stem cell renewal and differentiation. Once such factors and signals will begin to be elucidated, new technologies will have to be envisaged where uniform differentiation of adult or embryonic stem cells along one differentiation pathway can be induced. Controlled differentiation of stem cells will require the engineering of niches and extracellular signal combinations that would amplify a particular signaling network and allow uniform and selective differentiation. Three recent advances in stem cell research open the possibility to approach engineering studies for cell replacement therapies. Fusion events between stem cells and adult cells or between adult and embryonic stem cells have been shown to result in altered fates and nuclear reprogramming of cell hybrids. Hematopoietic stem cells were shown to require Wnt signaling in order to renew. The purification of Wnt proteins would allow their use as exogenous purified cytokines in attempts to amplify stem cells before bone marrow transplantation. The homeodomain protein Nanog has been shown to be crucial for the embryonic stem cell renewal and pluripotency. However, the cardinal question of how stemness is preserved in the early embryo and adult stem cells remains opened. [source] Stem cell generation and choice of fate: role of cytokines and cellular microenvironmentJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2000S.N. Constantinescu Hematopoietic stem cells (HSC) have provided a model for the isolation, enrichment and transplantation of stem cells. Gene targeting studies in mice have shown that expression of the thrombopoietin receptor (TpoR) is linked to the accumulation of HSCs capable to generate long-term blood repopulation when injected into irradiated mice. The powerful increase in vivo in HSC numbers by retrovirally transduced HOX4B, a homeotic gene, along with the role of the TpoR, suggested that stem cell fate, renewal, differentiation and number can be controlled. The discovery of the precise region of the mouse embryo where HSCs originate and the isolation of supporting stromal cell lines open the possibility of identifying the precise signals required for HSC choice of fate. The completion of human genome sequencing coupled with advances in gene expression profiling using DNA microarrays will enable the identification of key genes deciding the fate of stem cells. Downstream from HSCs, multipotent hematopoietic progenitor cells appear to co-express a multiplicity of genes characteristic of different blood lineages. Genomic approaches will permit the identification of the select group of genes consolidated by the commitment of these multipotent progenitors towards one or the other of the blood lineages. Studies with neural stem cells pointed to the unexpected plastic nature of these cells. Isolation of stem cells from multiple tissues may suggest that, providing the appropriate environment/signal, tissues could be regenerated in the laboratory and used for transplantation. A spectacular example of influence of the environment on cell fate was revealed decades ago by using mouse embryonic stem cells (ES). Injected into blastocysts, ES cells contribute to the formation of all adult tissues. Injected into adult mice, ES cells become cancer cells. After multiple passages as ascites, when injected back into the blastocyst environment, ES- derived cancer cells behaved again as ES cells. More recently, the successful cloning of mammals and reprogramming of transferred nuclei by factors in the cytoplasm of oocytes turned back the clock by showing that differentiated nuclei can be "re-booted" to generate again the stem cells for different tissues. [source] Bone marrow-derived stem cells in liver repair: 10 years down the lineLIVER TRANSPLANTATION, Issue 2 2010Eleanor S. Gilchrist Hematopoietic stem cells have potential in the field of regenerative medicine because of their capacity to form cells of different lineages. Bone marrow stem cells have been shown to contribute to parenchymal liver cell populations, and although this may not be functionally significant, it has sparked interest in the field of autologous stem cell infusion as a possible treatment for cirrhosis. In this review, we will examine the evidence for the contribution of bone marrow-derived cells to populations of liver cells and for the functional contribution of bone marrow-derived cells to both liver fibrosis and repair. The mechanisms by which cells are trafficked from the bone marrow to the liver are complex; the stromal derived factor-1/CXC receptor 4 axis is central to this process. There are limited data in liver injury, but we will examine findings from the bone marrow transplantation literature and discuss their relevance to liver disease. Stromal derived factor-1 also has a role in endogenous liver stem cell accumulation. Some groups have already started infusing autologous bone marrow cells into patients with cirrhosis. We will review these trials in the context of the basic science that we have discussed, and we will consider targets for investigation in the future. Liver Transpl 16:118,129, 2010. © 2010 AASLD. [source] Hematopoietic stem cells mobilized by granulocyte colony-stimulating factor partly contribute to liver graft regeneration after partial orthotopic liver transplantationLIVER TRANSPLANTATION, Issue 7 2006Feng Liu On the basis of the recently recognized potential of hematopoietic stem cells (HSCs) to give rise to hepatocytes, we investigated whether HSCs mobilized by granulocyte colony-stimulating factor (G-CSF) or G-CSF per se could contribute to faster recovery and promote tissue reparation after rats' (cross-sex) partial orthotopic liver transplantation (PLTx). Sex-mismatched (female to male) syngeneic rat PLTx was established. The recipients were repeatedly administrated recombinant G-CSF for 5 consecutive days before (G-CSF + PLTx group) and after PLTx (PLTx + G-CSF group). Compared with those in PLTx group, CD34+ cells in peripheral blood and portal tract region increased from day 1 to 7 after transplantation in G-CSF + PLTx group and from day 3 to 14 after transplantation in PLTx + G-CSF group, respectively, which suggested that CD34+ HSCs were mobilized and migrated into liver graft. Compared with that in G-CSF + PLTx and PLTx groups, there was a higher survival rate in the PLTx + G-CSF group. On day 3 after surgery, the level of aspartate aminotransferase and alanine aminotransferase were lower, whereas the mitosis index, proliferating cell nuclear antigen,positive nuclei, bromodeoxyuridine (BrdU) incorporation, and graft-to-recipient weight ratio were higher in the PLTx + G-CSF group. In contrast, these parameters had no significant difference between G-CSF + PLTx and PLTx groups. To define the origin of proliferating cells reconstituting liver after injury, sry+ (sex-determining region for Y chromosome) and sry+/cytokeratin 19+ (CK19) cells were quantitated. Higher percentage of sry+ and sry+/CK19+ cells in PLTx + G-CSF was detected than in G-CSF + PLTx groups on day 14 after surgery, although the liver engraftment rate still remained rather low. Some of the sry+/CK19+ cells in the portal tract areas were similar to hepatic oval cells/cholangiocytes. In conclusion, G-CSF administration after PLTx greatly improved survival rate and liver regeneration of partial graft, partly by its mobilizing HSCs into the injured liver to differentiate into hepatocytes through hepatic oval cells'/cholangiocytes' engraftment. Liver Transpl 12:1129,1137, 2006. © 2006 AASLD. [source] Trafficking of Murine Hematopoietic Stem and Progenitor Cells in Health and Vascular DiseaseMICROCIRCULATION, Issue 6 2009CHRISTIAN SCHULZ ABSTRACT Hematopoietic stem cells (HSCs) possess the unique capacity for self-renewal and differentiation into various hematopoietic cell lineages. Here we summarize the processes that underlie their mobilization and directed migration from bone marrow into peripheral tissues and back to the bone marrow compartment. We specifically focus on the potential role of hematopoietic stem and progenitor cell (HSPC) migration in vascular diseases and review data from recent studies on mice. A better understanding of the mechanisms that guide HSPCs to vascular tissues will be critical for the development of novel therapeutic strategies to prevent or reverse cardiovascular diseases. [source] Insights into human CD34+ hematopoietic stem/progenitor cells through a systematically proteomic survey coupled with transcriptomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2006Feng Liu Abstract Hematopoietic stem cells are capable of self-renewal and differentiation into different hematopoietic lineages. To gain a comprehensive understanding of hematopoietic stem/progenitor cells, a systematic proteomic survey of human CD34+ cells collected from human umbilical cord blood was performed, in which the proteins were separated by 1- and 2-DE, as well as by nano-LC, and subsequently identified by MS. A total of 370,distinct proteins identified from those cells provided new insights into the potential of the stem/progenitor cells because the nerve, gonad, and eye-associated proteins were reliably identified. Interestingly, the transcripts of 133 (35.9%) identified proteins were not found by the prevalent transcriptome approaches, although several selected transcripts could be detected by RT-PCR. Moreover, the heterogeneity of 33,proteins identified from 2-DE was attributable primarily to post-translational processes rather than to alternative splicing at transcriptional level. Furthermore, the biosyntheses of 15,proteins identified in this study appears not to be completely interrupted in spite of the fact that corresponding antisense RNAs were found in the existing transcriptome data. The integrated proteomic and transcriptomic analyses employed here provided a unique view of the human stem/progenitor cells. [source] Sustained and stable hematopoietic donor-recipient mixed chimerism after unrelated cord blood transplantation for adult patients with severe aplastic anemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2005P. Mao Abstract:, We evaluated the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT (cord blood stem cell transplantation). Nine patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60 mg/kg) and ALG (120 mg/kg). The prophylaxis of GVHD consisted of standard CsA and MTX. Patients have a media age of 25.3 yr (range: 15,37), and a median weight of 57.2 kg (range: 52.5,60) at the time of transplantation. Cord blood searches were all conducted at Guangzhou Cord Blood Bank. The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Engrafted evidence has been found in seven patients involved by biomolecular analyses showing donor-recipient mixed chimerism post-transplant which was stable and persistent. After a median follow up of 32.2 months (range: 4,69), seven patients were alive and disease free. This study shows that durable donor-recipient stable mixed chimerism can be achieved by unrelated CBSCT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation. [source] Expression of a non-DNA-binding Ikaros isoform exclusively in B cells leads to autoimmunity but not leukemogenesisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2007Heather Wojcik Abstract Ikaros is a transcriptional regulator whose function is essential for B cell development. It is expressed in the hematopoietic stem cell (HSC) through the mature B cell stage. Using genetically engineered mice in which the endogenous Ikaros gene is disrupted, it has been shown that a lack of Ikaros leads to a block in B cell development and that its severe diminution results in a hyperresponsive B cell compartment. Ikaros expression within the HSC has led to speculation as to whether the role of Ikaros in B cell biology is largely accomplished prior to B cell specification. In addition, widespread expression of Ikaros in hematopoietic cells leads to the possibility that some or all of the observed defects are not B cell autonomous. In this report, we demonstrate that over-expression of a dominant interfering Ikaros isoform exclusively in B cells has profound effects on mature B cell function. We provide evidence that continued high-level expression of Ikaros is essential for homeostasis of peripheral lymphocytes and maintenance of B cell tolerance. We also show that deregulation of Ikaros activity does not rapidly result in B cell leukemogenesis as it does with 100% penetrance within the T cell lineage. [source] Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult ratsHEPATOLOGY, Issue 2 2002Li Yin Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. [source] Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regenerationJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 12 2007Iraz T Aydin Abstract Background and Aim:, The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Methods:, Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. Results:, The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. Conclusion:, The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration. [source] High-dose chemotherapy with autologous hematopoietic stem cell rescue for stage 4B retinoblastoma,PEDIATRIC BLOOD & CANCER, Issue 1 2010Ira J. Dunkel MD Abstract Background Stage 4b retinoblastoma (central nervous system metastatic disease) has been lethal in virtually all cases reported. Here we describe a series of eight patients treated with intensive chemotherapy, defined as the intention to include high-dose chemotherapy with autologous hematopoietic stem cell rescue. Procedure Induction chemotherapy included cyclophosphamide and/or carboplatin with a topoisomerase inhibitor. High-dose chemotherapy regimens were carboplatin and thiotepa with or without etoposide (n,=,3) or carboplatin, etoposide, and cyclophosphamide (n,=,2). Results Seven patients had leptomeningeal disease and one patient had only direct extension to the CNS via the optic nerve. Three patients had stage 4b disease at the time of original diagnosis of the intra-ocular retinoblastoma; five had later onset at a median of 12 months (range 3,69 months). One patient died of toxicity (septicemia and multi-organ system failure) during induction and two had disease progression prior to high-dose chemotherapy. Five patients received high-dose chemotherapy at a median of 6 months (range 4,6) post-diagnosis of stage 4b disease. Two patients survive event-free at 40 and 101 months; one was irradiated following recovery from the high-dose chemotherapy. Conclusions Intensive multimodality therapy may be beneficial for some patients with stage 4b retinoblastoma. Longer follow-up will determine whether it has been curative. Pediatr Blood Cancer 2010;55:149,152. © 2010 Wiley-Liss, Inc. [source] Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluationTHE JOURNAL OF GENE MEDICINE, Issue 8 2010Elodie Robert-Richard Abstract Background Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients. Methods RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose. Results We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34+ cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo. Conclusions The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation. Copyright © 2010 John Wiley & Sons, Ltd. [source] Partial phenotypic correction and immune tolerance induction to enzyme replacement therapy after hematopoietic stem cell gene transfer of ,-glucosidase in Pompe diseaseTHE JOURNAL OF GENE MEDICINE, Issue 4 2009Gaëlle Douillard-Guilloux Abstract Background Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid ,-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. Even if enzyme replacement therapy (ERT) has already proven some efficacy, its results remain heterogeneous in skeletal muscle, especially in cross reactive immunological material (CRIM)-negative patients. We investigated for the first time the use of hematopoietic stem cell (HSC) gene therapy in a murine model of GSDII. Methods Deficient HSC were transduced with a lentiviral vector expressing human GAA or enhanced green fluorescent protein (GFP) under the control of the retroviral MND promoter and transplanted into lethally irradiated GSDII mice. Animals were then subjected to an ERT protocol for 5 weeks and monitored for metabolic correction and GAA-induced immune reaction. Results GAA was expressed as a correctly processed protein, allowing a complete enzymatic correction in transduced deficient cells without toxicity. Seventeen weeks after transplantation, a partial restoration of the GAA enzymatic activity was observed in bone marrow and peripheral blood cells of GSDII mice, allowing a significant glycogen clearance in skeletal muscle. ERT induced a robust antibody response in GFP-transplanted mice, whereas no immune reaction could be detected in GAA-transplanted mice. Conclusions Lentiviral vector-mediated HSC gene therapy leads to a partial metabolic correction and induces a tolerance to ERT in GSDII mice. This strategy could enhance the efficacy of ERT in CRIM-negative Pompe patients. Copyright © 2009 John Wiley & Sons, Ltd. [source] Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansionTHE JOURNAL OF GENE MEDICINE, Issue 2 2005David Bryder Abstract Background Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. Methods The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. Results We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. Conclusions These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells. Copyright © 2004 John Wiley & Sons, Ltd. [source] Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactorTHE JOURNAL OF GENE MEDICINE, Issue 12 2004Dao Pan Abstract Background One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. Methods In comparison with a conventional bag transduction protocol, a ,closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34+ progenitor cells (PBPCMPS) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. Results A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPCMPS over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8,14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPCMPS -LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). Conclusions MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer. Copyright © 2004 John Wiley & Sons, Ltd. [source] Single-cell image analysis to assess ABC-transporter,mediated efflux in highly purified hematopoietic progenitorsCYTOMETRY, Issue 4 2002H.G.P. Raaijmakers Abstract Background Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. Methods We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter,mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). Results The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein,mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 ± 0.35 and 1.14 ± 0.11, respectively; P = 0.01). P-glycoprotein,mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 ± 0.19 and 1.28 ± 0.18, respectively). Conclusion The described method is a valuable tool for assessing ABC-transporter,mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells. Cytometry 49:135,142, 2002. © 2002 Wiley-Liss, Inc. [source] Gene transfer by electroporation into hemogenic endothelium in the avian embryoDEVELOPMENTAL DYNAMICS, Issue 6 2010Catalina Ana Rosselló Abstract Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained by a population of self-renewing multipotent hematopoietic stem cells (HSCs) throughout the life of an organism. Cells with definitive HSC properties appear in the mid-gestation embryo as dense clusters of cells budding from the floor of the aorta, and that of the vitelline and umbilical arteries in the aorta-gonads-mesonephros region. Attempts to genetically modify the aortic floor from which these HSCs arise have been unsuccessful in the mouse, since the regulation of gene expression in the hemogenic endothelium is largely unknown. Here we report the implementation of gene transfer by electroporation into dorsal aortic endothelial cells in the chick embryo. This approach provides a quick and reproducible method of generating gain/loss-of-function models to investigate the function of genes involved in HSC birth. Developmental Dynamics 239:1748,1754, 2010. © 2010 Wiley-Liss, Inc. [source] Novel agents to override imatinib resistance mechanismsDRUG DEVELOPMENT RESEARCH, Issue 7 2008Asumi Yokota Abstract Chronic myelogenous leukemia (CML) is a disorder of hematopoietic stem cells that results from the Philadelphia chromosome (Ph) created through translocation of human chromosomes 9 and 22. The resulting Bcr-Abl fusion protein has constitutively high tyrosine kinase activity that causes transformation of hematopoietic stem cells. Imatinib mesylate (IM) was developed as a specific Bcr-Abl kinase inhibitor and is efficacious in treating Ph-chromosome-positive (Ph+) leukemias such as CML and Ph+ acute lymphoblastic leukemia (ALL). Within a few years of its introduction to the clinic, IM has dramatically altered the first-line therapy for CML. Although most newly diagnosed CML patients in the chronic phase (CP) achieved durable responses when treated with IM, resistance to IM has become a major problem in patients with advanced-stage disease. The most important mechanism of IM resistance are point mutations within the Abl kinase domain; therefore, there is an urgent need for novel agents that can inhibit mutated Bcr-Abl. In this review, we describe novel Bcr-Abl tyrosine kinase inhibitors, the so-called "Super Gleevec" inhibitors. Drug Dev Res 69:398,406, 2008. © 2008 Wiley-Liss, Inc. [source] Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependentEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2010Seiji Mishima Abstract Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine-differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast-differentiated B10 (Ost-B10) as a feeder layer and examined ex vivo expansion of CD34+CD38, HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost-B10 cells exhibited similar effects on total HSC expansion; however, Ost-B10 demonstrated a higher potency in CD34+CD38, cell-specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony-forming cell assay and long-term culture initiating cell assay revealed that Ost-B10 displayed multipotent differentiation and enabled long-term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost-B10 cells compared with the B10 cells. CD34+CD38, cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost-B10, whereas expansion of CD34+CD38, cells was decreased in Ost-B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast-differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway. [source] The canonical Wnt signaling pathway plays an important role in lymphopoiesis and hematopoiesisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Frank Abstract The evolutionarily conserved canonical Wnt-,-catenin-T cell factor (TCF)/lymphocyte enhancer binding factor (LEF) signaling pathway regulates key checkpoints in the development of various tissues. Therefore, it is not surprising that a large body of gain-of-function and loss-of-function studies implicate Wnt-,-catenin signaling in lymphopoiesis and hematopoiesis. In contrast, recent papers have reported that Mx-Cre-mediated conditional deletion of ,-catenin and/or its homolog ,-catenin (plakoglobin) did not impair hematopoiesis or lymphopoiesis. However, these studies also report that TCF reporter activity remains active in ,-catenin- and ,-catenin-deficient hematopoietic stem cells and all cells derived from these precursors, indicating that the canonical Wnt signaling pathway was not abrogated. Therefore, these studies in fact show that the canonical Wnt signaling pathway is important in hematopoiesis and lymphopoiesis, even though the molecular basis for the induction of the reporter activity is currently unknown. In this perspective, we provide a broad background to the field with a discussion of the available data and create a framework within which the available and future studies may be evaluated. [source] PRDX4, a member of the peroxiredoxin family, is fused to AML1 (RUNX1) in an acute myeloid leukemia patient with a t(X;21)(p22;q22)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Yanming Zhang The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) ,, which forms a heterodimer with CBF , that acts as a transcriptional activating factor. CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations. We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia,M2. Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples. The involvement of AML1 was confirmed by fluorescence in situ hybridization studies. Using 3, RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4. RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts. PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed. Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation. PRDX4 plays a regulatory role in the activation of the transcription factor NF-,B and is significantly down-regulated in acute promyelocytic leukemia. This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia. © 2004 Wiley-Liss, Inc. [source] Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axonsGLIA, Issue 1 2001Masanori Sasaki Abstract The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue ,-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34+ hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS. GLIA 35:26,34, 2001. © 2001 Wiley-Liss, Inc. [source] The effects of STI571 on antigen presentation of dendritic cells generated from patients with chronic myelogenous leukemiaHEMATOLOGICAL ONCOLOGY, Issue 2 2003Naoko Sato Abstract Chronic myelogenous leukemia is caused by the acquisition of the reciprocal (9;22)(q34;q11) chromosomal translocation in hematopoietic stem cells. The fusion protein showed higher and aberrant tyrosine kinase activity. The inhibition of the tyrosine kinase activity of the protein represents a specific therapeutic strategy for bcr/abl-expressing leukemias. STI571 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the Abl protein tyrosine kinase. In this study, we evaluated the effects of STI571 on antigen presentation of dendritic cells generated from the patients with CML. The data showed that by the addition of STI571 the dendritic cells derived from CML clone showed an increased expression of CD1a, CD83, CD80 and CD86 by flow cytometry analysis and showed more intense abilities of allogeneic antigen presentation by mixed leukocyte culture, compared with the control cells without STI571. Our results suggested that STI571 not only has a direct cytotoxic effect on bcr-abl gene rearranged cells but also an indirect effect associated with increased anti-leukemic immunological function due to an intensified antigen presentation. Copyright © 2003 John Wiley & Sons, Ltd. [source] The aging of early B-cell precursorsIMMUNOLOGICAL REVIEWS, Issue 1 2005David Allman Summary:, B-cell genesis in the bone marrow declines with advancing age. In this review, we discuss our current understanding of why B-cell production rates decline with age with a special emphasis on why age-related factors might target very early lymphoid precursors. We consider the impact of aging on cytokine responsiveness and how current models for lineage relationships for very early B- and T-cell precursors might influence interpretations of experiments addressing age-associated declines in B- and T-cell differentiation. This discussion centers on the notion that aging affects events associated with the process by which hematopoietic stem cells are guided toward the B-cell pathway. Finally, we present a model in which the age-associated loss of early B-cell precursors is linked to suboptimal function of key transcriptional regulators of very early B-cell development. [source] Microenvironmental influences on human B-cell developmentIMMUNOLOGICAL REVIEWS, Issue 1 2000F. E. Bertrand Summary: Mammalian B-cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement , culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B-cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints. [source] The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responsesIMMUNOLOGICAL REVIEWS, Issue 1 2000Shuhji Seki Summary: The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells. Hepatocytes produce acute phase proteins and complement in bacterial infections. Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)-12. IL-12 and other monokines (IL-18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon-g and thereby acquire cytotoxicity against tumors and microbe-infected cells. These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases. However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place. Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury. NK1.1 Ag+ T cells and another T-cell subset with an intermediate T-cell receptor, CD122+CD8+ T cells, can develop independently of thymic epithelial cells. Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein. NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease. [source] Selected Stro-1-enriched bone marrow stromal cells display a major suppressive effect on lymphocyte proliferationINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1 2009A. NASEF Summary Mesenchymal stem cells (MSCs) have an immunosuppressive effect and can inhibit the proliferation of alloreactive T cells in vitro and in vivo. Cotransplantation of MSCs and hematopoietic stem cells (HSCs) from HLA-identical siblings has been shown to reduce the incidence of acute graft- vs.-host disease. MSCs are heterogeneous and data on the inhibitory effects of different MSC subsets are lacking. The antigen Stro1 is a marker for a pure primitive MSC subset. We investigated whether Stro-1-enriched induce a more significant suppressive effect on lymphocytes in a mixed lymphocyte reaction (MLR), and whether this action is related to a specific gene expression profile in Stro-1-enriched compared to other MSCs. We demonstrated that the Stro-1-enriched population elicits a significantly more profound dose-dependent inhibition of lymphocyte proliferation in a MLR than MSCs. One thousand expanded Stro-1-enriched induced an inhibitory effect comparable to that of 10 times as many MSCs. Inhibition by Stro-1-enriched was more significant in contact-dependent cultures than in noncontact-dependant cultures at higher ratio. The Stro-1-enriched inhibitory effect in both culture types was linked to increased gene expression for soluble inhibitory factors such as interleukin-8 (IL-8), leukemia inhibitory factor (LIF), indoleamine oxidase (IDO), human leukocyte antigen-G (HLA-G), and vascular cell adhesion molecule (VCAM1). However, tumor growth factor-,1 (TGF-,) and IL-10 were only up-regulated in contact-dependant cultures. These results may support using a purified Stro-1-enriched population to augment the suppressive effect in allogeneic transplantation. [source] Restoration of Bone Mass and Strength in Glucocorticoid-Treated Mice by Systemic Transplantation of CXCR4 and Cbfa-1 Co-Expressing Mesenchymal Stem Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2009Chun-Yang Lien Abstract Transplantation of gene-modified mesenchymal stem cells (MSCs) in animals for bone regeneration therapy has been evaluated extensively in recent years. However, increased endosteal bone formation by intravenous injection of MSCs ectopically expressing a foreign gene has not yet been shown. Aside from the clearance by lung and other tissues, the surface compositions of MSCs may not favor their bone marrow (BM) migration and engraftment. To overcome these hurdles, a gene encoding the chemokine receptor largely responsible for stromal-derived factor-1 (SDF-1)-mediated BM homing and engraftment of hematopoietic stem cells (HSCs), CXCR4, was transduced into mouse C3H10T1/2 cells by adenovirus infection. A dose-dependent increase of CXCR4 surface expression with a parallel enhanced chemotaxis toward SDF-1 in these cells after virus infection was clearly observed. Higher BM retention and homing of CXCR4-expressing MSCs were also found after they were transplanted by intramedullary and tail vein injections, respectively, into immunocompetent C3H/HeN mice. Interestingly, a full recovery of bone mass and a partial restoration of bone formation in glucocorticoid-induced osteoporotic mice were observed 4 wk after a single intravenous infusion of one million CXCR4-expressing C3H10T1/2 cells. In the meantime, complete recovery of bone stiffness and strength in these animals was consistently detected only after a systemic transplantation of CXCR4 and Cbfa-1 co-transduced MSCs. To our knowledge, this is the first report to show unequivocally the feasibility of ameliorating glucocorticoid-induced osteoporosis by systemic transplantation of genetically manipulated MSCs. [source] | ||||||||||||||||||||||||||||||||||