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Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies. 相似文献
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《Cell communication & adhesion》2013,20(3):45-55
AbstractNatural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies. 相似文献
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In vitro generation of functional dendritic cells differentiated from CD34 negative cells isolated from human umbilical cord blood
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Yo Seph Park Changsik Shin Han Sung Hwang Martin Zenke Dong Wook Han Young Sun Kang Kisung Ko Yoonkyung Do Kinarm Ko 《Cell biology international》2015,39(9):1080-1086
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造血干细胞(HSCs)是血液系统中的一类成体干细胞群,具有自我更新和多谱系分化两个基本特征。造血干细胞移植(HSCT)可以治疗退行性疾病和多种血液系统疾病。脐带血来源造血干细胞(CB HSCs)是降低HLA配型要求的突破点,但单份脐带血中HSCs数量不能满足使用要求,为了获得足够数量的CB HSCs,体外扩增是一种可行的方法。近几年,学者们探索了多种体外扩增方法,包括优化细胞生长因子混合物、与基质细胞共培养及加入小分子化合物(SMCs)激动剂等。目前应用细胞因子联合小分子的扩增方法在多个临床试验中获得成功。本文对目前体外扩增CB HSCs的研究进展做一综述。 相似文献
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Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion 总被引:2,自引:0,他引:2
Li N Eljaafari A Bensoussan D Wang Y Latger-Cannard V Serrurier B Boura C Kennel A Stoltz J Feugier P 《Cytotherapy》2006,8(4):335-342
BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion. 相似文献
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BACKGROUND: CD133 is a newly developed hematopoietic stem cell marker but little is known about its function. Whether CD133(+) cell selection provides any advantage over CD34(+) selection for hematopoietic stem cell isolation and transplantation is unclear. The present study compared colony formation and endothelial cell differentiation of these two cell types from umbilical cord blood (UCB). METHODS: Mononuclear cells from the same UCB samples were used for both CD133(+) and CD34(+) cell selection. Cells with 97.1% purity were incubated in semi-solid culture medium containing stem cell growth factor (SCGF) and G-CSF or erythropoietin (EPO). Purified cells were also cultured in M199 containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). RESULTS: CD34(+) and CD133(+) cells produced similar numbers of CFU-GM colonies (median 43.25 and 30.5, respectively; P>0.2). However, a greater than four-fold difference in BFU-E colony formation was observed from CD34(+) cells compared with CD133(+) cells (median 35 and 8, respectively; P<0.04). CD34(+) cells gave rise to endothelial-like cells when stimulated with VEGF, bFGF and IGF-1. CD133(+) cells were unable produce this cell type under the same conditions. DISCUSSION: CD133(+) cells produced smaller BFU-E colonies and were unable to differentiate into mature endothelial cells. CD34(+) cells contained endothelial progenitors that could differentiate into mature cells of this lineage. Based on these data, it appears that CD133 offers no distinct advantage over CD34 as a selective marker for immunoaffinity-based isolation of hematopoietic stem cells and endothelial progenitor cells. 相似文献
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In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions. 相似文献
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Gibson JS Speake PF Muzyamba MC Husain F Luckas MC Ellory JC 《Biochimica et biophysica acta》2001,1512(2):231-238
The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease. 相似文献
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Blake JM Nicoud IB Weber D Voorhies H Guthrie KA Heimfeld S Delaney C 《Cytotherapy》2012,14(7):818-822
Background aimsCD34+ enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1–2 billion cells, raising a question regarding the optimal tubing set for CBU CD34+ enrichment. We compared CD34+ cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS.MethodsForty-six freshly collected CBU (≤ 36 h) were processed for CD34+ enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34+ cell content.ResultsTwo-sample t-tests of mean CD34+ recovery and viability revealed significant differences in favor of LS (CD34+ recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34+ purity, demonstrated statistically significant correlations only with the tubing set used and age of unit.ConclusionsFor CD34+ enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument. 相似文献
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Objectives: Ex vivo expansion is a feasible strategy, which may overcome limitation of the very low frequency of haematopoietic stem/progenitor cells, in umbilical cord blood (UCB). However, both quality of cells and safety of expanded population are critical issues to be addressed for their clinical application. Hence, in this study, we evaluated genetic stability of UCB‐derived CD34+ cells during ex vivo culture, based on karyotype analysis, as well as its effect on cell proliferation characteristics. Materials and methods: CD34+ cells were isolated from human UCB samples by immunomagnetic separation and were expanded ex vivo over a 28‐day period. Expansion of total nucleate cells, CD34+ cells and CD34+ CD38? cells was investigated. Karyotype analysis of the expanded cells from six randomly selected UCB samples was performed to evaluate their genetic stability. Results: Chromosomal abnormality of expanded cells mainly appeared by day 14, but was seldom sustained until day 28. None of the chromosomal abnormal samples displayed neoplastic proliferation, and expanded cells with altered chromosomes did not show obvious transformation phenomena according to soft agar assay. Conclusions: Ex vivo expansion could lead to occurrence of chromosomal abnormality, although here it did not produce excessive proliferative advantage of the expended cells. Importantly, chromosomal alteration seemed not to be inheritable and unlikely to result in malignant transformation. However, further in‐depth evaluation of potential clinical risks of chromosomal abnormality is warranted. 相似文献
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CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood. 总被引:6,自引:0,他引:6
Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants. 相似文献
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Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis. 相似文献
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Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood. 相似文献
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Background aimsCord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.MethodsThis is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34+ hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.ResultsOptimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.ConclusionsThis study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested. 相似文献
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Kamonnaree Chotinantakul Chavaboon Dechsukhum Duangnapa Dejjuy Wilairat Leeanansaksiri 《Cellular & molecular biology letters》2013,18(2):263-283
Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34+ cells (CB-CD34+ cells), freshly isolated CB-CD34+ cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34+ cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34+ cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34+ cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34+ cells and the cytokine cocktail were shown to promote recruitment of CD31+-endothelial cells in the wounds. Both the CB-CD34+ cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34+ cells or freshly isolated CB-CD34+ cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34+ cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34+ cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation. 相似文献
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Ueda Y Hagihara M Gansuvd B Yu Y Masui A Okamoto A Higuchi A Tazume K Kato S Hotta T 《Cancer immunology, immunotherapy : CII》2003,52(10):625-631
Purpose The first objective of this study was to investigate in vitro effects of -galactosylceramide (GalCer) on the proliferation of umbilical cord blood (UCB) natural killer T (NKT) cells and enhancement of their cytotoxicity. The second one is to examine whether purified NKT cells could affect the cytotoxicity of UCB-NK cells either in the presence or absence of dendritic cells (DCs).Methods Mononuclear cells (MNCs) from UCB were cultured for 2 weeks in the presence of IL-2 (100 U/ml), with or without GalCer. The effect of neutralizing monoclonal antibodies (MoAb) against TCRV24 and CD1d was also examined. TCRV24 V11 double positive NKT cells were purified by FACS sorter and then cocultured with syngeneic isolated UCB–CD56+NK cells in either the presence or absence of DCs. The cytotoxicity against various malignant cell targets and cytokine production was determined.Results The addition of GalCer induced human NKT cells to proliferate in UCB-MNCs to a greater extent than in adult PB-MNCs. However, it suppressed the cytotoxic activity against malignant cell targets. Anti-TCRV24 and CD1d MoAb recovered the cytotoxicity by inhibiting the proliferation of UCB-NKT cells. NKT cells cocultured with auto-DCs significantly increased NK cell cytotoxicity against K562, and Raji cells and produced IFN- at much higher levels than UCB-NKT cells alone.Conclusion In UCB samples, GalCer–pulsed DCs and NKT cells acted together to enhance NK cytotoxicity in vitro. 相似文献