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Haematopoietic Stem Cells (haematopoietic + stem_cell)
Terms modified by Haematopoietic Stem Cells Selected AbstractsCombined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidenceHISTOPATHOLOGY, Issue 2 2008F Zhang Aims:, Combined hepatocellular cholangiocarcinoma (CHC) is a rare form of primary liver cancer, showing a mixture of hepatocellular and biliary features. Data suggest that most CHC arise from hepatic progenitor cells (HPCs). The aim was to investigate the origin of CHC. Methods and results:, Twelve cases of CHC were studied by immunohistochemistry for hepatocytic (hepPar1, ,-fetoprotein), cholangiocytic cytokeratin [(CK) 7, CK19], hepatic progenitor cell (OV-6), haematopoietic stem cell (c-kit, CD34), as well as CD45 and chromogranin-A markers. The combination of double-fluorescence immunostaining consisted of HepPar1 with CK19, and c-kit with OV-6. All 12 cases demonstrated more or less transitional areas, with strands/trabeculae of small, uniform, oval-shaped cells including scant cytoplasm and hyperchromatic nuclei embedded within a thick, desmoplastic stroma; however, two cases were found to consist entirely of such transitional areas. Simultaneous co-expression of hepPar1 and CK7, or CK19, was demonstrated in 10/12 (83.3%) cases of CHC. c-kit expression was noted in 10/12 (83.3%) cases, of which 7/10 (70%) showed co-expression of OV-6. Conclusions:, The results suggest that CHC are of HPC origin, supporting the concept that human hepatocarcinogenesis may originate from the transformation of HPCs. [source] New light on the biology and developmental potential of haematopoietic stem cells and progenitor cellsJOURNAL OF INTERNAL MEDICINE, Issue 4 2009M. Sigvardsson Abstract. Even though stem cells have been identified in several tissues, one of the best understood somatic stem cells is the bone marrow residing haematopoietic stem cell (HSC). These cells are able to generate all types of blood cells found in the periphery over the lifetime of an animal, making them one of the most profound examples of tissue-restricted stem cells. HSC therapy also represents one of the absolutely most successful cell-based therapies applied both in the treatment of haematological disorders and cancer. However, to fully explore the clinical potential of HSCs we need to understand the molecular regulation of cell maturation and lineage commitment. The extensive research effort invested in this area has resulted in a rapid development of the understanding of the relationship between different blood cell lineages and increased understanding for how a balanced composition of blood cells can be generated. In this review, several of the basic features of HSCs, as well as their multipotent and lineage-restricted offspring, are addressed, providing a current view of the haematopoietic development tree. Some of the basic mechanisms believed to be involved in lineage restriction events including activities of permissive and instructive external signals are also discussed, besides transcription factor networks and epigenetic alterations to provide an up-to-date view of early haematopoiesis. [source] Mathematical modelling of the impact of haematopoietic stem cell-delivered gene therapy for HIVTHE JOURNAL OF GENE MEDICINE, Issue 12 2009John M. Murray Abstract Background Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention. Methods Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. Results For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log10 and 1 log10, respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm3 compared to 96 cells/mm3 for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log10 and 0.86 log10 at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. Conclusions The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV. Copyright © 2009 John Wiley & Sons, Ltd. [source] Haematopoietic stem cell niche in DrosophilaBIOESSAYS, Issue 8 2007Ute Koch Development and homeostasis of the haematopoietic system is dependent upon stem cells that have the unique ability to both self-renew and to differentiate in all cell lineages of the blood. The crucial decision between haematopoietic stem cell (HSC) self-renewal and differentiation must be tightly controlled. Ultimately, this choice is regulated by the integration of intrinsic signals together with extrinsic cues provided by an exclusive microenvironment, the so-called haematopoietic niche. Although the haematopoietic system of vertebrates has been studied extensively for many decades, the specification of the HSC niche and its signals involved are poorly understood. Much of our current knowledge of how niches regulate long-term maintenance of stem cells is derived from studies on Drosophila germ cells. Now, two recently published studies by Mandal et al.1 and Krezmien et al.2 describe the Drosophila haematopoietic niche and signal transduction pathways that are involved in the maintenance of haematopoietic precursors. Both reports emphasize several features that are important for controlling stem cell behavior and show parallels to both the vertebrate haematopoietic niche as well as the Drosophila germline stem cell niches in ovary and testis. The findings of both papers shed new light on the specific interactions between haematopoietic progenitors and their microenvironment. BioEssays 29:713,716, 2007. © 2007 Wiley Periodicals, Inc. [source] Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2002Dimitrios Tzoanopoulos Summary. Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN-,) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK,STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor-1 (IRF-1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF-1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF-1 in CML is currently unknown. Therefore, mutational analysis of IRF-1 was performed and its expression pattern was also studied in CML patients. We studied IRF-1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full-length IRF-1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full-length IRF-1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full-length IRF-1 mRNA was observed. These findings demonstrate that, in CML patients, IRF-1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full-length IRF-1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease. [source] Inorganic arsenic induces necrosis of human CD34-positive haematopoietic stem cellsENVIRONMENTAL TOXICOLOGY, Issue 2 2008Laurent Vernhet Abstract Inorganic arsenic is a major environmental contaminant known to exert immunosuppressive effects. In this study, we report toxicity of As2O3, a trivalent inorganic form, toward isolated human hematopoietic CD34+ progenitor cells. Our results demonstrate that low concentrations of As2O3 (0.1,5 ,M) inhibit in vitro proliferation of CD34+ cells and their differentiation into various hematological cell lineages. These effects were associated with the induction of a necrotic process independent of caspases and likely related to mitochondrial damage. We conclude that As2O3 can impair in vitro human hematopoiesis by decreasing survival of CD34+ progenitor cells. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Stem cells in the lung parenchyma and prospects for lung injury therapyEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2006C. C. Yen Abstract Until recently, it was thought that only embryonic stem cells were pluripotent and that adult stem cells were restricted in their differentiative and regenerative potential to become the tissues in which they reside. However, the discovery that adult stem cells in one tissue can contribute to the formation of other tissues, especially after injury or cell damage, implies that stem cells have developmental plasticity. For example, haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) from bone marrow can be used to regenerate diverse tissues at distant sites, including the lung. This article reviews the character of stem cells in the lung parenchyma and focuses on the potential uses of adult stem cells in research of lung injury and lung disease therapies. [source] Adult stem cell plasticity: will engineered tissues be rejected?INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2004Te-Chao Fang Summary The dogma that adult tissue-specific stem cells remain committed to supporting only their own tissue has been challenged; a new hypothesis, that adult stem cells demonstrate plasticity in their repertoires, is being tested. This is important because it seems possible that haematopoietic stem cells, for example, could be exploited to generate and perhaps deliver cell-based therapies deep within existing nonhaematopoietic organs. Much of the evidence for plasticity derives from histological studies of tissues from patients or animals that have received grafts of cells or whole organs, from a donor bearing (or lacking) a definitive marker. Detection in the recipient of appropriately differentiated cells bearing the donor marker is indicative of a switch in phenotype of a stem cell or a member of a transit amplifying population or of a differentiated cell. In this review, we discuss evidence for these changes occurring but do not consider the molecular basis of cell commitment. In general, the extent of engraftment is low but may be increased if tissues are damaged. In model systems of liver regeneration, the repeated application of a selection pressure increases levels of engraftment considerably; how this occurs is unclear. Cell fusion plays a part in regeneration and remodelling of the liver, skeletal muscle and even regions of the brain. Genetic disease may be amenable to some forms of cell therapy, yet immune rejection will present challenges. Graft- vs. -host disease will continue to present problems, although this may be avoided if the cells were derived from the recipient or they were tolerized. Despite great expectations for cellular therapies, there are indications that attempts to replace missing proteins could be confounded simply by the development of specific immunity that rejects the new phenotype. [source] Automated separation of cord blood units in top and bottom bags using the Compomat G4INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2006P. SOLVES Summary Cord blood (CB) has become a real alternative source of haematopoietic stem cells for bone marrow reconstitution in a variety of malignant disorders. As a response to this increasing activity, CB banks have been developed to guarantee the quality of processed CB units. Volume reduction of CB units maximizes storage space and also has other advantages. The aim of this study was to develop a program for the volume reduction of CB in the Compomat G4 device. We also compared two different top and bottom systems for CB fractionation (Compomat G4 and Optipress II). We empirically designed three different programs for volume reduction of CB with Compomat G4: two for final BC volume of 41 ml (CB1 and CB2) and the other one for buffy coat (BC) volume of 25 ml (CB3). Significantly worse recoveries were achieved for CB processed with program CB3. A RBC depletion of ,50%, ,60% and ,70% were achieved for 67%, 39% and 9% of all units respectively. When comparing Compomat G4 and Optipress II, total nucleated cell recovery was similar for both methods, while lymphocytes recovery was significantly better for Optipress II. [source] Myelodysplastic syndrome associated with trisomy 2INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2005M. HELLER Summary Clinical course and cytogenetic analysis suggest that myelodysplasia (MDS) is one step in a multistep model of malignant transformation of haematopoietic stem cells to acute myeloid leukaemia (AML). We report a further case of MDS associated with trisomy 2, and comment on the significance of the cytogenetic abnormality, which as a sole abnormality only occurs in MDS, but is found in combination with other chromosomal abnormalities in AML. Previous reports on balanced and unbalanced chromosomal abnormalities associated with therapy related MDS and therapy related AML suggest that trisomy 2 is an early chromosomal abnormality in leukaemogenesis. [source] New light on the biology and developmental potential of haematopoietic stem cells and progenitor cellsJOURNAL OF INTERNAL MEDICINE, Issue 4 2009M. Sigvardsson Abstract. Even though stem cells have been identified in several tissues, one of the best understood somatic stem cells is the bone marrow residing haematopoietic stem cell (HSC). These cells are able to generate all types of blood cells found in the periphery over the lifetime of an animal, making them one of the most profound examples of tissue-restricted stem cells. HSC therapy also represents one of the absolutely most successful cell-based therapies applied both in the treatment of haematological disorders and cancer. However, to fully explore the clinical potential of HSCs we need to understand the molecular regulation of cell maturation and lineage commitment. The extensive research effort invested in this area has resulted in a rapid development of the understanding of the relationship between different blood cell lineages and increased understanding for how a balanced composition of blood cells can be generated. In this review, several of the basic features of HSCs, as well as their multipotent and lineage-restricted offspring, are addressed, providing a current view of the haematopoietic development tree. Some of the basic mechanisms believed to be involved in lineage restriction events including activities of permissive and instructive external signals are also discussed, besides transcription factor networks and epigenetic alterations to provide an up-to-date view of early haematopoiesis. [source] New Insights in Vascular Development: Vasculogenesis and Endothelial Progenitor CellsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009S. Käßmeyer Summary In the course of new blood vessel formation, two different processes , vasculogenesis and angiogenesis , have to be distinguished. The term vasculogenesis describes the de novo emergence of a vascular network by endothelial progenitors, whereas angiogenesis corresponds to the generation of vessels by sprouting from pre-existing capillaries. Until recently, it was thought that vasculogenesis is restricted to the prenatal period. During the last decade, one of the most fascinating innovations in the field of vascular biology was the discovery of endothelial progenitor cells and vasculogenesis in the adult. This review aims at introducing the concept of adult vasculogenesis and discusses the efforts to identify and characterize adult endothelial progenitors. The different sources of adult endothelial progenitors like haematopoietic stem cells, myeloid cells, multipotent progenitors of the bone marrow, side population cells and tissue-residing pluripotent stem cells are considered. Moreover, a survey of cellular and molecular control mechanisms of vasculogenesis is presented. Recent advances in research on endothelial progenitors exert a strong impact on many different disciplines and provide the knowledge for functional concepts in basic fields like anatomy, histology as well as embryology. [source] A new bone to pick: osteoblasts and the haematopoietic stem-cell nicheBIOESSAYS, Issue 6 2004Jiang Zhu Two recent publications highlight the role of bone-forming cells, the osteoblasts, in controlling the development of neighboring haematopoietic stem cells (HSCs).1,2 Using two distinct transgenic mouse models, one using the conditional deletion of the Bone Morphogenetic Protein Receptor 1A (BMPR1A) gene, the other using over-expression of an active PTH/PTHrP receptor (PPR) mutant within osteoblasts, the authors show parallel, concordant increases in the generation of trabecular osteoblasts and the number of HSCs. In situ staining showed that rarely cycling HSCs sporadically attach to endosteal osteoblasts, while in vitro assays indicated that ligation of Jag1 on osteoblasts by Notch1 on HSCs promotes HSC proliferation. These two independent works have revived and revitalized the notion that osteoblasts are a major, defining component of the HSC niche within the bone marrow (BM). This minireview discusses these results in the context of other recent studies of mesenchymal cells within the BM microenvironment, presents one potential unified model of the functional anatomy of the BM HSC niche, and highlights new questions raised by these and other studies of osteoblasts and HSCs. BioEssays 26:595,599, 2004. © 2004 Wiley Periodicals, Inc. [source] Differential distribution of haematopoietic and nonhaematopoietic progenitor cells in intralesional and extralesional keloid: do keloid scars provide a niche for nonhaematopoietic mesenchymal stem cells?BRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010S.A. Iqbal Summary Background, Keloid disease is a benign, quasineoplastic disease with a high recurrence rate. Mesenchymal-like stem cells (MLSC) have previously been demonstrated in keloid scars and may be involved in keloid pathobiology. However, as these cells have only been examined by single colour fluorescence activated cell sorting (FACS) alone, they need to be more comprehensively characterized so that the key cellular contributors to keloid scars can be better understood. Objectives, To identify and characterize MLSC in intralesional and extralesional keloid, and to distinguish haematopoietic stem cells (HSC) from mesenchymal stem cells (MSC). Methods and patients, Punch biopsies from intralesional (top, middle and margin) and extralesional keloid scar sites were obtained from 17 patients with a keloid. Multicolour FACS analysis using antibodies specific for HSC markers CD34 and CD117 and MSC markers CD13, CD29, CD44 and CD90 was performed on freshly isolated keloid scar cells and on passage 0 and 1 cells. This was complemented by real-time quantitative polymerase chain reaction (PCR) and immunohistological in situ analyses. Results, Keloid scars contain distinct subpopulations of MLSCs. Cells positive for CD13, CD29, CD44 and CD90 were found to be significantly (P < 0·05) higher in the top and middle compartments of keloid scars compared with extralesional skin, where cells positive for CD34, CD90 and CD117 (representing HSCs) predominated. A unique population of CD34+ cells (cells positive for CD13, CD29, CD34, CD44 and CD90) were found in keloid scars and in extralesional skin. FACS and quantitative PCR analysis showed that many of the MSC markers were progressively downregulated and all HSC markers were lost during extended keloid fibroblast culture up to passage 1. Conclusion, We have found distinct subpopulations of haematopoietic and nonhaematopoietic MSC in keloid scars, whereby HSC accumulate extralesionally, while keloids seem to provide a niche environment for nonhaematopoietic MSC. Future therapy of keloids may have to target differentially both stem cell populations in order to deprive these tumours of their regenerative cell pools. [source] Improving the outcome of cord blood transplantation: use of mobilized HSC and other cells from third party donorsBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2009Manuel N. Fernández Summary We developed the strategy of umbilical cord blood transplants (UCBT) with co-infusion of a limited number of highly purified mobilized haematopoietic stem cells (MHSC) from a human leucocyte antigen (HLA) unrestricted third party donor (TPD). Short post-transplant periods of neutropenia were usually observed in adults with haematological neoplasms receiving UCBT with a relatively low cell content and 0,3 HLA mismatches after myeloablative conditioning. This resulted from an early and initially predominant engraftment of the TPD,MHSC. After a variable period of double complete TPD + UCB chimerism, final full UCB chimerism was achieved (cumulative incidence >90%) within 100 d. Early recovery of the circulating neutrophils resulting from the ,bridge transplant' of the TPD,MHSC reduced the incidence of serious neutropenia-related infections, also facilitating the use of drugs with myelosuppressive side effects to combat other infections. The observed incidence of graft- versus -host disease and relapses was low, with overall and disease-free survival curves comparable to those of HLA identical sibling transplants. Post-transplant recovery of natural killer cells occurred soon after the transplant and B cells recovered around 6 months, but T-cell recovery took more than 1 year. Available data show that T cell recovery derives from UCB,HSC through thymic differentiation and that cytomegalovirus (CMV)-specific lymphocytes develop following CMV reactivations. [source] Improving outcomes of cord blood transplantation: HLA matching, cell dose and other graft- and transplantation-related factorsBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2009Vanderson Rocha Summary The use of unrelated umbilical cord blood (UCB) as an alternative source of haematopoietic stem cells transplantation (HSCT) has been widely used for patients lacking a human leucocyte antigen (HLA) matched donor. One of the disadvantages of using UCB is the limited number of haematopoietic stem cells and, consequently, delayed engraftment and increased risk of early mortality. Many approaches have been investigated in the attempt to improve engraftment and survival. Among those, studies analysing prognostic factors related to patients, disease, donor and transplantation have been performed. Variable factors have been identified, such as factors related to donor choice (HLA, cell dose and others) and transplantation (conditioning and graft- versus -host disease prophylaxis regimens). This review will focus on the interactions between HLA, cell dose and other modifiable factors related to the UCB unit selection and transplantation that may improve outcomes after UCB transplantation. [source] Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitroBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2007Caroline J. Marshall Summary The transforming growth factor- , -related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34+ cells with different levels of c-Kit expression and multipotency were identified. CD34+/c-Kithigh cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34+/c-Kitlow cells are Flk1+/CD45neg and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34+/c-Kitlow cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34+/c-Kithigh cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34+/c-Kithigh haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34+/c-Kithigh progenitors cultured with BMP4 also generated adherent colonies typical of c-Kitlow cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34+ AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche. [source] Engraftment of NOD/SCID/,cnull mice with multilineage neoplastic cells from patients with juvenile myelomonocytic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2005Yoichi Nakamura Summary Several lines of evidence indicate the clonal nature of juvenile myelomonocytic leukaemia (JMML), involving myeloid, erythroid, megakaryocyte and B-lymphoid lineages. However, it is unclear whether the T-lymphocyte lineage is involved. We demonstrated that cells from six patients with JMML repopulated in non-obese diabetic/severe combined immunodeficient/,cnull mice and differentiated into granulocytes, monocytes, erythrocytes, B lymphocytes, T lymphocytes and natural killer cells. The percentage of human CD45 antigen-positive cells ranged from 41% to 73% in the murine bone marrow 12 weeks after transplantation. To examine the involvement of lymphocyte subpopulations, we purified human CD3+, CD19+ and CD56+ cells from murine bone marrow cells transplanted from a patient with monosomy 7. Fluorescence in situ hybridization (FISH) showed the clonal marker in 96,100% of purified CD3+, CD19+ and CD56+ subpopulations. These findings support the concept that JMML originates in transplantable multilineage haematopoietic stem cells. This novel murine xenotransplant model should be useful for investigating the nature of stem cells and testing new therapies for patients with JMML. [source] Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterizationBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Marie-Hélène Rodriguez Summary Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII. [source] Spleen neoangiogenesis in patients with myelofibrosis with myeloid metaplasiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Giovanni Barosi Summary Neoangiogenesis is an integral component of bone marrow myeloproliferation in patients with myelofibrosis with myeloid metaplasia (MMM). As extramedullary haematopoiesis is a constitutive feature of MMM, we studied spleen neoangiogenesis by a computerized image analysis in MMM patients. Compared with five normal subjects, spleen CD34-staining capillary vascular density (CVD) was 2·1,3·03 times higher than the upper range of normal in six of the 15 (40%) MMM patients. CD8-staining sinusoidal vascular density (SVD) was constantly normal or lesser than normal and was inversely correlated with CVD (R = ,0·53; P = 0·04). In MMM patients who did not receive cytoreductive or radiation therapy in the month before splenectomy (n = 9), the CVD was a significant determinant of spleen size (R = 0·88; P = 0·04). In MMM patients, the number of spleen CD34+ haematopoietic stem cells was increased from 1·2 to 98 times the upper limit of normal, and predicted the expansion of CVD (R = 0·57; P = 0·03). A population of cells expressing the CD34+/CD133+/VEGFR-2+ angiopoietic phenotype was present in the blood and spleen of five of seven patients. These results document that neoangiogenesis is an integral component of spleen re-localization of haematopoietic stem cells and suggest a cellular mechanism for spleen neoangiogenesis. [source] Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterizationBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2003Lindolfo da Silva Meirelles Summary., In spite of the attention given to the study of mesenchymal stem cells (MSCs) derived from the bone marrow (BM) of humans and other species, there is a lack of information about murine MSCs. We describe the establishment of conditions for the in vitro expansion of plastic-adherent cells from murine BM for over 50 passages, and provide their characterization regarding morphology, surface marker profile and growth kinetics. These cells were shown to differentiate along osteogenic and adipogenic pathways, and to support the growth and differentiation of haematopoietic stem cells, and were thus operationally defined as murine mesenchymal stem cells (mMSCs). mMSCs were positive for the surface markers CD44, CD49e, CD29 and Sca-1, and exhibited a homogeneous, distinctive morphology. Their frequency in the BM of adult BALB/c and C57Bl/6 mice, normal or knockout for the , -L-iduronidase (IDUA) gene, was preliminarily estimated to be 1 per 11 300,27 000 nucleated cells. The emergence of a defined methodology for the culture of mMSCs, as well as a comprehensive understanding of their biology, will make the development of cellular and genetic therapy protocols in murine models possible, and provide new perspectives in the field of adult stem cells research. [source] CXCR4 chemokine receptors (CD184) and ,4,1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)BRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2003Jan A. Burger Summary. Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell,stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28·7 ± 12%vs 18·1 ± 6·1% of input cells within 24 h, mean ± SD, n = 8), whereas 9·4 ± 12·6% (mean ± SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks ,4,1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that ,4,1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment. [source] Detection of tryptase,, chymase+ cells in human CD34+ bone marrow progenitorsCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2004Y. Shimizu Summary Background Mast cells (MCs) arise from haematopoietic stem cells. We have recently reported that CD34+ progenitors derived from human bone marrow (BM) develop into tryptase+, chymase+ MCs when cultured in the presence of recombinant human stem cell factor (rhSCF) and recombinant human IL-6 (rhIL-6). In an experiment for the expression of chymase during differentiation, chymase+ cells were detected in human BM, but tryptase+ cells were not found. Objective The purpose of this study was to show the appearance of chymase+ cells in CD34+ cells with an origin different from MC differentiation. Methods CD34+ cells from human BM were sorted with anti-CD117 monoclonal antibody (mAb), and cytospins of CD34+, CD34+CD117+, or CD34+CD117, were prepared. These cells were cultured with rhSCF+rhIL-6 for 12 weeks. Some of the cells were subjected to either histological stain with Wright,Giemsa or immunocytochemistry with anti-chymase mAb. Real-time RT-PCR was also performed to compare the transcriptional level of chymase from each cell preparation. Results Chymase was expressed in CD34+ cells as well as human MCs by immunocytochemistry. Substantial CD34+CD117, cells, but not CD34+CD117+ cells, were stained immunocytochemically with anti-chymase mAb. For 1 week culture with rhSCF+rhIL-6, no cells expressed chymase in any preparation. Real-time RT-PCR revealed positivity for chymase mRNA in CD34+ cells, but it reduced at 1 week of culture, and increased as cells developed into MCs. Chymase mRNA in CD34+CD117+ cells was negligible compared with that in CD34+CD117,. Tryptase mRNA was below the detectable level in CD34+ cells, and increased along with MC differentiation. After 12 weeks of culture, CD34+CD117+ developed predominantly into MCs, whereas CD34+CD117, developed into monocytes/macrophages. Conclusion Our findings suggested that chymase is present not only in MCs but also in CD34+CD117, BM progenitors, but that its origin is different from the MC lineage. [source] The role of the cellular prion protein in the immune systemCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2006J. D. Isaacs Summary Prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrPC) is expressed widely in the immune system, in haematopoietic stem cells and mature lymphoid and myeloid compartments in addition to cells of the central nervous system. It is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocyte. Furthermore, antibody cross-linking of surface PrP modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signalling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Although PrP,/, mice have been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self-renewal of haematopoietic stem cells. Here, we consider the evidence for a distinctive role for PrPC in the immune system and what the effects of anti-prion therapeutics may be on immune function. [source] Angiotensin I-Converting Enzyme And Metabolism Of The Haematological Peptide N -Acetyl-Seryl-Aspartyl-Lysyl-ProlineCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2001Michel Azizi SUMMARY 1. Angiotensin I-converting enzyme (ACE) has two homologous active N- and C-terminal domains and displays activity towards a broad range of substrates. The tetrapeptide N -acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) has been shown to be hydrolysed in vitro by ACE and to be a preferential substrate for its N-terminal active site. This peptide reversibly prevents the recruitment of pluripotent haematopoietic stem cells and normal early progenitors into the S-phase. 2. Angiotensin I-converting enzyme inhibitors, given as a single dose to normal subjects or during long-term treatment in hypertensive patients, result in plasma AcSDKP levels five- to six-fold higher and urine concentrations 40-fold higher than those of control subjects and/or patients. Thus, AcSDKP is a natural peptide hydrolysed by the N-terminal domain of ACE in vivo. In addition, ACE may be implicated in the process of haematopoietic stem cell regulation by permanently degrading this natural circulating inhibitor of cell entry into the S-phase. 3. Besides hydrolysis by ACE, the second very effective mechanism by which AcSDKP is cleared from plasma is glomerular filtration. Because of its high sensitivity and specificity, the measurement of AcSDKP in plasma and urine provides a valuable tool in screening specific inhibitors of the N-terminal domain of ACE and in monitoring ACE inhibition during chronic treatment. 4. The long-term consequences of AcSDKP accumulation are not known. During chronic ACE inhibition in rats, AcSDKP levels slightly increase in organs with high ACE content (kidneys, lungs). To significantly increase its concentration in target haematopoietic organs (the extracellular fraction of bone marrow), AcSDKP has to be infused on top of a captopril-based treatment. 5. A selective inhibitor of the N-domain of ACE in vitro and in vivo has been identified recently. The phosphinic peptide RXP 407 does not interfere with blood pressure regulation, but does increase, dose dependently, plasma concentrations of AcSDKP in mice, in contrast with lisinopril, which affects the metabolism of both AcSDKP and angiotensin I. N-Terminal-selective ACE inhibitors may be used to selectively control AcSDKP metabolism in target haematopoietic organs. This new therapeutic strategy may be of value for protecting haematopoietic cells from the toxicity of cancer chemotherapy. [source] | |||||||||||||||||||||||||