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Sample records for human cd34 positive

  1. CD34-positive interstitial cells of the human detrusor

    DEFF Research Database (Denmark)

    Rasmussen, Helle; Hansen, Alastair; Smedts, Frank;

    2007-01-01

    Interstitial cells of Cajal (ICC) are well described in the bowel wall. They are c-kit positive and play a role as pacemaker cells. Similar c-kit-positive cells have recently been described in the human bladder. The aim of this study was to characterize interstitial cells of the bladder detrusor...... using a panel of antibodies directed against CD117/c-kit, CD34, CD31, S100, tryptase, neurofilament, NSE, Factor-VIII and GFAP. A striking finding was an interstitial type of cell which is CD34 immunoreactive (CD34-ir) but CD117/c-kit negative. The cells have a tentacular morphology, enveloping...... and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  2. Human Umbilical Cord Blood CD34-Positive Cells as Predictors of the Incidence and Short-Term Outcome of Neonatal Hypoxic-Ischemic Encephalopathy: A Pilot Study

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    Nasr Eldin, Mohamed Hassan; Amer, Hanaa A.; Abdelhamid, Adel E.; El Houssinie, Moustafa; Ibrahim, Abir

    2017-01-01

    Background and Purpose Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of neurological handicap in developing countries. Human umbilical cord blood (hUCB) CD34-positive (CD34+) stem cells exhibit the potential for neural repair. We tested the hypothesis that hUCB CD34+ stem cells and other cell types [leukocytes and nucleated red blood cells (NRBCs)] that are up-regulated during the acute stage of perinatal asphyxia (PA) could play a role in the early prediction of the occurrence, severity, and mortality of HIE. Methods This case-control pilot study investigated consecutive neonates exposed to PA. The hUCB CD34+ cell count in mononuclear layers was assayed using a flow cytometer. Twenty full-term neonates with PA and 25 healthy neonates were enrolled in the study. Results The absolute CD34+ cell count (p=0.02) and the relative CD34+ cell count (CD34+%) (p<0.001) in hUCB were higher in the HIE patients (n=20) than the healthy controls. The hUCB absolute CD34+ cell count (p=0.04), CD34+% (p<0.01), and Hobel risk scores (p=0.04) were higher in patients with moderate-to-severe HIE (n=9) than in those with mild HIE (n=11). The absolute CD34+ cell count was strongly correlated with CD34+% (p<0.001), Hobel risk score (p=0.04), total leukocyte count (TLC) (p<0.001), and NRBC count (p=0.01). CD34+% was correlated with TLC (p=0.02). Conclusions hUCB CD34+ cells can be used to predict the occurrence, severity, and mortality of neonatal HIE after PA. PMID:28079317

  3. Vasoprotective effects of human CD34+ cells: towards clinical applications

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    Lerman Amir

    2009-07-01

    Full Text Available Abstract Background The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury. Methods Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later. Results Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 ± 0.18, and 1.71 ± 0.18 for CD34, and saline-treated vessels, respectively P Conclusion Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.

  4. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

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    Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

    2012-08-01

    The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

  5. Loss of CD34 expression in aging human choriocapillaris endothelial cells.

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    Elliott H Sohn

    Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

  6. Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker

    Institute of Scientific and Technical Information of China (English)

    Parveen Nyamath; Ayesha AM; Aejaz Habeeb; Sanjeev Khosla; Aleem A Khan; CM Habibullah

    2007-01-01

    AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker.METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation.RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture.CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

  7. Decitabine suspends human CD34+ cell differentiation and proliferation during lentiviral transduction.

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    Uchida, Naoya; Hsieh, Matthew M; Platner, Charlotte; Saunthararajah, Yogen; Tisdale, John F

    2014-01-01

    Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20-24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.

  8. Decitabine suspends human CD34+ cell differentiation and proliferation during lentiviral transduction.

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    Naoya Uchida

    Full Text Available Efficient ex vivo transduction of hematopoietic stem cells (HSCs is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20-24 weeks after transplantation, compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.

  9. Circulating human CD34(+) progenitor cells modulate neovascularization and inflammation in a nude mouse model

    NARCIS (Netherlands)

    van der Strate, B. W. A.; Popa, E. R.; Schipper, M.; Brouwer, L. A.; Hendriks, M.; Harmsen, M. C.; van Luyn, M. J. A.

    2007-01-01

    CD34(+) progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34(+) cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34(+) cells were incorporated in Matrigel,

  10. Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

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    Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

    2011-02-25

    Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature

  11. Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

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    Laurie Chicha

    Full Text Available BACKGROUND: Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC and induced pluripotent stem cells (iPSC into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. METHODOLOGY/PRINCIPAL FINDINGS: ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB CD34(+ cells. ESC-derived CD34(+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(- cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. CONCLUSIONS/SIGNIFICANCE: Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and i

  12. Loss of CD117 (c-kit)- and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction.

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    Streutker, C J; Huizinga, J D; Campbell, F; Ho, J; Riddell, R H

    2003-02-01

    Chronic idiopathic intestinal pseudo-obstruction is a syndrome in which symptoms of intestinal obstruction are present in the absence of mechanical obstruction. Lack of normal pacemaker activity, usually generated by the interstitial cells of Cajal (ICC), could account for the apparent obstruction. ICC are normally located around and between the myenteric plexus ganglia and within muscle and also in the deep muscular plexus of the small bowel and the submuscular plexus of the large intestine, just within the circular muscle. ICC can be demonstrated immunohistochemically with CD117 (c-kit) as well as with CD34, although this is less specific. CD34 also stains a population of fibroblasts that are intimately associated with ICC. To determine whether there is a relative deficiency of ICC and CD34-positive fibroblasts in patients with chronic idiopathic intestinal pseudo-obstruction, tissue from 30 patients of large intestine and eight patients with small intestine pseudo-obstruction was obtained. Controls (large intestinal specimens from 12 patients, small intestinal specimens from six patients) were chosen from resections for Crohn's disease and colorectal neoplasia, both with and without dilatation. Examination of pseudo-obstruction cases identified 10 patients (nine large intestinal and one small intestinal) in which both CD117 and CD34 were absent or severely reduced in all three of the examined areas. In contrast, the control cases, including those with preobstructive dilatation, showed relatively constant ICC staining. These results suggest that there is a proportion of pseudo-obstruction cases in which the ICC are markedly reduced. These results also demonstrate that, in these cases, loss of the kit immunoreactivity is correlated with the loss of CD34 staining: this indicates that both the ICC and the CD34-positive fibroblasts associated with the ICC are absent. These findings will allow surgical pathologists to identify this subpopulation of patients with CIIP

  13. CD34 immunoreactivity and interstitial cells of Cajal in the human and mouse gastrointestinal tract

    DEFF Research Database (Denmark)

    Vanderwinden, J M; Rumessen, J J; De Laet, M H;

    2000-01-01

    Immunoreactivity for the tyrosine kinase receptor Kit (Kit-ir) is an established marker for the interstitial cells of Cajal (ICC) of the gut. Recently, the presence of CD34 immunoreactivity (CD34-ir) has been reported in Kit-ir ICC around the myenteric plexus in human small intestine. Conversely,......-localization. The ontogeny and function of CD34-ir cells in the gut, as well as the origin of gastrointestinal stromal tumors, remain unclear....

  14. Circulating CD34-positive cells, glomerular filtration rate and triglycerides in relation to hypertension.

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    Shimizu, Yuji; Sato, Shimpei; Koyamatsu, Jun; Yamanashi, Hirotomo; Nagayoshi, Mako; Kadota, Koichiro; Maeda, Takahiro

    2015-11-01

    Serum triglycerides have been reported to be independently associated with the development of chronic kidney disease (CKD), which is known to play a role in vascular disturbance. On the other hand, circulating CD34-positve cells, including endothelial progenitor cells, are reported to contribute to vascular repair. However, no studies have reported on the correlation between triglycerides and the number of CD34-positive cells. Since hypertension is well known factor for vascular impairment, the degree of correlation between serum triglycerides and circulating CD34-positve cells should account for hypertension status. We conducted a cross-sectional study of 274 elderly Japanese men aged ≥ 60 years (range 60-79 years) undergoing general health checkups. Multiple linear regression analysis of non-hypertensive subjects adjusting for classical cardiovascular risk factors showed that although triglyceride levels (1SD increments; 64 mg/dL) did not significantly correlate with glomerular filtration rate (GFR) (β = -2.06, p = 0.163), a significant positive correlation was seen between triglycerides and the number of circulating CD34-positive cells (β = 0.50, p = 0.004). In hypertensive subjects, a significant inverse correlation between triglycerides and GFR was observed (β = -2.66, p = 0.035), whereas no significant correlation between triglycerides and the number of circulating CD34-positive cells was noted (β = -0.004, p = 0.974). Since endothelial progenitor cells (CD34-positive cells) have been reported to contribute to vascular repair, our results indicate that in non-hypertensive subjects, triglycerides may stimulate an increase in circulating CD34-positive cells (vascular repair) by inducing vascular disturbance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Effect of Weight Reduction on Cardiovascular Risk Factors and CD34-positive Cells in Circulation

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    Nina A Mikirova, Joseph J Casciari, Ronald E Hunninghake, Margaret M Beezley

    2011-01-01

    Full Text Available Being overweight or obese is associated with an increased risk for the development of non-insulin-dependent diabetes mellitus, hypertension, and cardiovascular disease. Dyslipidemia of obesity is characterized by elevated fasting triglycerides and decreased high-density lipoprotein-cholesterol concentrations. Endothelial damage and dysfunction is considered to be a major underlying mechanism for the elevated cardiovascular risk associated with increased adiposity. Alterations in endothelial cells and stem/endothelial progenitor cell function associated with overweight and obesity predispose to atherosclerosis and thrombosis.In our study, we analyzed the effect of a low calorie diet in combination with oral supplementation by vitamins, minerals, probiotics and human chorionic gonadotropin (hCG, 125-180 IUs on the body composition, lipid profile and CD34-positive cells in circulation.During this dieting program, the following parameters were assessed weekly for all participants: fat free mass, body fat, BMI, extracellular/intracellular water, total body water and basal metabolic rate. For part of participants blood chemistry parameters and circulating CD34-positive cells were determined before and after dieting.The data indicated that the treatments not only reduced body fat mass and total mass but also improved the lipid profile. The changes in body composition correlated with the level of lipoproteins responsible for the increased cardiovascular risk factors. These changes in body composition and lipid profile parameters coincided with the improvement of circulatory progenitor cell numbers.As the result of our study, we concluded that the improvement of body composition affects the number of stem/progenitor cells in circulation.

  16. Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

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    Miyazaki, T; Matsuda, I; Oguri, M; Amaya, H; Kiyosaki, M; Hamada, A; Tamaki, S; Tashiro, E; Kudo, Y; Taniguchi, O; Nakamura, T; Tomoyasu, S

    2001-01-01

    We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

  17. Low oxygen tension favored expansion and hematopoietic reconstitution of CD34(+) CD38(-) cells expanded from human cord blood-derived CD34(+) Cells.

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    Wang, Ziyan; Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Fan, Jinli; Tan, Wen-Song

    2016-07-01

    Oxygen tension is an important factor that regulates hematopoietic stem cells (HSCs) in both in vivo hematopoietic microenvironment and ex vivo culture system. Although the effect of oxygen tension on ex vivo expansion of HSCs was extensively studied, there were no clear descriptions on physiological function and gene expression analysis of HSCs under different oxygen tensions. In this study, the effects of oxygen tension on ex vivo expansion characteristics of human umbilical cord blood (UCB)-derived CD34(+) cells are evaluated. Moreover, the physiological function of expanded CD34(+) cells was assessed by secondary expansion ability ex vivo and hematopoietic reconstitution ability in vivo. Also, genetic profiling was applied to analyze the expression of genes related to cell function. It was found that low oxygen tension favored expansion of CD34(+) CD38(-) cells. Additionally, CD34(+) cells expanded under low oxygen tension showed better secondary expansion ability and reconstitution ability than those under atmospheric oxygen concentration. Finally, the genetic profiling of CD34(+) CD38(-) cells cultured under low oxygen tension was more akin to freshly isolated cells. These results collectively demonstrate that low oxygen tension was able to better maintain both self-renewal and hematopoietic reconstitution potential and may lay an experimental basis for clinical transplantation of HSCs.

  18. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

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    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  19. SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development

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    Rhys J.P. Skelton

    2014-07-01

    Full Text Available The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5GFP was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5GFP/w human embryonic stem cells (hESCs. Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5posCD34pos population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5posSIRPApos to NKX2-5posSIRPAposVCAM1pos. Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.

  20. In vitro differentiation of cultured human CD34+ cells into astrocytes

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    Katari Venkatesh

    2013-01-01

    Full Text Available Background: Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. Aim: The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. Materials and Methods: Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco′s modified eagle′s medium. Thus, cultured cells were induced with astrocyte defined medium (ADM and in the differentiated astrocytes serine/threonine protein kinases (STPK and glutamine synthetase (GLUL activities were estimated. The expression of glial fibrillary acidic protein (GFAP and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR. Results: The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb and GFAP (2.9 kb in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. Conclusion: In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

  1. In vitro transdifferentiation of human cultured CD34+ stem cells into oligodendrocyte precursors using thyroid hormones.

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    Venkatesh, Katari; Srikanth, Lokanathan; Vengamma, Bhuma; Chandrasekhar, Chodimella; Prasad, Bodapati Chandra Mouleshwara; Sarma, Potukuchi Venkata Gurunadha Krishna

    2015-02-19

    The extent of myelination on the axon promotes transmission of impulses in the neural network, any disturbances in this process results in the neurodegenerative condition. Transplantation of oligodendrocyte precursors that supports in the regeneration of axons through myelination is an important step in the restoration of damaged neurons. Therefore, in the present study, the differentiation of human CD34+ stem cells into oligodendrocytes was carried out. The pure human CD34+ culture developed from the stem cells obtained from a peripheral blood of a donor were subjected to oligodendrocyte differentiation medium (ODM). The ODM at a concentration of 40ng/ml thyroxine, 40ng/ml 3,3',5-tri-iodo-thyronine showed distinct morphological changes from day 6 to 9 with cells exhibiting conspicuous stellate morphology and extensive foot processes. The real-time PCR analysis showed prominent expression of Olig2, CNPase, PDGFRα and PLP1/DM20 in the differentiated cells confirming the formed cells are oligodendrocyte precursors. The expression of these genes increased from days 6 to 9 corresponding to the morphological changes observed with almost no expression of GFAP+ cells. The distinct CNPase activity was observed in these differentiated cells compared to normal CD34+ stem cells correlating with results of real-time PCR conclusively explains the development of oligodendrocytes from human CD34+ stem cells.

  2. CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors

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    Jerusa Martins Carvalho

    Full Text Available CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6 CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.

  3. Effect of intracranial transplantation of CD34+ cells derived from human umbilical cord blood in rats with cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    LIU Hai-ying; ZHANG Qing-jun; LI Hong-jun; HAN Zhong-chao

    2006-01-01

    @@ As a source of transplantable stem cells, the CD34+ subpopulation in human umbilical cord blood (HUCB) has been used extensively to treat some hematopoietic system diseases. However,whether CD34+ cells hold the therapeutic potential to cerebral ischemia is unknown. The purpose of this study was to observe the recovery of neural function after transplantation of CD34+ cells derived from HUCB into ischemic cerebral tissue in rats.

  4. Growth and differentiation of eosinophils from human peripheral blood CD 34+ cells.

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    Shalit, M

    1997-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte and neutrophil colonies. IL5 alone did not support colony growth. In contrast GM-CSF and IL3 alone or together supported the generation of more than 50% eosinophil colonies. Addition of IL5 increased the fraction of eosinophil colonies to over 70%. Under the best conditions (IL3 + GM-CSF + IL5), the addition of interferon-a or LPS inhibited colony growth by 51% and 58%, respectively. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5 responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF, washed, and plated with IL5 alone. Only when progenitors were grown at least 3 days, could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/104 cells plated at day 3 and 134 colonies/104 cells at day 7). Growth of CD34+ in liquid culture for 28 days in the presence of IL3, GM-CSF and IL5 resulted in almost 250 fold increase in cell number, yielding a population of 83% maturing eosinophils. We used our culture system and the sensitive technique of RT-PCR to analyze the kinetics of production of mRNA transcripts encoding several eosinophil proteins. Freshly isolated CD34+ cells contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. At day 3 of culture no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased

  5. Selective in vitro expansion and efficient retroviral transduction of human CD34(+) CD38(-) haematopoietic stem cells

    NARCIS (Netherlands)

    Ng, YY; Bloem, AC; van Kessel, B; Lokhorst, H; Logtenberg, T; Staal, FJT

    2002-01-01

    Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34(+) CD38(-) cells from purified CD34(+) cells upon stimulation with Flt3-ligand, stem cell factor and thromb

  6. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells.

    Science.gov (United States)

    Sunitha, Manne Mudhu; Srikanth, Lokanathan; Santhosh Kumar, Pasupuleti; Chandrasekhar, Chodimella; Sarma, Potukuchi Venkata Gurunadha Krishna

    2016-10-01

    Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.

  7. Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

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    Vêncio Ricardo Z

    2007-06-01

    Full Text Available Abstract Background Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer. Methods CD34+/CD133+ progenitor cells were purified from human umbilical cord blood and expanded in vitro. Correlated molecular changes were analyzed by gene expression profiling using microarrays covering up to 55,000 transcripts. Genes regulated during progenitor cell expansion were identified and functionally classified. Aberrant expression of such genes in cancer was indicated by in silico SAGE. Differential expression of selected genes was assessed by real-time PCR in hematopoietic cells from chronic myeloid leukemia patients and healthy individuals. Results Several genes and signaling pathways not previously associated with ex vivo expansion of CD133+/CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes was detected during conditions of enhanced progenitor cell expansion. Quantitative real-time PCR analysis confirmed down-regulation of several newly described cancer-associated genes in CD133+/CD34+ cells, including DOCK4 and SPARCL1 tumor suppressors, and parallel results were verified when comparing their expression in cells from chronic myeloid leukemia patients Conclusion Our findings reveal potential molecular targets for oncogenic transformation in CD133+/CD34+ cells and strengthen the link between deregulation of stem/progenitor cell expansion and the malignant process.

  8. CD34(-) cells at the apex of the human hematopoietic stem cell hierarchy have distinctive cellular and molecular signatures.

    Science.gov (United States)

    Anjos-Afonso, Fernando; Currie, Erin; Palmer, Hector G; Foster, Katie E; Taussig, David C; Bonnet, Dominique

    2013-08-01

    In addition to well-characterized CD34(+) hematopoietic stem and progenitor cells (HSPCs), the human hematopoietic stem cell (HSC) hierarchy contains a rare CD34(-) population with severe combined immunodeficiency-repopulating capacity. However, little is known about the molecular characteristics of these CD34(-) cells or their relationship to the CD34(+) populations. Here, we show that the self-renewing Lin(-)CD34(-)CD38(-)CD93(hi) population contains cells that not only function as HSCs, but can also be placed above the CD34(+) populations in the hematopoietic hierarchy. These cells have an active Notch pathway, in which signaling through Delta4 is crucial for maintenance of the primitive state, and combined signals from Jagged1 and TGF-β are important in controlling its quiescence. They are also refractory to proliferative signals and show a repressed canonical Wnt pathway, in part regulated by Notch. Overall, therefore, CD34(-) cells represent an immature and quiescent human HSC population maintained through a distinctive network of cellular signaling interactions.

  9. Intrathecal Administration of Autologous CD34 Positive Cells in Patients with Past Cerebral Infarction: A Safety Study

    Science.gov (United States)

    Wang, Liming; Ji, Haijie; Li, Ming; Zhou, Jianjun; Bai, Wen; Zhong, Zhanqiang; Li, Na; Zhu, Delin; Zhang, Zijia; Liu, Yongjun; Wu, Mingyuan

    2013-01-01

    Regenerative strategies in treatment of stroke have great potential. The goal of the current study was to investigate safety of intrathecal administration of autologous CD34 positive cells in treatment of patients with poststroke. A total of eight male patients with a history of stroke were enrolled. The patients were treated subcutaneously with 5 μg/kg body weight rhG-CSF for 5 consecutive days, and then leukapheresis was performed to concentrate cells for CD34 positive immunoselection. All patients underwent intrathecal administration of CD34 positive cells via lumbar puncture. The primary outcome was safety evaluation for 12-month followup. In addition, behavioral function was evaluated with NIH stroke scale and Barthel index 1, 6, and 12 months after the last treatment, respectively. There were no major adverse events, and abnormal changes of blood tests during the whole treatment process included intrathecal administration and 12-month followup. The main message from the current study was that administration of G-CSF-mobilized autologous CD34 positive cells in patients with poststroke was safe. Future studies with larger population and control group are needed to confirm the safety and investigate the efficacy. PMID:24187628

  10. Growth Induction and Low-Oxygen Apoptosis Inhibition of Human CD34+ Progenitors in Collagen Gels

    Directory of Open Access Journals (Sweden)

    Daniele Avitabile

    2013-01-01

    Full Text Available Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment. In the present contribution, we show that embedding human cord blood-derived CD34+ cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.

  11. Liposome-mediated Functional Expression of Multiple Drug Resistance Gene in Human Bone Marrow CD34+ Cells

    Institute of Scientific and Technical Information of China (English)

    曹文静; 邹萍

    2004-01-01

    Summary: The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34+ cells was observed. Human normal bone marrow CD34+ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34+ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34 + cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34+ cells was obviously higher than that in non-transfected CD34+ cells. The amount of P-gp in non-transfected CD34+ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34 + cells.

  12. Mobilization of human CD34+ CD133+ and CD34+ CD133(-) stem cells in vivo by consumption of an extract from Aphanizomenon flos-aquae--related to modulation of CXCR4 expression by an L-selectin ligand?

    Science.gov (United States)

    Jensen, Gitte S; Hart, Aaron N; Zaske, Lue A M; Drapeau, Christian; Gupta, Niraj; Schaeffer, David J; Cruickshank, J Alex

    2007-01-01

    The goal of this study was to evaluate effects on human stem cells in vitro and in vivo of an extract from the edible cyanobacterium Aphanizomenon flos-aquae (AFA) enriched for a novel ligand for human CD62L (L-selectin). Ligands for CD62L provide a mechanism for stem cell mobilization in conjunction with down-regulation of the CXCR4 chemokine receptor for stromal derived factor 1. Affinity immunoprecipitation was used to identify a novel ligand for CD62L from a water extract from AFA. The effects of AFA water extract on CD62L binding and CXCR4 expression was tested in vitro using human bone marrow CD34+ cells and the two progenitor cell lines, KG1a and K562. A double-blind randomized crossover study involving 12 healthy subjects evaluated the effects of consumption on stem cell mobilization in vivo. An AFA extract rich in the CD62L ligand reduced the fucoidan-mediated externalization of the CXCR4 chemokine receptor on bone marrow CD34+ cells by 30% and the CD62L+ CD34+ cell line KG1A by 50% but did not alter the CXCR4 expression levels on the CD34(-) cell line K562. A transient, 18% increase in numbers of circulating CD34+ stem cells maximized 1 hour after consumption (P<.0003). When 3 noncompliant volunteers were removed from analysis, the increase in CD34+ cells was 25% (P<.0001). AFA water extract contains a novel ligand for CD62L. It modulates CXCR4 expression on CD34+ bone marrow cells in vitro and triggers the mobilization of CD34+ CD133+ and CD34+ CD133(-) cells in vivo.

  13. Pericardial patch angioplasty heals via an Ephrin-B2 and CD34 positive cell mediated mechanism.

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    Xin Li

    Full Text Available OBJECTIVE: Pericardial patches are commonly used in vascular surgery to close arteriotomies. The mechanism of early healing after patch implantation is still not well defined. We used a rat aortic patch model to assess pericardial patch healing and examined Ephrin-B2, a marker of arterial identity, expression within the post-implantation patch. We also determined whether endothelial progenitor cells (EPC are associated with early patch healing in the arterial environment. METHODS: Wistar rats (200-250 grams underwent infrarenal aortic arteriotomy and then closure via bovine or porcine pericardial patch angioplasty. Control groups included subcutaneously implanted patches. Patches were harvested at 0-30 days and analyzed by histology, immunohistochemistry, immunofluorescence and Western blot as well as quantitative PCR. RESULTS: Prior to implantation, pericardial patches are largely composed of collagen and are acellular. Following arterial implantation, increasing numbers of CD68-positive cells as well as Ephrin-B2 and CD34 dual-positive cells are found within both bovine and porcine pericardial patches, whereas the infiltrating cells are negative for vWF and α-actin. Porcine patches have a luminal monolayer of cells at day 7, compared to bovine patches that have fewer luminal cells. Subcutaneously implanted patches do not attract Ephrin-B2/CD34-positive cells. By day 30, both bovine and porcine pericardial patches develop a neointima that contains Ephrin-B2, CD34, and VEGFR2-positive cells. CONCLUSION: Both CD68-positive and Ephrin-B2 and CD34 dual-positive cells infiltrate the pericardial patch early after implantation. Arteriotomy closure via pericardial patch angioplasty shows patch adaptation to the arterial environment that may involve a foreign body response as well as localization of EPC. Arterial remodeling of pericardial patches support endothelialization and may represent a paradigm of healing of scaffolds used for tissue engineering.

  14. Angptl4 maintains in vivo repopulation capacity of CD34+ human cord blood cells.

    Science.gov (United States)

    Blank, Ulrika; Ehrnström, Birgitta; Heinz, Niels; Nilsson, Eva; Brun, Ann; Baum, Christopher; Schiedlmeier, Bernhard; Karlsson, Stefan

    2012-09-01

    Methods to expand hematopoietic stem cells (HSCs) ex vivo encompass an attractive approach that would substantially broaden the clinical applicability of HSCs derived from cord blood (CB). Recently, members of the angiopoietin-like (Angptl) family of growth factors were shown to expand both murine and human HSCs. Specifically, Angptl5 has been implicated in the expansion of human NOD/SCID-repopulating cells (SRCs) ex vivo. Here, we sought to evaluate the potential of additional Angptls to expand human SRCs from CB. Additionally, the purpose of this study was to evaluate the reproducibility of Angptl-mediated expansion of SRCs across independent experiments. Human CD34(+) cells from CB were cultured in vitro for eleven or 8 d in the presence or absence of Angptls. The reconstitution capacity of expanded cells was subsequently measured in vivo by transplantation into NOD/SCID or NSG mice and compared with that of uncultured cells. We report here that Angptl4 functions to maintain SRC activity of CD34(+) CB-derived cells ex vivo as assayed in NOD/SCID and NSG mice. However, all Angptls tested, including Angptl1, Angptl4, and Angptl5, were associated with variation between experiments. Our findings indicate that Angptl4 and Angptl5 can lead to increased engraftment capacity of SRCs, but more frequently, these factors are associated with maintenance of SRC activity during ex vivo culture. Thus, Angptl-mediated expansion of SRCs ex vivo is associated with more interexperimental variation than previously thought. We conclude that Angptls would be useful in instances where there is a need to maintain HSCs ex vivo, such as during transduction for gene therapy applications. © 2012 John Wiley & Sons A/S.

  15. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    YiZhanga; ChangdongLi; XiaoxiaJiang; ShuangxiZhang; YingWu; BingLiu; PeihsienTang; NingMao

    2005-01-01

    Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ ceils from UCB in vitro.Materials and Methods. Human placenta MPC was isolated and characterized by morphologic,immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculturewith placeutal MPC. Suitable aliquots of cells were used to monitor cell production, elonogenie activity, and tong-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay.Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin,and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and α-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes.In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of eytokines, was significantly enhanced: CD34+ cells by 14.89±2.32 fold; colonyforming cell (CFC) by 36.73±5.79 told; and LTC-IC by 7.43±2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli.Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.

  16. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.

    Science.gov (United States)

    Sumiyoshi, Teiko; Holt, Nathalia G; Hollis, Roger P; Ge, Shundi; Cannon, Paula M; Crooks, Gay M; Kohn, Donald B

    2009-12-01

    Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell

  17. Hypocellular Plaque-Like CD34-Positive Dermal Fibroma (Medallion-Like Dermal Dendrocyte Hamartoma) Presenting as a Skin-Colored Dermal Nodule.

    Science.gov (United States)

    Mutgi, Krishna A J; Chitgopeker, Pooja; Ciliberto, Heather; Stone, Mary S

    2016-01-01

    Plaque-like CD34-positive dermal fibromas, also known as medallion-like dermal dendrocyte hamartomas (MDDHs), are a recently recognized group of congenital and acquired spindle cell neoplasms that may appear histologically similar to dermatofibrosarcoma protuberans. Recognizing the clinical heterogeneity of this neoplasm and the subtle pathologic differences are crucial to making the correct diagnosis and avoiding the aggressive surgical intervention required to treat a dermatofibrosarcoma protuberans. We present the case of a 9-year-old girl with an acquired variant of a plaque-like CD34-positive dermal fibroma without clinical epidermal change. Our case expands the clinical spectrum to include an acquired variant of a plaque-like CD34-positive dermal fibroma without clinical epidermal change. Examination of more cases is needed to determine whether all clinical variants are truly subtypes of the same neoplasm or represent distinct CD34-positive spindle cell proliferations.

  18. Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Honglian Jin

    2014-01-01

    Full Text Available Red blood cell (RBC supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.

  19. Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors.

    Science.gov (United States)

    Shalit, M; Sekhsaria, S; Malech, H L

    1995-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5

  20. Local transplantation of ex vivo expanded bone marrow-derived CD34-positive cells accelerates fracture healing.

    Science.gov (United States)

    Kawakami, Yohei; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Matsumoto, Tomoyuki; Kuroda, Ryosuke; Shoji, Taro; Fukui, Tomoaki; Masuda, Haruchika; Akimaru, Hiroshi; Mifune, Yutaka; Kuroda, Tomoya; Horii, Miki; Yokoyama, Ayumi; Kurosaka, Masahiro; Asahara, Takayuki

    2012-01-01

    Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

  1. In Vitro Generation of Human XCR1(+) Dendritic Cells from CD34(+) Hematopoietic Progenitors.

    Science.gov (United States)

    Balan, Sreekumar; Dalod, Marc

    2016-01-01

    Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells which play a key role in orchestrating immune defenses. Most of the information gained on human DC biology was derived from studies conducted with DCs generated in vitro from peripheral blood CD14(+) monocytes (MoDCs) or from CD34(+) hematopoietic progenitors. Recent advances in the field revealed that these types of in vitro-derived DCs strikingly differ from the DC subsets that are naturally present in human lymphoid organs, in terms of global gene expression, of specialization in the sensing of different types of danger signals, and of the ability to polarize T lymphocytes toward different functions. Major efforts are being made to better characterize the biology and the functions of lymphoid organ-resident DC subsets in humans, as an essential step for designing innovative DC-based vaccines against infections or cancers. However, this line of research is hampered by the low frequency of certain DC subsets in most tissues, their fragility, and the complexity of the procedures necessary for their purification. Hence, there is a need for robust procedures allowing large-scale in vitro generation of human DC subsets, under conditions allowing their genetic or pharmacological manipulation, to decipher their functions and their molecular regulation. Human CD141(+)CLEC9A(+)XCR1(+) DCs constitute a very interesting DC subset for the design of immunotherapeutic treatments against infections by intracellular pathogens or against cancer, because these cells resemble mouse professional cross-presenting CD8α(+)Clec9a(+)Xcr1(+) DCs. Human XCR1(+) DCs have indeed been reported by several teams to be more efficient than other human DC subsets for cross-presentation, in particular of cell-associated antigens but also of soluble antigens especially when delivered into late endosomes or lysosomes. However, human XCR1(+) DCs are the rarest and perhaps the most fragile of the human DC

  2. Identification of Cancer Stem Cell Subpopulations of CD34+ PLC/PRF/5 That Result in Three Types of Human Liver Carcinomas

    Science.gov (United States)

    Park, Su Cheol; Nguyen, Ngoc Tue; Eun, Jong Ryeol; Zhang, Yanling; Jung, Yong Jin; Tschudy-Seney, Benjamin; Trotsyuk, Artem; Lam, Alexander; Ramsamooj, Rajendra; Zhang, Yanghong; Theise, Neil D.; Zern, Mark A.

    2015-01-01

    CD34+ stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34+ stem cells. Here, we determined that a population of CD34+ cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSCs) by forming HLCs in immunodeficient mice with as few as 100 cells. Moreover, the CD34+ PLC subpopulation cells had an advantage over CD34− PLCs at initiating tumors. Three types of HLCs were generated from CD34+ PLC: hepatocellular carcinomas (HCCs); cholangiocarcinomas (CC); and combined hepatocellular cholangiocarcinomas (CHCs). Tumors formed in mice transplanted with 12 subpopulations and 6 progeny subpopulations of CD34+ PLC cells. Interestingly, progenies with certain surface antigens (CD133, CD44, CD90, or EPCAM) predominantly yielded HCCs. CD34+ PLCs that also expressed OV6 and their progeny OV6+ cells primarily produced CHC and CC. This represents the first experiment to demonstrate that the OV6+ antigen is associated with human CHC and CC. CD34+ PLCs that also expressed CD31 and their progeny CD31+ cells formed CHCs. Gene expression patterns and tumor cell populations from all xenografts exhibited diverse patterns, indicating that tumor-initiating cells (TICs) with distinct antigenic profiles contribute to cancer cell heterogeneity. Therefore, we identified CD34+ PLC cells functioning as LCSCs generating three types of HLCs. Eighteen subpopulations from one origin had the capacity independently to initiate tumors, thus functioning as TICs. This finding has broad implications for better understanding of the multistep model of tumor initiation and progression. Our finding also indicates that CD34+ PLCs that also express OV6 or CD31 result in types of HLCs. This is the first report that PLC/PRF/5 subpopulations expressing CD34 in combination with particular antigens defines categories of HLCs, implicating a

  3. Identification of cancer stem cell subpopulations of CD34(+) PLC/PRF/5 that result in three types of human liver carcinomas.

    Science.gov (United States)

    Park, Su Cheol; Nguyen, Ngoc Tue; Eun, Jong Ryeol; Zhang, Yanling; Jung, Yong Jin; Tschudy-Seney, Benjamin; Trotsyuk, Artem; Lam, Alexander; Ramsamooj, Rajendra; Zhang, Yanghong; Theise, Neil D; Zern, Mark A; Duan, Yuyou

    2015-04-15

    CD34(+) stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34(+) stem cells. Here, we determined that a population of CD34(+) cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSCs) by forming HLCs in immunodeficient mice with as few as 100 cells. Moreover, the CD34(+) PLC subpopulation cells had an advantage over CD34(-) PLCs at initiating tumors. Three types of HLCs were generated from CD34(+) PLC: hepatocellular carcinomas (HCCs); cholangiocarcinomas (CC); and combined hepatocellular cholangiocarcinomas (CHCs). Tumors formed in mice transplanted with 12 subpopulations and 6 progeny subpopulations of CD34(+) PLC cells. Interestingly, progenies with certain surface antigens (CD133, CD44, CD90, or EPCAM) predominantly yielded HCCs. CD34(+) PLCs that also expressed OV6 and their progeny OV6(+) cells primarily produced CHC and CC. This represents the first experiment to demonstrate that the OV6(+) antigen is associated with human CHC and CC. CD34(+) PLCs that also expressed CD31 and their progeny CD31(+) cells formed CHCs. Gene expression patterns and tumor cell populations from all xenografts exhibited diverse patterns, indicating that tumor-initiating cells (TICs) with distinct antigenic profiles contribute to cancer cell heterogeneity. Therefore, we identified CD34(+) PLC cells functioning as LCSCs generating three types of HLCs. Eighteen subpopulations from one origin had the capacity independently to initiate tumors, thus functioning as TICs. This finding has broad implications for better understanding of the multistep model of tumor initiation and progression. Our finding also indicates that CD34(+) PLCs that also express OV6 or CD31 result in types of HLCs. This is the first report that PLC/PRF/5 subpopulations expressing CD34 in combination with particular antigens defines categories of

  4. CD34-positive interstitial cells of the human detrusor

    DEFF Research Database (Denmark)

    Rasmussen, Helle; Hansen, Alastair; Smedts, Frank;

    2007-01-01

    and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  5. CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

    Science.gov (United States)

    Hoban, Megan D; Lumaquin, Dianne; Kuo, Caroline Y; Romero, Zulema; Long, Joseph; Ho, Michelle; Young, Courtney S; Mojadidi, Michelle; Fitz-Gibbon, Sorel; Cooper, Aaron R; Lill, Georgia R; Urbinati, Fabrizia; Campo-Fernandez, Beatriz; Bjurstrom, Carmen F; Pellegrini, Matteo; Hollis, Roger P; Kohn, Donald B

    2016-09-01

    Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

  6. In vitro study of the influence of GST-π gene transfer on drug-resistance of human cord blood CD34 + cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective: To investigate the influence of GST-π gene transfer on drug-resis-tance of human cord blood CD34+ cells. Methods:CD34+ cells were purified from cord blood fromnormal full-term pregnancy. Gene transduction into human cord blood CD34 + cells was carried outusing GST-π gene containing retrovirus vector. The GST-π gene expression in transduced CD34 +cell was confirmed by RT-PCR. After confirmation of GST-π gene transfer, the transfected CD34 +cells were cultured by colony assay in the presence of carboplatin. Results:GST-π mRNA was de-tected in 30% of CFU-GM derived from GST-π gene transduced CD34+ cells. In vitro drug resis-tance test showed that the number of CFU-GM formed was significantly higher (2 ~ 3 fold) in GST-π gene transduced CD34+ cells than untransduced CD34+ cells. Conclusion:GST-π gene transfercan confer resistance to hematopoietic progenitors against carboplatin in vitro.

  7. Myelotoxicity of trichothecenes and apoptosis: an in vitro study on human cord blood CD34+ hematopoietic progenitor.

    Science.gov (United States)

    Le Dréan, G; Auffret, M; Batina, P; Arnold, F; Sibiril, Y; Arzur, D; Parent-Massin, D

    2005-12-01

    Previous studies have revealed that hematological disorders associated with trichothecenes intoxication in humans could result from hematopoiesis inhibition. The most frequent and potent trichothecene mycotoxins are T-2 toxin and deoxynivalenol (DON), respectively. Apoptosis induction by these two toxins was investigated in vitro on human hematopoietic progenitors (CD34+ cells). Hoechst coloration, DNA fragmentation and annexin-V/PI labeling in flow cytometry showed that T-2 toxin, in contrast to DON, induced apoptosis in CD34+ cells. T-2 toxin effect was dose- and time-dependent with a significant increase of apoptotic cells as early as 3h after incubation at 10(-7) M and a maximum reached at 12 h. This observation evidenced the high sensitivity of hematopoietic progenitors to T-2 toxin. The inhibition of T-2 toxin-induced apoptosis by a pan-caspase inhibitor (Z-VAD-fmk) suggested the involvement of caspases. The proportional increase of caspase-3 specific activity (DEVDase) with T-2 toxin concentration confirmed its role in the process. After incubation of CD34+ cells with T-2 toxin, in conditions that induced apoptosis, clonal expansion of granulo-monocytes, erythrocytes and megakaryocytes precursors was dose-dependently inhibited. The hematological effects observed in T-2 toxin mycotoxicosis could then be assigned to hematopoiesis inhibition by apoptosis. Different mechanisms that need to be further elucidated are involved in DON myelotoxicity.

  8. CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon gamma (IFN-gamma)-inducible protein 10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report the novel finding that CXCR3 is also...... expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up...... for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood...

  9. Ex vivo expansion of Primate CD34+ Cells isolated from Bone Marrow and Human Bone Marrow Mononuclear Cells using a Novel Scaffold

    Directory of Open Access Journals (Sweden)

    Devaprasad D

    2009-01-01

    Full Text Available Bone marrow derived CD34+ cells have been in clinical application in patients with haematological malignancies. One of the major problems with this treatment is the non-availability of matched donors or the necessity of multiple transfusions depending upon the pathology. Recently evidences have been accumulating to prove the safety and efficacy of autologous CD34+ cells in diseases such as myocardial dysfunction, peripheral vascular diseases and neurological certain conditions. However there are only a few reports in the literature on ex vivo expansion of the bone marrow derived CD34+ cells. We have in two different studies proven that isolated CD34+ cells from baboon bone marrow and non-isolated BMMNCs from human bone marrow could be expanded with increase in percentage of CD34+ cells using a novel scaffold.

  10. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

    Science.gov (United States)

    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.

  11. Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

    Institute of Scientific and Technical Information of China (English)

    张毅; 唐佩弦; 金滢; 李秀森; 张双喜; 吴英; 毛宁

    2001-01-01

    To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold.Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, I.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.

  12. Expression of lumican related to CD34 and VEGF in the articular disc of the human temporomandibular joint.

    Directory of Open Access Journals (Sweden)

    N. Kiga

    2010-07-01

    Full Text Available Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ disc is made up of fibrocartilage with an extracellular matrix (ECM composed of collagen and proteoglycans. The existence and behaviour of lumican have not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy. In both normal and deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblastlike cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells.

  13. The Protective Effect of Human Umbilical Cord Blood CD34+ Cells and Estradiol against Focal Cerebral Ischemia in Female Ovariectomized Rat: Cerebral MR Imaging and Immunohistochemical Study.

    Directory of Open Access Journals (Sweden)

    Ching-Chung Liang

    Full Text Available Human umbilical cord blood derived CD34+ stem cells are reported to mediate therapeutic effects in stroke animal models. Estrogen was known to protect against ischemic injury. The present study wished to investigate whether the protective effect of CD34+ cells against ischemic injury can be reinforced with complemental estradiol treatment in female ovariectomized rat and its possible mechanism. Experiment 1 was to determine the best optimal timing of CD34+ cell treatment for the neuroprotective effect after 60-min middle cerebral artery occlusion (MCAO. Experiment 2 was to evaluate the adjuvant effect of 17β-estradiol on CD34+ cell neuroprotection after MCAO. Experiment 1 showed intravenous infusion with CD34+ cells before MCAO (pre-treatment caused less infarction size than those infused after MCAO (post-treatment on 7T magnetic resonance T2-weighted images. Experiment 2 revealed infarction size was most significantly reduced after CD34+ + estradiol pre-treatment. When compared with no treatment group, CD34+ + estradiol pre-treatment showed significantly less ADC reduction at 2 h and 2 d, less CBF reduction at 2 h and less hyperperfusion at 2 d. The immunoreactivity of c-Fos, c-Jun and GFAP was attenuated, and BDNF showed significant recovery from 2 h to 2 d after MCAO, especially after CD34+ + estradiol pre-treatment. The present study suggests pre-treatment with CD34+ cells with complemental estradiol can be most protective against ischemic injury, which may act through stabilization of cerebral hemodynamics and normalization of the expressions of immediate early genes and BDNF.

  14. Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model.

    Science.gov (United States)

    Kim, Jaehyup; Zanjani, Esmail D; Jeanblanc, Christine M; Goodrich, A Daisy; Hematti, Peiman

    2013-08-01

    Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

  15. Effect of Human WEE1 and Stem Cell Factor on Human CD34+ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

    Institute of Scientific and Technical Information of China (English)

    Ping LEI; Yong HE; Wenfang SHI; Jilin PENG; Sha WU; Huifen ZHU; Jianguo CHEN; Guanxin SHEN

    2007-01-01

    Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE1 (WEE1Hu) and proliferation-stimulating stem cell factor (SCF) was generated. In this study, we selected human umbilical cord blood CD34+ cells as the in vitro model in an attempt to investigate whether WEE1Hu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay,colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEE1Hu and SCF in CD34+ cells provided the cells with some protection. These findings suggest that the expression of WEE1Hu and SCF might rescue CD34+ cells from chemotherapyinduced myelosuppression.

  16. Imatinib and nilotinib inhibit hematopoietic progenitor cell growth, but do not prevent adhesion, migration and engraftment of human cord blood CD34+ cells.

    Directory of Open Access Journals (Sweden)

    Ludovic Belle

    Full Text Available BACKGROUND: The availability of tyrosine kinase inhibitors (TKIs has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34(+ cells. DESIGN AND METHODS: After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null mice. RESULTS: TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34(+ cell cycle entry. Adhesion of CD34(+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT. CONCLUSIONS: These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.

  17. 人脐血CD34+细胞在NOD/SCID小鼠上有效重建造血系统%Transplanting human umbilical cord blood CD34 + cells reconstitutes hematopoieticsystem in NOD/SCID mice

    Institute of Scientific and Technical Information of China (English)

    贾新涛; 穆媛媛; 贾潇潇; 胡文华; 邓飞

    2011-01-01

    目的 探讨人脐血CD34+造血干细胞在非肥胖糖尿病/重症联合免疫缺陷(no obese diabetic/severe combined Immunodeficiency,NOD/SCID)小鼠模型上造血重建的作用.方法 利用密度梯度离心法,从新鲜脐血中分离出单个核细胞,利用免疫磁珠分选法筛选CD34+造血干细胞,经尾静脉输注入经亚致死剂量照射后的NOD/SCID小鼠体内,移植后3、7、10、14 d分别用断尾法取小鼠外周血,血常规计数外周血动态变化情况;移植后4、6、8、10周,取其外周血,运用PCR法检测外周血中人特异性Alu基因的表达情况.结果 免疫磁珠分选得到的CD34+细胞浓度达91.2%,照射后小鼠骨髓腔内有核细胞和巨细胞数量明显减少或消失,达到清髓目的.移植后第3天移植组小鼠外周血各系细胞均明显低于正常组(P<0.01),移植后第7天,移植组小鼠外周血象开始恢复,明显高于阴性对照组[白细胞:(3.90±0.53)×109/L vs (1.30±0.18)×109/L,血红蛋:(139.8±5.0)g/L vs (79.8±11.0)g/L,白血小板:(253.0±17.5)×109/L vs (52.0±6.9)×109/L,(P<0.01)],移植后第10天,移植组小鼠外周血象恢复到辐照前水平,与正常组无差别.移植4周后,PCR方法 在小鼠外周血中可检测到人特异Alu基因序列,未移植组小鼠照射后2周内全部死亡.结论 经照射后的NOD/SCID小鼠通过人脐血CD34+细胞植入可建立起人鼠嵌合模型.NOD/SCID小鼠经照射后,不破坏骨髓造血微环境及造血基质,可用于异基因细胞移植模型.人脐血CD34+细胞移植入NOD/SCID小鼠,其造血系统能有效重建.%Objective To investigate the hematopoietic reconstitution by human umbilical cord bloodCD34 + cells in no obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Methods Mono-nuclear cells (MNCs) were isolated from human fresh cord blood by gradient centrifugation, and then CD34 +hematopoietic stem cells were selected by magnetic activated cell sorting. The selected cells were

  18. Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A series of retroviral vectors encoding human mdr1 gene alone as well as in combination with either human mgmt gene or human mutant Ser31-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34+ cells. It has been shown that expression of dual drug resistance genes in transduced cells confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in an in vivo model in which transduced hematopoietic cells will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cells more effectively.

  19. CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells.

    Science.gov (United States)

    Noh, Eui Kyu; Ra, Jae Sun; Lee, Seong Ae; Kwon, Byoung S; Han, In Seob

    2005-12-31

    A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

  20. Complementary populations of human adipose CD34+ progenitor cells promote growth, angiogenesis, and metastasis of breast cancer.

    Science.gov (United States)

    Orecchioni, Stefania; Gregato, Giuliana; Martin-Padura, Ines; Reggiani, Francesca; Braidotti, Paola; Mancuso, Patrizia; Calleri, Angelica; Quarna, Jessica; Marighetti, Paola; Aldeni, Chiara; Pruneri, Giancarlo; Martella, Stefano; Manconi, Andrea; Petit, Jean-Yves; Rietjens, Mario; Bertolini, Francesco

    2013-10-01

    Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.

  1. Non-invasive tracking of human haemopoietic CD34{sup +} stem cells in vivo in immunodeficient mice by using magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Niemeyer, Markus; Jacobs, Volker R.; Timmer, Sebastian; Kiechle, Marion [Technische Universitaet Muenchen, Department of Gynaecology, Klinikum rechts der Isar, Munich (Germany); Oostendorp, Robert A.J.; Hippauf, Sandra; Bekker-Ruz, Viktoria [Technische Universitaet Muenchen, Department of Oncology, Klinikum rechts der Isar, Munich (Germany); Kremer, Markus [Technische Universitaet Muenchen, Department of Pathology, Klinikum rechts der Isar, Munich (Germany); Baurecht, Hansjoerg [Technische Universitaet Muenchen, Department of Statistics, Klinikum rechts der Isar, Institute for Medical Statistics and Epidemiology, Munich (Germany); Ludwig, Georg; Rummeny, Ernst J. [Technische Universitaet Muenchen, Department of Radiology, Klinikum rechts der Isar, Munich (Germany); Piontek, Guido [Technische Universitaet Muenchen, Department of Neuropathology, Munich (Germany); Beer, Ambros J. [Technische Universitaet Muenchen, Department of Radiology, Klinikum rechts der Isar, Munich (Germany); Technische Universitaet Muenchen, Department of Nuclear Medicine, Munich (Germany)

    2010-09-15

    To assess migration of CD34{sup +} human stem cells to the bone marrow of athymic mice by using magnetic resonance (MR) imaging and Resovist, a contrast agent containing superparamagnetic iron oxide (SPIO) particles. All animal and human procedures were approved by our institution's ethics committee, and women had given consent to donate umbilical cord blood (UCB). Balb/c-AnN Foxn1{sup nu}/Crl mice received intravenous injection of 1 x 10{sup 6} (n = 3), 5 x 10{sup 6} (n = 3) or 1 x 10{sup 7} (n = 3) human Resovist-labelled CD34{sup +} cells; control mice received Resovist (n = 3). MR imaging was performed before, 2 and 24 h after transplantation. Signal intensities of liver, muscle and bone marrow were measured and analysed by ANOVA and post hoc Student's t tests. MR imaging data were verified by histological and immunological detection of both human cell surface markers and carboxydextran-coating of the contrast agent. CD34{sup +} cells were efficiently labelled by Resovist without impairment of functionality. Twenty-four hours after administration of labelled cells, MR imaging revealed a significant signal decline in the bone marrow, and histological and immunological analyses confirmed the presence of transplanted human CD34{sup +} cells. Intravenously administered Resovist-labelled CD34{sup +} cells home to bone marrow of mice. Homing can be tracked in vivo by using clinical 1.5-T MR imaging technology. (orig.)

  2. CD34+ cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells of Cajal

    DEFF Research Database (Denmark)

    Vanderwinden, J M; Rumessen, J J; De Laet, M H;

    1999-01-01

    and confocal microscopy. CD34 immunoreactivity identified previously unrecognized cells closely adjacent to, but distinct from, the Kit immunoreactive ICC. These CD34 immunoreactive cells expressed the fibroblast marker prolyl 4-hydroxylase-whereas ICC did not-and were also distinct from smooth muscle cells...

  3. Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

    Science.gov (United States)

    Yin, Ping; Ono, Masanori; Moravek, Molly B; Coon, John S; Navarro, Antonia; Monsivais, Diana; Dyson, Matthew T; Druschitz, Stacy A; Malpani, Saurabh S; Serna, Vanida A; Qiang, Wenan; Chakravarti, Debabrata; Kim, J Julie; Bulun, Serdar E

    2015-04-01

    Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

  4. Combination of CD34-positive cell subsets with infarcted myocardium-like matrix stiffness: a potential solution to cell-based cardiac repair.

    Science.gov (United States)

    Zhang, Shuning; Ma, Xin; Yao, Kang; Zhu, Hong; Huang, Zheyong; Shen, Li; Qian, Juying; Zou, Yunzeng; Sun, Aijun; Ge, Junbo

    2014-06-01

    Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell-based cardiac repair. Bone marrow-derived CD34-positive cells, a well-characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue-like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time-dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness-dependent differentiation of the unselected bone marrow-derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell-based cardiac repair. However, the difference in tissue stiffness-dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34-positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium-like matrix stiffness compared with CD34-negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell-based cardiac repair.

  5. Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice.

    Science.gov (United States)

    Wiekmeijer, Anna-Sophia; Pike-Overzet, Karin; Brugman, Martijn H; Salvatori, Daniela C F; Egeler, R Maarten; Bredius, Robbert G M; Fibbe, Willem E; Staal, Frank J T

    2014-06-01

    Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

  6. IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE CDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8′ 105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

  7. [Effects of human mesenchymal stem cells and fibroblastoid cell line as feeder layers on expansion of umbilical cord blood CD34(+) cells in vitro].

    Science.gov (United States)

    Ma, Li-Jun; Gao, Lei; Zhou, Hong; Qiu, Hui-Ying; Hu, Xiao-Xia; Xie, Lin-Na; Wang, Jian-Min

    2006-10-01

    To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC

  8. Human adipose CD34+ CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues.

    Science.gov (United States)

    Ferraro, Giuseppe A; De Francesco, Francesco; Nicoletti, Gianfranco; Paino, Francesca; Desiderio, Vincenzo; Tirino, Virginia; D'Andrea, Francesco

    2013-05-01

    Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi-potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co-expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34(-) CD90(-) cells and was able to differentiate in vitro into adipocytes (PPARγ(+) and adiponectin(+)) and endothelial cells (CD31(+) VEGF(+) Flk1(+)). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34(+) /CD90(+) stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34(+) /CD90 ASCs are extremely useful for regenerative medicine.

  9. Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a, CD34, and CD36

    Directory of Open Access Journals (Sweden)

    Bin Mao

    2016-11-01

    Full Text Available The development of human erythroid cells has been mostly examined in models of adult hematopoiesis, while their early derivation during embryonic and fetal stages is largely unknown. We observed the development and maturation of erythroblasts derived from human pluripotent stem cells (hPSCs by an efficient co-culture system. These hPSC-derived early erythroblasts initially showed definitive characteristics with a glycophorin A+ (GPA+ CD34lowCD36− phenotype and were distinct from adult CD34+ cell-derived ones. After losing CD34 expression, early GPA+CD36− erythroblasts matured into GPA+CD36low/+ stage as the latter expressed higher levels of β-globin along with a gradual loss of mesodermal and endothelial properties, and terminally suppressed CD36. We establish a unique in vitro model to trace the early development of hPSC-derived erythroblasts by serial expression of CD34, GPA, and CD36. Our findings may provide insight into the understanding of human early erythropoiesis and, ultimately, therapeutic potential.

  10. MicroRNAs as markers for neurally committed CD133+/CD34+ stem cells derived from human umbilical cord blood.

    Science.gov (United States)

    Hafizi, Maryam; Atashi, Amir; Bakhshandeh, Behnaz; Kabiri, Mahboubeh; Nadri, Samad; Hosseini, Reza Haji; Soleimani, Masoud

    2013-04-01

    Neural differentiation of the CD133+/CD34+ subpopulation of human umbilical cord blood stem cells was investigated, and neuro-miR (mir-9 and mir-124) expression was examined. An efficient induction protocol for neural differentiation of hematopoietic stem cells together with the exclusion of retinoic acid in this process was also studied. Transcription of some neural markers such as microtubule-associated protein-2, beta-tubulin III, and neuron-specific enolase was evaluated by real-time PCR, immunocytochemistry, and western blotting. Increased expression of neural indicators in the treated cells confirmed the appropriate neural differentiation, which supported the high efficiency of our defined neuronal induction protocol. Verified high expression of neuro-miRNAs along with neuronal specific proteins not only strengthens the regulatory role of miRNAs in determining stem cell fate but also introduces these miRNAs as novel indicators of neural differentiation. These data highlight the prominent therapeutic potential of hematopoietic stem cells for use in cell therapy of neurodegenerative diseases.

  11. Human XCR1+ dendritic cells derived in vitro from CD34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells.

    Science.gov (United States)

    Balan, Sreekumar; Ollion, Vincent; Colletti, Nicholas; Chelbi, Rabie; Montanana-Sanchis, Frédéric; Liu, Hong; Vu Manh, Thien-Phong; Sanchez, Cindy; Savoret, Juliette; Perrot, Ivan; Doffin, Anne-Claire; Fossum, Even; Bechlian, Didier; Chabannon, Christian; Bogen, Bjarne; Asselin-Paturel, Carine; Shaw, Michael; Soos, Timothy; Caux, Christophe; Valladeau-Guilemond, Jenny; Dalod, Marc

    2014-08-15

    Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1(+) DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1(+) human DC. Assessment of the immunoactivation potential of XCR1(+) human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1(+) and XCR1(-) human DC in CD34(+) progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1(-) CD34-DC are similar to canonical MoDC, whereas XCR1(+) CD34-DC resemble XCR1(+) blood DC (bDC). XCR1(+) DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1(+) DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1(+) CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1(+) bDC. Hence, it is feasible to generate high numbers of bona fide XCR1(+) human DC in vitro as a model to decipher the functions of XCR1(+) bDC and as a potential source of XCR1(+) DC for clinical use.

  12. Delta-tocotrienol suppresses radiation-induced microRNA-30 and protects mice and human CD34+ cells from radiation injury.

    Directory of Open Access Journals (Sweden)

    Xiang Hong Li

    Full Text Available We reported that microRNA-30c (miR-30c plays a key role in radiation-induced human cell damage through an apoptotic pathway. Herein we further evaluated radiation-induced miR-30 expression and mechanisms of delta-tocotrienol (DT3, a radiation countermeasure candidate, for regulating miR-30 in a mouse model and human hematopoietic CD34+ cells. CD2F1 mice were exposed to 0 (control or 7-12.5 Gy total-body gamma-radiation, and CD34+ cells were irradiated with 0, 2 or 4 Gy of radiation. Single doses of DT3 (75 mg/kg, subcutaneous injection for mice or 2 μM for CD34+ cell culture were administrated 24 h before irradiation and animal survival was monitored for 30 days. Mouse bone marrow (BM, jejunum, kidney, liver and serum as well as CD34+ cells were collected at 1, 4, 8, 24, 48 or 72 h after irradiation to determine apoptotic markers, pro-inflammatory cytokines interleukin (IL-1β and IL-6, miR-30, and stress response protein expression. Our results showed that radiation-induced IL-1β release and cell damage are pathological states that lead to an early expression and secretion of miR-30b and miR-30c in mouse tissues and serum and in human CD34+ cells. DT3 suppressed IL-1β and miR-30 expression, protected against radiation-induced apoptosis in mouse and human cells, and increased survival of irradiated mice. Furthermore, an anti-IL-1β antibody downregulated radiation-induced NFκBp65 phosphorylation, inhibited miR-30 expression and protected CD34+ cells from radiation exposure. Knockdown of NFκBp65 by small interfering RNA (siRNA significantly suppressed radiation-induced miR-30 expression in CD34+ cells. Our data suggest that DT3 protects human and mouse cells from radiation damage may through suppression of IL-1β-induced NFκB/miR-30 signaling.

  13. Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

    Science.gov (United States)

    Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

    2016-01-28

    Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function.

  14. RELATIONSHIP BETWEEN CD34/CD45 POSITIVE CELLS CONCENTRATION IN BLOOD OF HEART RECIPIENTS AND LEVEL OF GRAFT VASCULOPATHY RISK BIOMARKERS

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    O. P. Shevchenko

    2013-01-01

    Full Text Available Aim. To determine the relationship between CD34/CD45 positive cells number in peripheral blood before and after heart transplantation (HTx and plasma level of the biomarkers. Materials and methods. We studied 27 pts. (23 men; 40 ± 13 years with heart failure caused by dilated (17 cases or ischemic cardiomyopathy (10 cases before and after HTx. CD34/CD45 positive cells were measured in peripheral blood before and 2–4 days after the operation by flow cytometry and expressed in number of the cells per 106 events, plasma level of the biomar- kers – by ELISA. Results. The number of CD34/CD45+ cells in pts. with heart failure (224 ± 166 was similar to those in healthy individuals (233 ± 120 and there was no significant difference in the cell number between pts. with dilated (253 ± 188 and ischemic cardiomyopathy (169 ± 100. The cell number did not correlate with age, sex, body weight, blood cells counts and preoperative levels of the biomarkers: PlGF, sCD40L, PAPP-A. In 2–5 days after HTx the cell number decreased to 103 ± 102. The cell number after HTx did not correlate with demographic and laboratory parameters, anesthesia, operation, ischemia and hypothermia duration, blood loss volume and preoperative levels of PlGF and PAPP-A but correlated with sCD40L level in pts. with ischemic cardiomyopathy. Conclusion: Circulating HSC number in patients with heart failure does not differ from those in healthy individuals and decreases after HTx. In patients with ischemic cardiomyopathy the cell number after transplantation associates with preoperative level of sCD40L – the negative predictor of vasculopathy. 

  15. Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

    Science.gov (United States)

    Albright, Emily R.

    2013-01-01

    Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34+ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34+ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34+ cells. However, in contrast to what occurs in primary CD34+ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34+ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed. PMID:23824798

  16. The Application of CD34 Histochemical Method in the Study of Micro-vascular Architecture of human Scar%CD34组化法在瘢痕微血管构筑中的应用研究

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    目的:探讨瘢痕微血管构筑在瘢痕形成、发展及临床防治中的意义。方法:利用CD34血管内皮细胞可靠标记显示瘢痕微血管构筑,以期研究不同类型的瘢痕,同一类型瘢痕的不同时期以及同一瘢痕内不同区域的微血管构筑间的差异。结果:不同类型的瘢痕间、瘢痕内不同区域及不同时期间瘢痕微血管密度均有明显统计学差异(p<0.01)。结论:增生性瘢痕内的微血管密度明显高于萎缩性瘢痕的微血管密度;瘢痕早期微血管密度高随着病程延长而逐渐减少;瘢痕交界处为较幼稚瘢痕,血管最为丰富,能代表整个瘢痕的生物学行为。因此可把此区作为研究瘢痕发生、发展与转归的重点。%Objective:To explore the clinical significance of human scar micro-vascular architecture in the formation, development and prevention and treatment of scar. Method: CD34 antibody staining method was used to study the micro-vascular architecture of human scars. Results: Micro vascular density of hyperplastic scar in the central area was higher than that in the peripheral area; micro-vascular density of hyperplastic scar was higher than that of atrophic scar; Scars of different periods had different micro-vascular density. (P,0.01). Conclusion: In the border of the scar, the micro-vascular density is the highest. There scar border is the focal area to be studied in order to make clear the course of the generation, development and the fate of the scars.

  17. Wnt1 Accelerates an Ex Vivo Expansion of Human Cord Blood CD34+CD38− Cells

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    Kamonnaree Chotinantakul

    2013-01-01

    Full Text Available Cord blood hematopoietic stem cells (CB-HSCs transplantation has been increasing gradually with facing the limitation of insufficient quantity of HSCs in each CB unit. Therefore, efficient expansion methods which can maintain stem cell characteristics are needed. In this study, umbilical CB-CD34+ cells were cultured in two different cytokine cocktails: 4 factors (4F = Flt3-L, SCF, IL-6, and TPO and 5 factors (5F = Wnt1 + 4F in both serum and serum-free media. The data revealed that the best condition to accelerate an expansion of CD34+CD38− cells was serum-free culture condition supplemented with 5F (5F KSR. This condition yielded 24.3 ± 2.1 folds increase of CD34+CD38− cells. The expanded cells exhibited CD34+ CD38− CD133+ CD71low CD33low CD3− CD19− markers, expressed nanog, oct3/4, c-myc, and sox2 genes, and maintained differentiation potential into lymphoid, erythroid and myeloid lineages. The achievement of CD34+CD38− cells expansion may overcome an insufficient quantity of the cells leading to the improvement of the stem cell transplantation. Altogether, our findings highlight the role of Wnt1 and the new culture condition in stimulating hematopoietic stem/progenitor cells expansion which may offer a new therapeutic avenue for cord blood transplantation, regenerative medicine, stem cell bank applications, and other clinical applications in the future.

  18. Cotransplantation of human umbilical cord-derived mesenchymal stem cells and umbilical cord blood-derived CD34⁺ cells in a rabbit model of myocardial infarction.

    Science.gov (United States)

    Li, Tong; Ma, Qunxing; Ning, Meng; Zhao, Yue; Hou, Yuelong

    2014-02-01

    The objective of the study is to investigate the effect of hypoxic preconditioning on the immunomodulatory properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and the effect of cotransplantation of hUC-MSCs and human umbilical cord blood (hUCB)-derived CD34(+) cells in a rabbit model of myocardial infarction. hUC-MSCs with or without hypoxic preconditioning by cobalt chloride were plated in a 24-well plate, and then cocultured with hUCB-CD34(+) cells and PBMCs for 96 h at 37 °C in a 5% CO₂ incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34(+) cells + PBMC, A2 = hUC-MSCs + hUCB-CD34(+) cells + PBMC, Negative Control = hUCB-CD34(+) cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured by ELISA. A rabbit model of MI was established using a modified Fujita method. The animals were then randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or hUC-MSCs + CD34(+) cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone. Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively. Stem cell differentiation and neovascularization in the infracted area were characterized for the presence of cardiac Troponin I (cTnI) and CD31 by immunohistochemical staining, and the extent of myocardial fibrosis was evaluated by hematoxylin and eosin (H&E) and Masson's trichrome. IFN-γ was 27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups, respectively, which indicated that hUCB-CD34(+) cells induced immune reaction of peripheral blood mononuclear cells, whereas both hUC-MSCs and HP-MSCs showed an immunosuppressive effect, which, however, was attenuated

  19. Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG mice.

    Directory of Open Access Journals (Sweden)

    Adam C Drake

    Full Text Available Increasing demand for human hematopoietic stem cells (HSCs in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+ CD133(+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/- (NSG mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+ CD133(+ fraction of expanded cells and that CD34(+ CD133(+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.

  20. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

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    Justyna Czapla

    Full Text Available The aim of the present study was to isolate mesenchymal stromal cells (MSC with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI. The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO. Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb. MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

  1. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice

    Science.gov (United States)

    Wiśniewska, Ewa; Jarosz-Biej, Magdalena; Smolarczyk, Ryszard; Cichoń, Tomasz; Głowala-Kosińska, Magdalena; Śliwka, Joanna; Garbacz, Marcin; Szczypior, Mateusz; Jaźwiec, Tomasz; Langrzyk, Agnieszka; Zembala, Michał; Szala, Stanisław

    2016-01-01

    Aims The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. Methods and Results MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. Conclusion In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart. PMID:27415778

  2. Dynamics of alpha-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34(+) cells in culture.

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    Mahajan, Milind C; Karmakar, Subhradip; Newburger, Peter E; Krause, Diane S; Weissman, Sherman M

    2009-10-01

    The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron. A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway. Our results

  3. Histone deacetylase inhibition enhances self renewal and cardioprotection by human cord blood-derived CD34 cells.

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    Ilaria Burba

    Full Text Available BACKGROUND: Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs, have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of "enhancement" strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit. PRINCIPAL FINDINGS: Pharmacologic inhibition of histone deacetylases (HDACs is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34(+ were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction. CONCLUSIONS: Our results show that HDAC blockade leads to phenotype changes in CD34(+ cells with enhanced self renewal and cardioprotection.

  4. Distinguishing neurofibroma from desmoplastic melanoma: the value of the CD34 fingerprint.

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    Yeh, Iwei; McCalmont, Timothy H

    2011-08-01

    We have observed 'fingerprint' immunopositivity in association with perineurioma and neurofibroma. A fingerprint consists of delicate, elongated areas of positive labeling that fall between collagen bundles, thereby creating a whorled configuration that is reminiscent of a human fingerprint. At present, the differential diagnosis between early desmoplastic melanoma and neurofibroma remains challenging in a subset of cases because of overlapping histopathological and immunohistochemical features. To assess whether fingerprint CD34 reactivity could be contributory in this context, we stained 50 desmoplastic melanomas and 50 neurofibromas with CD34. Fingerprint CD34 labeling was present in greater than 30% of the proliferation in 96% (n = 48) of neurofibromas and in only 4% (n = 2) of desmoplastic melanomas. Over two-thirds of the neurofibromas exhibited a CD34 fingerprint involving more than 60% of their surface area. In the two cases of desmoplastic melanoma that showed CD34 fingerprint positivity, the staining was patchy and involved less than 60% of the tumor. In partially staining neurofibromas, areas without a CD34 fingerprint tended to occur in central lobular areas. We conclude that CD34 fingerprint immunoreactivity is useful in distinguishing neurofibroma from early desmoplastic melanoma, especially if the fingerprint involves more than 60% of a tumor's cross-sectional area.

  5. Efficient Reprogramming of Human Cord Blood CD34 + Cells for Formation of Induced Pluripotent Stem Cells with Non-integrating Plasmid System%非整合型质粒重编程脐带血CD34+细胞形成诱导性多潜能干细胞的研究

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    张权娥; 刘淑平; 刘延风; 张鸿雁; 袁卫平; 陈桂彬; 李彦欣; 许静

    2013-01-01

    This study was to establish the episomal vector reprogramming method to reprogram iPSC from human cord blood (CB) CD34 + cells.The non-integrating plasmids of pEB-C5 and pEB-Tg were transfected into short-term cultured CB CD34 + cells by using the nucleofector,so as to demonstrate efficient reprogramming of CB CD34 + cells.Within 14 days of one-time transfection by two plasmids together,up to 200 iPSC-like colonies per 2 million transfected CB CD34 + cells were generated.The results showed that the pluripotency of iPSC-derived CB CD34 + cells was similar to that of hESC and the karyotypes of iPSC were normal.In addition,no vector integration was found in iPSC of 9th and 10th passages.Furthermore,hiPSC formed teratoma with three embryonic germ layers.It is concluded that the integration-free method to generate human iPSC from CB CD34 + cells is reliable and can provide new ways for both research and future clinical applications.%本研究探索和优化非整合质粒的方法,将人脐带血来源的CD34+(CB CD34+)细胞进行重编程,建立无病毒的iPS技术体系.利用细胞核转染仪将质粒pEB-C5和pEB-Tg转入短暂培养后的CB CD34+细胞中,使其进行重编程形成iPSC,14 d后观察到2×106 CB CD34+细胞中约有200个类似ES细胞特征的克隆出现,并对产生的iPSC进行体内外多能性鉴定及细胞核型检测.结果表明,重编程后的CB CD34+细胞的多能性基因表达与ESC类似,细胞核型正常,外源基因无插入,且具有体内分化成三胚层的全能性.结论:利用非整合型质粒的方法重编程CB CD34+细胞形成iPSC,该方法无外源基因插入、重复性好、操作简单、且效率较高,为建立相对安全的iPSC提供了一个行之有效的途径,为深入探索iPSC应用于临床药物的筛选、组织工程和再生医学研究等提供了很好的研究材料.

  6. Quantification of nucleated cells, CD34-positive cells and CFU-GM colonies in single bone marrow samples and bone marrow harvests derived from healthy children.

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    Schündeln, Michael M; Walde, Gabriele; Basu, Oliver; Havers, Werner; Kremens, Bernhard

    2014-05-01

    Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

  7. Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses.

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    Jong-Ho Kim

    Full Text Available Adipose-derived stem cells (ADSCs have the potential to differentiate into various cell lineages and they are easily obtainable from patients, which makes them a promising candidate for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, hTERT-immortalized CD34+ and CD34- mouse ADSC lines (mADSCshTERT tagged with GFP were established. We evaluated the proliferation capacity, multi-differentiation potential, and secretory profiles of CD34+ and CD34- mADSCshTERT in vitro, as well as their effects on cardiac function and systemic inflammation following transplantation into a rat model of acute myocardial infarction (AMI to assess whether these cells could be used as a novel cell source for regeneration therapy in the cardiovascular field. CD34+ and CD34- mADSCshTERT demonstrated phenotypic characteristics and multi-differentiation potentials similar to those of primary mADSCs. CD34+ mADSCshTERT exhibited a higher proliferation ability compared to CD34- mADSCshTERT, whereas CD34- mADSCshTERT showed a higher osteogenic differentiation potential compared to CD34+ mADSCshTERT. Primary mADSCs, CD34+, and CD34- mADSCshTERT primarily secreted EGF, TGF-β1, IGF-1, IGF-2, MCP-1, and HGFR. CD34+ mADSCshTERT had higher secretion of VEGF and SDF-1 compared to CD34- mADSCshTERT. IL-6 secretion was severely reduced in both CD34+ and CD34- mADSCshTERT compared to primary mADSCs. Transplantation of CD34+ and CD34- mADSCshTERT significantly improved the left ventricular ejection fraction and reduced infarct size compared to AMI-induced rats after 28 days. At 28 days after transplantation, engraftment of CD34+ and CD34- mADSCshTERT was confirmed by positive Y chromosome staining, and differentiation of CD34+ and CD34- mADSCshTERT into endothelial cells was found in the infarcted myocardium. Significant decreases were observed in circulating IL-6 levels in CD34+ and CD34- mADSCshTERT groups compared to the AMI

  8. Failure of effector function of human CD8+ T Cells in NOD/SCID/JAK3⁻/⁻ immunodeficient mice transplanted with human CD34+ hematopoietic stem cells.

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    Yoshinori Sato

    Full Text Available Humanized mice, which are generated by transplanting human CD34+ hematopoietic stem cells into immunodeficient mice, are expected to be useful for the research on human immune responses. It is reported that antigen-specific T cell responses occur in immunodeficient mice transplanted with both human fetal thymus/liver tissues and CD34+ fetal cells, but it remains unclear whether antigen-specific T cell responses occur in those transplanted with only human CD34+ hematopoietic stem cells (HSCs. Here we investigated the differentiation and function of human CD8+ T cells reconstituted in NOD/SCID/Jak3⁻/⁻ mice transplanted with human CD34+ HSCs (hNOK mice. Multicolor flow cytometric analysis demonstrated that human CD8+ T cells generated from the CD34+ HSCs comprised only 3 subtypes, i.e., CD27(highCD28+CD45RA+CCR7+, CD27+CD28+CD45RA⁻CCR7+, and CD27+CD28+CD45RA⁻CCR7⁻and had 3 phenotypes for 3 lytic molecules, i.e., perforin(Per⁻granzymeA(GraA⁻granzymeB(GraB⁻, Per⁻GraA+GraB⁻, and Per(lowGraA+GraB+. These CD8+ T cells failed to produce IFN-γ and to proliferate after stimulation with alloantigens. These results indicate that the antigen-specific T cell response cannot be elicited in mice transplanted with only human CD34+ HSCs, because the T cells fail to develop normally in such mice.

  9. Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects.

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    Fracchiolla, Nicola Stefano; Todoerti, Katia; Bertazzi, Pier Alberto; Servida, Federica; Corradini, Paolo; Carniti, Cristiana; Colombi, Antonio; Cecilia Pesatori, Angela; Neri, Antonino; Deliliers, Giorgio Lambertenghi

    2011-04-28

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34+ cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34+ cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.

  10. Autophagy Proteins ATG5 and ATG7 Are Essential for the Maintenance of Human CD34(+) Hematopoietic Stem-Progenitor Cells.

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    Gomez-Puerto, Maria Catalina; Folkerts, Hendrik; Wierenga, Albertus T J; Schepers, Koen; Schuringa, Jan Jacob; Coffer, Paul J; Vellenga, Edo

    2016-06-01

    Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can be considered to be a general cellular housekeeping process, it has become clear that it may also play cell type-dependent functional roles. In this study, we analyzed the functional importance of autophagy in human hematopoietic stem/progenitor cells (HSPCs), and how this is regulated during differentiation. Western blot-based analysis of LC3-II and p62 levels, as well as flow cytometry-based autophagic vesicle quantification, demonstrated that umbilical cord blood-derived CD34(+) /CD38(-) immature hematopoietic progenitors show a higher autophagic flux than CD34(+) /CD38(+) progenitors and more differentiated myeloid and erythroid cells. This high autophagic flux was critical for maintaining stem and progenitor function since knockdown of autophagy genes ATG5 or ATG7 resulted in reduced HSPC frequencies in vitro as well as in vivo. The reduction in HSPCs was not due to impaired differentiation, but at least in part due to reduced cell cycle progression and increased apoptosis. This is accompanied by increased expression of p53, proapoptotic genes BAX and PUMA, and the cell cycle inhibitor p21, as well as increased levels of cleaved caspase-3 and reactive oxygen species. Taken together, our data demonstrate that autophagy is an important regulatory mechanism for human HSCs and their progeny, reducing cellular stress and promoting survival. Stem Cells 2016;34:1651-1663.

  11. Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Yi(

    2001-01-01

    [1]Luens. K. M., Travis, M. A., Chen, B. P. et al., Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy+Lin cells with preserved ability to engraft SCID-hu bone, Blood, 1998, 91: 1206-1215.[2]Piacibello, W., Sanavio, F., Garetto, L. et al., Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood, Blood, 1997, 89(8)L 2644-2653.[3]Zhang Yi, Tang Peixian, Li Xiusen et al., Expression of human FL ligand and thrombopoietin genes in a bone marrow stromal cell line by internal ribosome entry site (IRES) sequence, Chin. J. Hematol. (in Chinese), 1999, 20(12): 624-627.[4]Zhou. S., Mao, N., Zhao, M. et al., Effect of recombinant FLT3 ligand (rhFL) on in vitro expansion of human cord blood CD34+ cells. Natl. Med. J. China, 1998, 78: 37-39.[5]Dexter. T. M.. Allen, T. D., Lajtha, L. G. et al., Conditions controlling the proliferation of hematopoietic stem cells in vitro,J. Cell Physiol.. 1997, 91: 335-344.[6]Petzer. A. L., Hogge, D. E.. Lansdorp, P. M. et al., Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium, Pro. Natl. Acad. Sci. USA, 1996, 93:1470-1474.[7]Sirchia, G., Rebulla, P., Placental/umbilical cord blood transplantation, Haematologica, 1999, 84: 738-747.[8]Wineman, J., Moore, K., Lemischka, I. et al., Functional heterogeneity of the hematopoietic microenvironment: Rare stromal elements maintain long-term repopulating stem cells, Blood, 1996, 87: 4082-4090.[9]Szilvassy, S. J., Weller, K. P., Lin, W. et al., Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells, Blood, 1996, 87:4618-4628.[10]Aiuti, A., Fredrich, C., Sieff, C. A. et al., Identification of distinct elements of the stromal

  12. Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke.

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    M Z Ratajczak

    2006-06-01

    Full Text Available The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day and 144 hrs (6th day since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM and erythroid (BFU-E-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.

  13. 房颤对外周血CD34+造血祖细胞的影响 及SDF-1/CXCR4在心房中的表达%Effect of atrial fibrillation on human CD34 + hematopoietic progenitor cells in circulation and expression of SDF-1/CXCR4 in atrium

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    李佳; 葛海龙; 陈光远; 高倩萍; 孙俊峰; 李元十; 富路

    2011-01-01

    目的:研究不同类型的房颤(AF)对人外周血CD34+造血祖细胞(CD34+ HPCs)的影响,以及持续心房快速起搏犬心肌基质细胞衍生因子-1(SDF-1)及其受体CXCR4的表达,初步探讨CD34+ HPCs及SDF-1/CXCR4在AF时心肌损伤修复中的作用.方法:应用流式细胞术测定阵发性AF患者组(a=35)、持续性AF患者组(n=35)及窦性心律者对照组(n=30)外周血中CD34+ HPCs的百分含量;并对持续性AF患者组中24例患者成功进行体外直流电复律后48 h,测定外周血中CD34+ HPCs的百分含量.另外,将成年健康杂种犬13条随机分为两组:即快速起搏组(n=7)和假手术组(n=6),均开胸后于右心耳缝植AOO型起搏器,快速起搏组以400次/min起搏6周,假手术组不起搏.应用RT-PCR测定左心耳和左心房CXCR4mRNA的表达水平,用蛋白质免疫印迹法检测左心房SDF-1蛋白的表达.结果:持续性AF患者组外周血中CD34+ HPCs的百分含量明显高于阵发性AF患者组和对照组(P<0.05);而后两组间无差别.持续性AF患者成功进行体外直流电复律后48 h,外周血中CD34+ HPCs的百分含量较复律前明显下降(P<0.05).快速起搏组犬左心耳和左心房CXCR4mRNA表达的水平明显高于假手术组(P<0.05),左心耳增高16.7%,左心房增高18.8%:SDF-1蛋白质表达的水平亦明显高于假手术组(P<0.01).结论:持续性AF患者外周血中CD34 HPCs的数量增加;心房快速起搏犬心房SDF-1/CXCR4的表达增加.CD34+ HPCs和SDF1/CXCR4可能参与了持续性AF患者心房损伤时心肌组织的修复过程.%AIM: To investigate the effect of different kinds of atrial fibrillation (AF) on human CD34 + hematopoietic cells ( HPCs) in circulation and on myocardial expression of SDF-1 and its receptor CXCR4 in canines with lasting rapid atrial pacing and to explore the role of CD34 + HPCs and SDF-1/ CXCR4 in repairing atrium during AF. METHODS; Included in our study were 100 subjects (35 with paroxysmal AF, 35

  14. Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors.

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    Verónica Ramos-Mejía

    Full Text Available Human induced pluripotent stem cells (hiPSC have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM. The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.

  15. A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures.

    Science.gov (United States)

    Chute, J P; Saini, A A; Kampen, R L; Wells, M R; Davis, T A

    1999-02-01

    Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34+CD38- cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34+CD38- cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34+CD38- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34+ cells and the CD34+CD38- subset before and after ex vivo expansion on PMVECs. Purified human CD34+ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34+ population and the CD34+CD38- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34+CD38+ subset were found to be in G1, and 51% were in G2/S/M phase of the cell cycle. More remarkably, 53% of the CD34+CD38- subset were in G1, and 17% were in G2/S/M phase after 7 days of PMVEC coculture. In contrast, only 22% of the CD34+CD38- subset remaining after 7 days of stroma-free culture were in G1, and 6% were in G2/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin

  16. Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor

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    Xianmei Meng; Rui-Jun Su; David J Baylink; Amanda Neises; Jason B Kiroyan; Wayne Yuk-Wai Lee; Kimberly J Payne

    2013-01-01

    The direct conversion of skin cells into somatic stem cells has opened new therapeutic possibilities in regenerative medicine.Here,we show that human induced mesenchymal stem cells (iMSCs) can be efficiently generated from cord blood (CB)-or adult peripheral blood (PB)-CD34+ cells by direct reprogramming with a single factor,OCT4.In the presence of a GSK3 inhibitor,16% of the OCT4-transduced CD34+ cells are converted into iMSCs within 2 weeks.Efficient direct reprogramming is achieved with both episomal vector-mediated transient OCT4 expression and lentiviral vector-mediated OCT4 transduction.The iMSCs express MSC markers,resemble bone marrow (BM)-MSCs in morphology,and possess in vitro multilineage differentiation capacity,yet have a greater proliferative capacity compared with BM-MSCs.Similar to BM-MSCs,the implanted iMSCs form bone and connective tissues,and are non-tumorigenic in mice.However,BM-MSCs do not,whereas iMSCs do form muscle fibers,indicating a potential functional advantage of iMSCs.In addition,we observed that a high level of OCT4 expression is required for the initial reprogramming and the optimal iMSC self-renewal,while a reduction of OCT4 expression is required for multilineage differentiation.Our method will contribute to the generation of patient-specific iMSCs,which could have applications in regenerative medicine.This discovery may also facilitate the development of strategies for direct conversion of blood cells into other types of cells of clinical importance.

  17. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34+ hematopoietic stem cells and for chondrogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    JIN-FUWANG; LI-JUANWANG; YI-FANWU; YINGXIANG; CHUN-GANGXIE; BING-BINGJIA; JENNYHARRINGTON; IANK.MCNIECE

    2005-01-01

    Background and Objectives. Human mesenchymal stem/progenitor cells (MSPC) ar pluripotent, being the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of hematopoietic stem/progenitor cells (HSPC) in umbilical cord blood (UCB) is well known, that of MSPC has been not fully evaluated. Design and Methods. In this study, we examined the immunophenotype, the supporting function in relation to exvivo expansion of hematopoietic stem progenitor cells and the chondrogenic differentiation of cultured cells with characteristics of MSPC from UCB. When UCB nucleated cells were isolated and 107 cells cultured in IMDM with 20% fetal bovine serum, the mean number of adherent fibroblastlike colonies was 3.5±0.7/106 monuclear cells. Results. UCB-derived MSPC could be expanded for at least 15 passages. In their undifferentiated state, UCB-derived MSPC were CD 13+, CD29+, CD90+, CD105+, CD166+, SH2+,SH3+, SH4+, CD45-, CD34-, and CD14-; they produced stem cell factor, interleukin 6 and tumor necrosis factor α.UCB-derived MSPC cultured in chondrogenic media differentiated into chondrogenic cells. UCB-derived MSPC supported the proliferation and differentiation of CD34+ cells from UCB in vitro. Interpretation and Conclusions. UCB-derived MSPC have the potential to support ex vivo expansion of HSPC and chondrogenic differentiation. UCB should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells and may provide a unique source of fetal cells for cellular and gene therapy.

  18. Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14 (+ monocytes and CD34 (+ cells.

    Directory of Open Access Journals (Sweden)

    Cyprian C Rossetto

    Full Text Available The parameters involved in human cytomegalovirus (HCMV latent infection in CD14 (+ and CD34 (+ cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+ and CD34 (+ cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs, RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+ monocytes followed by next generation sequencing (ChIP-Seq were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE, which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+ monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.

  19. Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

    Science.gov (United States)

    Lagresle-Peyrou, Chantal; Benjelloun, Fatine; Hue, Christophe; Andre-Schmutz, Isabelle; Bonhomme, Delphine; Forveille, Monique; Beldjord, Kheira; Hacein-Bey-Abina, Salima; De Villartay, Jean-Pierre; Charneau, Pierre; Durandy, Anne; Fischer, Alain; Cavazzana-Calvo, Marina

    2008-02-01

    Severe combined immunodeficiency (SCID) caused by mutation of the recombination-activating gene 1 (RAG1) or Artemis gene lead to the absence of B- and T-cell differentiation. The only curative treatment is allogeneic bone marrow (BM) transplantation, which displays a high survival rate when an HLA compatible donor is available but has a poorer prognosis when the donor is partially compatible. Consequently, gene therapy may be a promising alternative strategy for these diseases. Here, we report that lentiviral gene-corrected BM CD34(+) cells (isolated from Artemis- or RAG1-deficient patients) sustain human B-cell differentiation following injection into non-obese diabetic/SCID (NOD-SCID) mice previously infused with anti-interleukin-2 receptor beta chain monoclonal antibody. In most of the mice BM, engrafted with Artemis-transduced cells, human B-cell differentiation occurred until the mature stage. The B cells were functional as human immunoglobulin M (IgM) was present in the serum. Following injection with RAG1-transduced cells, human engraftment occurred in vivo but B-cell differentiation until the mature stage was less frequent. However, when it occurred, it was always associated with human IgM production. This overall approach represents a useful tool for evaluating gene transfer efficiency in human SCID forms affecting B-cell development (such as Artemis deficiency) and for testing new vectors for improving in vivo RAG1 complementation.

  20. CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood......Ab blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig...

  1. Retroviral transfer of the nlsLacZ gene into human CD34+ cell populations and into TF-1 cells: future prospects in gene therapy.

    Science.gov (United States)

    Bagnis, C; Gravis, G; Imbert, A M; Herrera, D; Allario, T; Galindo, R; Lopez, M; Pavon, C; Sempere, C; Mannoni, P

    1994-11-01

    Few data are available concerning behavior of reimplanted human hematopoietic cells after autologous stem cell transplantation. This paper reports the possibility to transfer gene markers coding for beta-galactosidase (beta-Gal) activity by retroviral vectors into a human leukemic growth factor-dependent cell line, TF-1, and into human hematopoietic progenitors isolated from peripheral blood or bone marrow. Using various combinations of retroviral vectors and packaging cell lines, we demonstrated high expression of a bacterial beta-Gal activity induced by the LacZ gene, the nlsLacZ gene, or the Sh-ble/LacZ gene, in human hematopoietic cells. The expression of the nlsLacZ construct was stable until the end of the culture in infected CD34+ cell-enriched cell populations, and a slow decrease of transgene expression was observed in a transduced TF-1 cell population during a 1-year long-term culture. Data obtained with the nlsLacZ gene demonstrate that both retroviral transfer and corresponding gene expression were not found to modify the pattern of cell proliferation and differentiation. These results open interesting prospectives for the use of the nlsLacZ gene to mark and follow the fate of progenitor cells isolated from patients with cancers prior to reimplantation.

  2. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Science.gov (United States)

    2011-01-01

    Background Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis

  3. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Huang Sheng-Shan

    2011-05-01

    Full Text Available Abstract Background Acute myeloid leukemia (AML is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1, DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to

  4. An optimized protocol for the generation and functional analysis of human mast cells from CD34(+) enriched cell populations.

    Science.gov (United States)

    Yin, Yuzhi; Bai, Yun; Olivera, Ana; Desai, Avanti; Metcalfe, Dean D

    2017-09-01

    The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×10(6) vs. 2.4±0.28×10(6), respectively, from 1.0×10(8) lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×10(6)), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward. Published by Elsevier B.V.

  5. Gene expression profiles of cryopreserved CD34{sup +} human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Sudo, Kazuhiro [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan); Yasuda, Jun, E-mail: yasuda-jun@umin.ac.jp [Omics Science Center, RIKEN, Yokohama (Japan); Department of Cell Biology, The JFCR-Cancer Institute (Japan); Nakamura, Yukio, E-mail: yukionak@brc.riken.jp [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan)

    2010-07-09

    Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34{sup +} UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-{beta}, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.

  6. Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

    Institute of Scientific and Technical Information of China (English)

    Zhong-Jing Su; Hai-Bin Chen; Jin-Kun Zhang; Lan Xu

    2005-01-01

    AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCTD) mice.METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect.RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%,47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80%vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01).CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

  7. Effects of human embryonic lung fibroblasts feeder layer transfected with leukemia inhibitory factor gene on the proliferation of umbilical cord blood CD34+ cells in vitro%转白血病抑制因子基因人胚肺成纤维细胞对脐血CD34+细胞体外扩增的影响

    Institute of Scientific and Technical Information of China (English)

    夏卫; 郁心; 姜健; 田丽娜; 陈瑶; 缪竞诚

    2012-01-01

    背景:单份脐血的造血细胞数量有限,难以满足成人的需要,如何有效地扩增脐血造血干/祖细胞是目前研究的热点.目的:构建人白血病抑制因子基因修饰的人胚肺成纤维细胞,观察转基因细胞对脐血CD34+造血干/祖细胞体外扩增的影响.方法:建立转人白血病抑制因子基因的饲养层细胞,用RT-PCR法和ELISA法鉴定目的基因的表达;采用免疫磁珠法分离脐血CD34+造血干/祖细胞,流式细胞术检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果;扩增后的造血干/祖细胞用跨膜迁移实验检测自发迁移率和基质细胞衍生因子1诱导迁移试验以鉴定体外扩增的造血干/祖细胞的归巢能力.结果与结论:成功建立转基因饲养层细胞,RT-PCR法和ELISA法证实有目的基因表达,与人白血病抑制因子转基因饲养层细胞共培养7 d后CD34+造血干/祖细胞可大量扩增,同时表面黏附分子的表达量仍较高.体外迁移实验显示与转基因饲养层细胞共培养的造血细胞的诱导迁移率明显高于对照组,可以较好地保持其归巢能力.因此转人白血病抑制因子基因的饲养层细胞可有效扩增脐血CD34+造血干/祖细胞,延缓其分化,并且体外扩增后仍保持较高的归巢能力.%BACKGROUND: The number of hematopoietic cels in single copy cord blood is limited, and it is difficult to meet the needs of adults. How to effectively amplify cord blood hematopoietic stem/progenitor cels (HSPCs) is a hot res earch issue currently. OBJECTIVE: To construct gene-modified human embryonic lung fibroblast cels using human leukemia inhibitory factor (hLIF) and to observe the effect of trans genic cells on the amplification of HSPCs in vitro.METHODS: The feeder layer trancfected with recombinat adenouirus Ad-hLIF was established and objective gene was detected by using RT-PCR and ELISA. Umbilical cord blood CD34+ HSPC were

  8. Expression map of the human exome in CD34+ cells and blood cells: increased alternative splicing in cell motility and immune response genes.

    Directory of Open Access Journals (Sweden)

    Sylvie Tondeur

    Full Text Available BACKGROUND: Hematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells. PRINCIPAL FINDINGS: Using Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php. At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi, a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes. CONCLUSION: The relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function.

  9. Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion.

    Science.gov (United States)

    Rodríguez-Pardo, Viviana M; Vernot, Jean Paul

    2013-03-01

    The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increased CD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

  10. Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid Mobilization for the Collection of Human CD34+ Cells in Healthy Volunteers.

    Science.gov (United States)

    Abraham, Michal; Pereg, Yaron; Bulvik, Baruch; Klein, Shiri; Mishalian, Inbal; Wald, Hanna; Eizenberg, Orly; Beider, Katia; Nagler, Arnon; Golan, Rottem; Vainstein, Abi; Aharon, Arnon; Galun, Eithan; Caraco, Yoseph; Or, Reuven; Peled, Amnon

    2017-08-23

    The potential of the high affinity CXCR4 antagonist BL-8040 as a monotherapy mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019). The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open label phase, in which 8 subjects received a single injection of BL-8040 (1mg/kg) and approximately 4hrs later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immune deficient mice. BL-8040 was found safe and well tolerated at all doses tested (0.5-1 mg/kg). The main treatment related AEs were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol and promethazine pre-treatment. In the first part of the study BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. 4hrs post dose, the count rose to a mean of 8, 37, 31 and 35cells/µL (placebo, 0.5, 0.75 and 1mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B and NK cells. In the second part the mean CD34+/kg collected were 11.6 x106 cells/kg. The graft composition was rich in immune cells. The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Copyright ©2017, American Association for Cancer Research.

  11. Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects.

    Science.gov (United States)

    Vandevoorde, C; Vral, A; Vandekerckhove, B; Philippé, J; Thierens, H

    2016-06-01

    As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end

  12. 抗TGF-β抗体对人脐血CD34+细胞体外扩增及黏附分子表达的影响%Effect of Anti-TGF-β Antibody on ex vivo Expansion and Expression of Adhesion Molecules of Human Cord Blood CD34 + Cells

    Institute of Scientific and Technical Information of China (English)

    钱筠; 刘复强; 吴轶苹; 王阳; 康文婷

    2005-01-01

    本研究的目的是证实抗TGF-β抗体在人脐血CD34+细胞体外扩增过程中可以增加CD34+细胞的扩增效率,并观察其对脐血CD34+细胞粘附分子表达的影响.从6份新鲜脐血标本中富集CD34+细胞,将其接种于含抗TGF-β抗体+SCF+Flt-3L+TPO+IL-3因子组合的SFEM无血清培养体系中,培养6天后,细胞计数,并用FACS检测CD34、c-kit(CD117)、CD11a、CD49d、CD33表达情况,同时进行集落分析.结果表明:①实验组有核细胞数、CD34+细胞数、CD34+c-kit+细胞数及CFU-GEMM分别扩增了41.82±13.49,15.62±6.95,13.36±6.12,11.07±4.05倍,明显高于对照组(P分别=0.001,0.002,0.003,0.002),其中实验组中更为早期的CD34+c-kitˉ亚群的扩增倍数更多(69.10±41.06),多于有核细胞、CD34+细胞、CD34+c-kit+细胞的扩增倍数(P=0.024).②TGF-β抗体对CD11a和CD49d的表达无明显影响(P值均大于0.05).结论:抗TGF-β抗体可协同早期干细胞生长因子有效扩增脐血CD34+细胞,同时不降低CD34+细胞黏附分子的表达.

  13. Encapsulated feeder cells within alginate beads for ex vivo expansion of cord blood-derived CD34(+) cells

    National Research Council Canada - National Science Library

    Pan, Xiuwei; Sun, Qiong; Cai, Haibo; Gao, Yun; Tan, Wensong; Zhang, Weian

    2016-01-01

    A co-culture system based on encapsulated feeder cells within alginate beads was developed through optimizing the detailed aspects of the cell culture system to expand CD34-positive (CD34(+)) cells ex vivo...

  14. Efficient reprogramming of human cord blood CD34+ cells into induced pluripotent stem cells with OCT4 and SOX2 alone.

    Science.gov (United States)

    Meng, Xianmei; Neises, Amanda; Su, Rui-Jun; Payne, Kimberly J; Ritter, Linda; Gridley, Daila S; Wang, Jun; Sheng, Matilda; Lau, K-H William; Baylink, David J; Zhang, Xiao-Bing

    2012-02-01

    The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

  15. CD34+ stem cells from umbilical cord blood

    Directory of Open Access Journals (Sweden)

    Alfio D’Agati

    2011-09-01

    Full Text Available We describe the relation between umbilical cord clamping time and two different enrichment system of CD34+ stem cells from umbilical cord blood with the proliferative ability and bone marrow reconstitution of the stem cells obtained. After an obstetrician performed the cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections using the Stem Sep method, or with a positive cells selection based on their surface CD34 antigens using the Mini Macs system. An excellent recovery of haematopoietic progenitors [Burst Forming Unit Erythroids (BFUE; Colony Forming Unit Granulocytes and Macrophages (CFU-GM; and Colony Forming Unit Granulocytes, Erythroids, Monocytes and Macrophages (CFU-GME], inversely related to the increase in clamping time, was performed with the Mini Macs system (54% of colonies, with 90% purity. With Stem Sep method, haematopoietic progenitor’s recovery was 35% (with 80% purity. By applying early clamping of umbilical cord blood we obtained a greater number of CD34+ cells and their clonogenic activity was increased with enrichment. This is a useful technique considering that the number of CD34+ stem cells usually contained from a unit of placental blood is enough for the transplant to a child, but not for an adult. Thus, using these methods, we can get a larger number of CD34+ stem cells which reduces the risk of Graft versus Host Disease also in adult patients, producing survival rates similar to those obtained with transplantation of bone marrow from unrelated donors.

  16. CD34+ stem cells from umbilical cord blood

    Directory of Open Access Journals (Sweden)

    Carlo Pafumi

    2011-10-01

    Full Text Available We describe the relation between umbilical cord clamping time and two different enrichment system of CD34+ stem cells from umbilical cord blood with the proliferative ability and bone marrow reconstitution of the stem cells obtained. After an obstetrician performed the cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections using the Stem Sep method, or with a positive cells selection based on their surface CD34 antigens using the Mini Macs system. An excellent recovery of haematopoietic progenitors [Burst Forming Unit Erythroids (BFUE; Colony Forming Unit Granulocytes and Macrophages (CFU-GM; and Colony Forming Unit Granulocytes, Erythroids, Monocytes and Macrophages (CFU-GME], inversely related to the increase in clamping time, was performed with the Mini Macs system (54% of colonies, with 90% purity. With Stem Sep method, haematopoietic progenitor’s recovery was 35% (with 80% purity. By applying early clamping of umbilical cord blood we obtained a greater number of CD34+ cells and their clonogenic activity was increased with enrichment. This is a useful technique considering that the number of CD34+ stem cells usually contained from a unit of placental blood is enough for the transplant to a child, but not for an adult. Thus, using these methods, we can get a larger number of CD34+ stem cells which reduces the risk of Graft versus Host Disease also in adult patients, producing survival rates similar to those obtained with transplantation of bone marrow from unrelated donors.

  17. CD34, acancer stem cell marker,in nasopharyngeal carcinoma celllines%恶性肿瘤干细胞标记物CD34在鼻咽癌细胞系的表达

    Institute of Scientific and Technical Information of China (English)

    张军红; 李青; 王春华; 吴秀云; 卢新阁; 王莉菲; 杨蕾蕾; 王秋红

    2016-01-01

    BACKGROUND:Previous research have confirmed that CD34 is closely related to oncogenesis, progress, recurrence, metastasis and drug-resistance of various cancers, but its role in nasopharyngeal carcinoma remains unclear. OBJECTIVE:Tosortcels positive and negative for CD34 in nasopharyngealcarcinoma cel lines and to detect cel proliferation and migration. METHODS:Expressionsof CD34 in nasopharyngeal carcinoma cel lines 5-8F, 6-10B, CNE1 and CNE2 were detected by flow cytometry. And CD34+and CD34-cels were sorted based on cel surfacemarkers for purity identification. Afterwards, proliferation and migrationof CD34+and CD34-celswere detected by MTT assay, colony-formation assay and scratch assay. RESULTS AND CONCLUSION:Al four nasopharyngeal carcinoma cel lines expressed CD34 in 0.1%-0.2%, and the level of CD34 was closely related to the cel growth density. The purity of CD34+cel was more than 98% in the sorted CD34+celpopulations, but no CD34+cels were found inthe sorted CD34-celpopulations.At 1, 3, 5 and 7 daystheproliferation rate of CD34+cel, populationswas significantly higher than that of CD34-cels (P  目的:分选鼻咽癌细胞株中CD34+与CD34-细胞的增殖和迁移能力。  方法:利用流式细胞术检测CD34在鼻咽癌细胞株5-8F、6-10B、CNE1、CNE2中的表达,根据细胞表面标志物分选出CD34+与CD34-细胞,进行纯度鉴定;通过体外MTT法、平板克隆形成实验以及划痕实验分析CD34+细胞与CD34-细胞增殖和迁移能力。  结果与结论:①4种鼻咽癌细胞株中CD34表达均恒定在0.1%-0.2%,且CD34水平与细胞的生长密度关系密切;②分选的CD34+细胞群中,CD34+细胞例能达到98%以上,在CD34-细胞群中未能检测到CD34+细胞;③CD34+细胞在第1,3,5,7天增殖速度显著高于CD34-细胞(P <0.05);④CD34+细胞克隆形成率显著高于CD34-细胞(P<0.05);⑤CD34+细胞向划痕区迁移的细胞明显多于CD34-细胞(P<0.05);⑥结果提示,CD

  18. The high-affinity CXCR4 antagonist BKT140 is safe and induces a robust mobilization of human CD34+ cells in patients with multiple myeloma.

    Science.gov (United States)

    Peled, Amnon; Abraham, Michal; Avivi, Irit; Rowe, Jacob M; Beider, Katia; Wald, Hanna; Tiomkin, Lena; Ribakovsky, Lena; Riback, Yossi; Ramati, Yaron; Aviel, Sigal; Galun, Eithan; Shaw, Howard Laurence; Eizenberg, Orly; Hardan, Izhar; Shimoni, Avichai; Nagler, Arnon

    2014-01-15

    CXCR4 plays an important role in the retention of stem cells within the bone marrow. BKT140 (4F-benzoyl-TN14003) is a 14-residue bio stable synthetic peptide, which binds CXCR4 with a greater affinity compared with plerixafor (4 vs. 84 nmol/L). Studies in mice demonstrated the efficient and superior mobilization and transplantation of stem cells collected with GCSF-BKT140, compared with those obtained when using stem cells obtained with each one of these mobilizing agent alone. These results have served as a platform for the present clinical phase I study. Eighteen patients with multiple myeloma who were preparing for their first autologous stem cell transplantation were included. Patients received a standard multiple myeloma mobilization regimen, consisting of 3 to 4 g/m(2) cyclophosphamide (day 0), followed by granulocyte colony-stimulating factor (G-CSF) at 5 μg/kg/d starting on day 5 and administered between 8 and 10 pm until the end of stem cell collection. A single injection of BKT140 (0.006, 0.03, 0.1, 0.3, and 0.9 mg/kg) was administered subcutaneously on day 10 in the early morning, followed by G-CSF 12 hours later. BKT140 was well tolerated at all concentrations, and none of the patients developed grade 3 and 4 toxicity. A single administration of BKT140 at the highest dose, 0.9 mg/kg, resulted in a robust mobilization and collection of CD34(+) cells (20.6 ± 6.9 × 10(6)/kg), which were obtained through a single apheresis. All transplanted patients received ∼5.3 × 10(6) CD34(+) cells/kg, which rapidly engrafted (n = 17). The median time to neutrophil and platelet recovery was 12 and 14 days, respectively, at the highest dose (0.9 mg/kg). When combined with G-CSF, BKT140 is a safe and efficient stem cell mobilizer that enabled the collection of a high number of CD34(+) cells in 1 and 2 aphaeresis procedures, resulting in successful engraftment. ©2013 AACR.

  19. Preparation of immunoliposomes directed against CD34 antigen as target.

    Science.gov (United States)

    Mercadal, M; Carrion, C; Domingo, J C; Petriz, J; Garcia, J; de Madariaga, M A

    1998-04-22

    The My-10 monoclonal antibody has facilitated the search of haematopoietic stem cells by recognizing selectively the human CD34 antigen. In the present work, My-10 immunoliposomes directed specifically against CD34+ cells were prepared, characterized and tested in vitro. Binding to target cells at 4 degreesC of immunoliposomes containing carboxyfluorescein as aqueous marker was evaluated by flow cytometry and fluorescence microscopy. These immunoliposomes demonstrated their capacity to bind specifically to CD34+ cells. Studies have shown that 9 antibodies/vesicle were sufficient to obtain a good binding efficiency. The product was stable over one month at 4 degreesC in terms of leakage of encapsulated carboxyfluorescein, particle size and antigen binding capacity.

  20. A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells

    Directory of Open Access Journals (Sweden)

    Sandra Ferry

    2011-09-01

    Full Text Available Abstract Background The finding of human umbilical cord blood as one of the most likely sources of hematopoietic stem cells offers a less invasive alternative for the need of hematopoietic stem cell transplantation. Due to the once-in-a-life time chance of collecting it, an optimum cryopreservation method that can preserve the life and function of the cells contained is critically needed. Methods Until now, slow-cooling has been the routine method of cryopreservation; however, rapid-cooling offers a simple, efficient, and harmless method for preserving the life and function of the desired cells. Therefore, this study was conducted to compare the effectiveness of slow- and rapid-cooling to preserve umbilical cord blood of mononucleated cells suspected of containing hematopoietic stem cells. The parameters used in this study were differences in cell viability, malondialdehyde content, and apoptosis level. The identification of hematopoietic stem cells themselves was carried out by enumerating CD34+ in a flow cytometer. Results Our results showed that mononucleated cell viability after rapid-cooling (91.9% was significantly higher than that after slow-cooling (75.5%, with a p value = 0.003. Interestingly, the malondialdehyde level in the mononucleated cell population after rapid-cooling (56.45 μM was also significantly higher than that after slow-cooling (33.25 μM, with a p value p value = 0.138. However, CD34+ enumeration was much higher in the population that underwent slow-cooling (23.32 cell/μl than in the one that underwent rapid-cooling (2.47 cell/μl, with a p value = 0.001. Conclusions Rapid-cooling is a potential cryopreservation method to be used to preserve the umbilical cord blood of mononucleated cells, although further optimization of the number of CD34+ cells after rapid-cooling is critically needed.

  1. Associations among Darbepoetin-α, CD34+ Cells and Cardiovascular Disease Events in Patients on Hemodialysis

    Directory of Open Access Journals (Sweden)

    Daisuke Sanada

    2012-09-01

    Full Text Available Background and Objectives: Erythropoiesis-stimulating agents (ESAs might moderate circulating CD34-positive hematopoietic stem (CD34+ cells. We assessed associations between ESA therapy and CD34+ cells and their impact on cardiovascular disease (CVD events in patients on prevalent hemodialysis (HD. Design, Setting, Participants and Measurements: We analyzed 95 patients on prevalent HD who received the ESAs epoetin-β (n = 22, darbepoetin-α (n = 60, or neither (control; no ESA, n = 13. Baseline values for CD34+ cells, high-sensitivity C-reactive protein, interleukin-6, vascular endothelial growth factor, inter-cellular adhesion molecule-1, and carotid intima-media thickness were determined. The numbers of CD34+/erythropoietin receptor (EPOR+ cells were determined in 35 and 8 patients in the darbepoetin-α and control groups, respectively. CD34+ cells were counted after 6 and 12 months of darbepoetin-α treatment (n = 35. All patients were followed up for a mean of 28 months. Results: Hemoglobin levels were lower, carotid intima-media thickness was more pronounced, and the ESA dose was higher in patients with a low, than with a high, CD34+ cell count. The ratio of CD34+/EPOR+ to CD34+ cells positively correlated with the darbepoetin-α dose. A low, but not a high, dose of darbepoetin-α for 6 and 12 months was associated with more CD34+ cells. Although high-dose darbepoetin-α therapy was an independent predictor of composite CVD events, this association disappeared when adjusted for the CD34+ cell count with other confounders. Conclusions: High-dose ESA therapy is associated with a low CD34+ cell count and comprises a risk factor for CVD events in patients on prevalent HD.

  2. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

    Science.gov (United States)

    Salles, Isabelle I; Thijs, Tim; Brunaud, Christine; De Meyer, Simon F; Thys, Johan; Vanhoorelbeke, Karen; Deckmyn, Hans

    2009-12-01

    Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

  3. Extra-acral cutaneous sclerosing perineurioma with CD34 fingerprint pattern.

    Science.gov (United States)

    Macarenco, Ricardo S; Cury-Martins, Jade

    2017-04-01

    Sclerosing perineuroma is a variant of extraneural perineurioma that, as a rule, occurs in acral sites. However, it has also been occasionally reported in non-acral regions. Recently, CD34 expression in a pattern reminiscent of the human fingerprint has been observed in a subset of perineuriomas, but this immunohistochemical finding has not been documented in non-acral sclerosing perineuriomas. We report a case of sclerosing perineurioma presenting CD34 expression in a fingerprint-like pattern on the skin of the neck (a previously unreported site for this neoplasm) of a 56-year-old man. In addition, alpha smooth-muscle actin showed a similar pattern of expression, suggesting that the cell population implicated in the remarkable immunolabeling is most probably fibroblastic/myofibroblastic. Other immunohistochemical findings included epithelial membrane antigen and claudin1-positive lesional cells, and the absence of S100, glucose transporter protein 1, MUC4 and desmin.

  4. Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

    Directory of Open Access Journals (Sweden)

    Fang Xiao

    Full Text Available Type 1 diabetes mellitus (T1DM is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

  5. Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells

    Directory of Open Access Journals (Sweden)

    Corneo Gianmarco

    2007-07-01

    Full Text Available Abstract Background Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting. Objective To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method. Methods We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, ΔNGFR. We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture. Results Overall recovery of CD34+ cells after culture was 128.5%; ΔNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after ΔNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7–1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and ΔNGFR selection. Conclusion We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.

  6. CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs).

    Science.gov (United States)

    Tasev, Dimitar; Konijnenberg, Lara S F; Amado-Azevedo, Joana; van Wijhe, Michiel H; Koolwijk, Pieter; van Hinsbergh, Victor W M

    2016-07-01

    Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(-) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(-) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(-): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells. However, during cell culture CD34(-) cells re-expressed CD34. Cell-seeding density, cell-cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(-) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function.

  7. Co-transplantation of human fetal thymus, bone and CD34(+) cells into young adult immunodeficient NOD/SCID IL2Rγ(null) mice optimizes humanized mice that mount adaptive antibody responses.

    Science.gov (United States)

    Chung, Yun Shin; Son, Jin Kyung; Choi, Bongkum; Joo, Sung-Yeon; Lee, Yong-Soo; Park, Jae Berm; Moon, Hana; Kim, Tae Jin; Kim, Se Ho; Hong, Seokmann; Chang, Jun; Kang, Myung-Soo; Kim, Sung Joo

    2015-04-01

    Both the thymus (T) and bone (B) are necessary hematopoietic niches in adult humans. We previously showed that co-transplantation of human fetal T and B tissues into neonatal immunodeficient NOD/SCID IL2Rγ(null) (NSG, N) mice facilitated hematopoiesis. However, transplantation into neonatal mice resulted in high frequency of early death, making it unrealistic for repetitive experiments. In this study, young adult N mice were pre-engrafted with T and B, T alone, B alone or no tissues. The animals were irradiated and injected with autologous fetal liver (FL)-derived CD34(+) cells (34). The resultant mice were TB34N, T34N, B34N and 34N, respectively, and challenged with T cell dependent antigens (Ags). The humanized TB34N mice showed best performance of these mouse models in many aspects resembling the adult human Ag-experienced spleen. The TB34N mice exhibited better hematopoietic reconstitution; balanced development of T- and B-cell, and common progenitor cells; follicular lymphoid structures with a functional germinal center (GC) enriched with follicular dendritic cells (FDCs) and plasma cells (PCs); secretion of hIgG in the sera in response to Ags at comparable levels to those of human; derivations of hIgG mAb-secreting hybridoma clones. Collectively, the humanized TB34N mice could develop an adaptive immunity that was capable of producing Ag-specific hIgG at a significant level via class switching. This unprecedented TB34N platform in humanized mice would be useful in dissecting human immunity, for generating human Abs and clinical applications.

  8. Human XCR1+ Dendritic Cells Derived In Vitro from CD34+ Progenitors Closely Resemble Blood Dendritic Cells, Including Their Adjuvant Responsiveness, Contrary to Monocyte-Derived Dendritic Cells

    OpenAIRE

    S. Balan; Ollion, V.; Colletti, N.; Chelbi, R.; Montanana-Sanchis, F.; LIU, H.; Vu Manh, T.-P.; Sanchez, C.; Savoret, J.; Perrot, I.; Doffin, A.-C.; Fossum, E.; Bechlian, D.; Chabannon, C.; Bogen, B

    2014-01-01

    Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1+ DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1+ human DC. Assessment of the immunoactivation potential of XCR1+ human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1+ and XCR1− human DC in CD3...

  9. Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency virus-infected patients: increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy

    DEFF Research Database (Denmark)

    Nielsen, S D; Afzelius, P; Dam-Larsen, S;

    1998-01-01

    The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4....... Furthermore, the fraction of naive CD4 cells increased. These findings have implications for the design of immunotherapy or gene therapy protocols....... and CD34 cells were measured. To examine the numbers of naive and memory type CD4 cells, CD4 cell coexpression of CD45RA and CD45RO was measured. Functionality of mobilized CD4 cells was examined by use of the proliferation assay and interleukin-2 ELISA. The number of CD34 cells increased from 1.50 to 20...

  10. Plerixafor for autologous CD34+ cell mobilization

    Directory of Open Access Journals (Sweden)

    Huda Salman

    2011-02-01

    Full Text Available Huda Salman, Hillard M LazarusDivision of Hematology-Oncology, Blood and Marrow Transplant Program, University Hospitals Case Medical Center, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USAAbstract: High-dose chemotherapy and autologous transplantation of hematopoietic cells is a crucial treatment option for hematologic malignancy patients. Current mobilization regimes often do not provide adequate numbers of CD34+ cells. The chemokine receptor CXCR4 and ligand SDF-1 are integrally involved in homing and mobilization of hematopoietic progenitor cells. Disruption of the CXCR4/SDF-1 axis by the CXCR4 antagonist, plerixafor, has been demonstrated in Phase II and Phase III trials to improve mobilization when used in conjunction with granulocyte colony-stimulating factor (G-CSF. This approach is safe with few adverse events and produces significantly greater numbers of CD34+ cells when compared to G-CSF alone. New plerixafor initiatives include use in volunteer donors for allogeneic hematopoietic cell transplant and in other disease targets.Keywords: plerixafor, autologous hematopoietic cell transplant, CD34, lymphoma, myeloma, granulocyte colony-stimulating factor (G-CSF

  11. Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2

    Directory of Open Access Journals (Sweden)

    Maho Nagasawa

    2017-08-01

    Full Text Available Innate lymphoid cells (ILCs have emerged as a key cell type involved in surveillance and maintenance of mucosal tissues. Mouse ILCs rely on the transcriptional regulator Inhibitor of DNA-binding protein 2 (Id2 for their development. Here, we show that Id2 also drives development of human ILC because forced expression of Id2 in human thymic progenitors blocked T cell commitment, upregulated CD161 and promyelocytic leukemia zinc finger (PLZF, and maintained CD127 expression, markers that are characteristic for human ILCs. Surprisingly CD5 was also expressed on these in vitro generated ILCs. This was not an in vitro artifact because CD5 was also found on ex vivo isolated ILCs from thymus and from umbilical cord blood. CD5 was also expressed on small proportions of ILC2 and ILC3. CD5+ ILCs were functionally immature, but could further differentiate into mature CD5− cytokine-secreting ILCs. Our data show that Id2 governs human ILC development from thymic progenitor cells toward immature CD5+ ILCs.

  12. Hepatitis B Virus Replication in CD34+ Hematopoietic Stem Cells From Umbilical Cord Blood.

    Science.gov (United States)

    Huang, Yanxin; Yan, Qin; Fan, Rongshan; Song, Shupeng; Ren, Hong; Li, Yongguo; Lan, Yinghua

    2016-05-18

    BACKGROUND Hepatitis B virus (HBV) is a hepatotropic virus that can infect extrahepatic tissue. Whether hematopoietic stem cells (HSCs) can be infected by HBV and serve as a potential virus reservoir is still unknown. In this study, the susceptibility of CD34+ HSCs to HBV was investigated. MATERIAL AND METHODS Cord blood-derived CD34+ HSCs were exposed to HBV in vitro, and immunocytochemistry, transmission electron microscopy, and RT-PCR were used to identify viral-related proteins and specific viral genomic sequences. Then, CD34+ HSCs were challenged by different titers of HBV, and intracellular and supernatant HBV DNA, and hepatitis B surface antigen (HBsAg) levels, were examined. In addition, CD34+ peripheral blood stem cells (PBSCs) from chronic HBV carriers were isolated and cultured, and HBV DNA levels were measured. RESULTS HBV-infected CD34+ cells showed positive signals for HBsAg by DAB staining and TRITC staining, and HBV particles were identified. RT-PCR results showed that the 403 bp PCR products corresponding to the amplified hepatitis B S gene fragment were observed in CD34+ HSCs infected by HBV. In addition, supernatant and intracellular HBV DNA increased with the proliferation of CD34+ HSCs. Similar results were obtained from intracellular HBsAg quantification tests. In addition, HBV DNA levels both in cells and in supernatants of CD34+ PBSCs increased proportionally, and the increments of HBV DNA in the supernatants paralleled those found in cells. CONCLUSIONS HBV can replicate in CD34+ HSCs in cord blood or peripheral blood of chronic HBV carriers.

  13. Supportive effects of stromal cells derived from human embryonic aorta-gonad-mesonepheros(AGM) region on the ability of umbilical cord blood CD34+ cells to form HPP-CFU%人AGM区基质细胞对脐血CD34+细胞形成HPP-CFU能力的支持作用

    Institute of Scientific and Technical Information of China (English)

    陈惠芹; 张涤华; 张绪超

    2007-01-01

    目的 探讨人主动脉-性腺-中肾(AGM)区基质细胞对脐血CD34+细胞形成高增殖潜能集落形成单位(HPP-CFU)能力的支持作用.方法 采用免疫磁珠方法分离人脐血CD34+细胞,接种于底层已制备好人AGM区基质细胞S1~S5饲养层的24孔板中,同时设无饲养层组及取自同期胚胎的躯干成纤维细胞hFT为对照,体外培养28天,每7天收获细胞行造血细胞集落培养.结果 5株人AGM区基质细胞hAGMS1~S5对HPP-CFU均有不同程度的扩增及维持作用,HPP-CFU在培养14天达到峰值,与无饲养层组、hFT组比较差异有统计学意义(P<0.05).hAGMS1~S5的造血支持作用组间比较差异亦有统计学意义(P<0.05),其中hAGMS3、S4优于S1、S2及S5.结论 人AGM区基质细胞S1~S5对脐血CD34+细胞形成HPP-CFU的能力具有支持作用,特别是hAGNS3、S4两株细胞具有更好的造血干性维持作用,为胚胎早期造血发生机制研究提供了重要的模式细胞和基础资料.

  14. CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

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    Mihalescu-Maingot Maria

    2010-02-01

    Full Text Available Abstract Background Tumor initiating cells (TICs provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs. Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

  15. Induction of various immune modulatory molecules in CD34(+) hematopoietic cells

    DEFF Research Database (Denmark)

    Umland, Oliver; Heine, Holger; Miehe, Michaela

    2004-01-01

    Lipopolysaccharide (LPS) has been shown to induce proliferation of human T-lymphocytes only in the presence of monocytes and CD34(+) hematopoietic cells (HCs) from peripheral blood. This finding provided evidence of an active role of CD34(+) HCs during inflammation and immunological events. To in...... cytokines including TNF-alpha, which may contribute to innate- and adaptive-immune processes........ To investigate mechanisms by which CD34(+) HCs become activated and exert their immune-modulatory function, we used the human CD34(+) acute myeloid leukemia cell line KG-1a and CD34(+) bone marrow cells (BMCs). We showed that culture supernatants of LPS-stimulated mononuclear cells (SUP(LPS)) as well as tumor...... revealed that T cell proliferation can be induced by TNF-alpha-stimulated KG-1a cells, which is preventable by blocking anti-ICAM-1 monoclonal antibodies. Our results demonstrate that CD34(+) HCs have the potential to express a variety of immune-regulatory mediators upon stimulation by inflammatory...

  16. CD34 and α smooth muscle actin distinguish verrucous hyperplasia from verrucous carcinoma.

    Science.gov (United States)

    Paral, Kristen M; Taxy, Jerome B; Lingen, Mark W

    2014-04-01

    This study evaluated the use of stromal biomarkers CD34 and α smooth muscle actin (α-SMA) to distinguish verrucous carcinoma (VC) from verrucous hyperplasia (VH). Thirteen VH, 15 VC, 20 squamous cell carcinoma (SCC), and 16 of uninvolved adjacent stroma specimens were analyzed for α-SMA and CD34 expression by immunohistochemistry. Stromal α-SMA positivity was observed in 100% (20 of 20) of the SCC and in 93% (14 of 15) of the VC, whereas none of the VH (0 of 13) or adjacent uninvolved stroma (0 of 16) demonstrated α-SMA reactivity. Stromal CD34 positivity was observed in 100% (13 of 13) of VH and adjacent stroma (16 of 16), while 20% (3 of 15) of VC and 11% (2 of 18) of SCC stroma expressed CD34. The SCC and VC groups differed significantly from the VH and uninvolved stroma groups for both α-SMA and CD34 expression (P < .0001). Stromal CD34 and α-SMA protein expression patterns may aid in distinguishing between VC and VH in challenging cases. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Double expression of CD34 and CD117 on bone marrow progenitors is a hallmark of the development of functional mast cell of Callithrix jacchus (common marmoset).

    Science.gov (United States)

    Nunomura, Satoshi; Shimada, Shin; Kametani, Yoshie; Yamada, Yuko; Yoshioka, Mino; Suemizu, Hiroshi; Ozawa, Manabu; Itoh, Toshio; Kono, Azumi; Suzuki, Ryuji; Tani, Kenzaburo; Ando, Kiyoshi; Yagita, Hideo; Ra, Chisei; Habu, Sonoko; Satake, Masanobu; Sasaki, Erika

    2012-09-01

    Mast cells (MCs) are developed from hematopoietic progenitor cells and play an important role in inflammation. Study of the kinetics of development and accumulation of primate MC in vivo is crucial for the control of human inflammatory diseases, as evolution of the immune system is quite rapid and inflammation including MC response is considered to be different between mouse and human. In the present study, we examined the development of MC from hematopoietic progenitors of Callithrix jacchus (common marmoset), an experimental animal of nonhuman primates. Bone marrow cells were fractionated for the expression of CD34 and CD117 by cell sorting. MCs were developed in vitro or by transplanting the cells to NOD/SCID/IL-2γc knockout (NOG) mice. In vitro culture of CD34(+)CD117(+) (double positive, DP) cells with stem cell factor could generate high-affinity Fc epsilon receptor (FcεR)-expressing CD117(+) cells with typical granules. The developed MC released β-hexosaminidase and produced leukotriene C(4) after the stimulation of FcεRI. Transplantation of DP cells gave rise to a marked expansion of CD34(-)CD45(+)CD117(+)FcεR(+) cells in NOG mice. They expressed transcripts encoding chymase 1 and tryptase β. Differentiation of CD34(-)CD117(+) cells to MCs was relatively limited compared with the DP cells, similarly to human MCs. These results suggest that this marmoset system provides a good model for human MC development.

  18. Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells.

    Science.gov (United States)

    Shi, Qiang; Schatten, Gerald; Hodara, Vida; Simerly, Calvin; VandeBerg, John L

    2013-02-01

    In this study, we used a large non-human primate model, the baboon, to establish a step-wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM-2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.

  19. CD34 Promotes Pathological Epi-Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

    Directory of Open Access Journals (Sweden)

    Martin J Siemerink

    Full Text Available The sialomucins CD34 and podocalyxin (PODXL are anti-adhesive molecules expressed at the luminal membrane of endothelial cells of small blood vessels and facilitate vascular lumen formation in the developing mouse aorta. CD34 transcript and protein levels are increased during human angiogenesis, its expression is particularly enriched on endothelial tip cell filopodia and CD34 is a marker for tip cells in vitro. Here, we investigated whether CD34 merely marks endothelial tip cells or has a functional role in tip cells and angiogenesis. We assessed that silencing CD34 in human microvascular endothelial cells has little effect on endothelial cell migration or invasion, but has a significant effect on vascular-endothelial growth factor-induced angiogenic sprouting activity in vitro. In vivo, the absence of CD34 reduced the density of filopodia on retinal endothelial tip cells in neonatal mice, but did not influence the overall architecture of the retinal vascular network. In oxygen-induced retinopathy, Cd34-/- mice showed normal intra-retinal regenerative angiogenesis but the number of pathological epi-retinal neovascular tufts were reduced. We conclude that CD34 is not essential for developmental vascularization in the retina, but its expression promotes the formation of pathological, invasive vessels during neovascularization.

  20. CD34 Promotes Pathological Epi-Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy

    Science.gov (United States)

    Siemerink, Martin J.; Dallinga, Marchien G.; Gora, Tomek; Cait, Jessica; Vogels, Ilse M. C.; Yetin-Arik, Bahar; Van Noorden, Cornelis J. F.; Klaassen, Ingeborg; McNagny, Kelly M.; Schlingemann, Reinier O.

    2016-01-01

    The sialomucins CD34 and podocalyxin (PODXL) are anti-adhesive molecules expressed at the luminal membrane of endothelial cells of small blood vessels and facilitate vascular lumen formation in the developing mouse aorta. CD34 transcript and protein levels are increased during human angiogenesis, its expression is particularly enriched on endothelial tip cell filopodia and CD34 is a marker for tip cells in vitro. Here, we investigated whether CD34 merely marks endothelial tip cells or has a functional role in tip cells and angiogenesis. We assessed that silencing CD34 in human microvascular endothelial cells has little effect on endothelial cell migration or invasion, but has a significant effect on vascular-endothelial growth factor-induced angiogenic sprouting activity in vitro. In vivo, the absence of CD34 reduced the density of filopodia on retinal endothelial tip cells in neonatal mice, but did not influence the overall architecture of the retinal vascular network. In oxygen-induced retinopathy, Cd34-/- mice showed normal intra-retinal regenerative angiogenesis but the number of pathological epi-retinal neovascular tufts were reduced. We conclude that CD34 is not essential for developmental vascularization in the retina, but its expression promotes the formation of pathological, invasive vessels during neovascularization. PMID:27352134

  1. Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system.

    Science.gov (United States)

    Boyer, Lucie; Robert, Amélie; Proulx, Chantal; Pineault, Nicolas

    2008-03-20

    Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end, we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells, and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo, while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence, we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase, the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings, two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly, these cocktails were either deprived of thrombopoietin or of stem cell factor, two cytokines known to favor megakaryopoiesis and HPC expansion, respectively. Based on these results, a short resource-efficient two-phase culture protocol for the production of Mks near purity (>95%) from human CD34+ CB cells has been established.

  2. Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency virus-infected patients: increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy

    DEFF Research Database (Denmark)

    Nielsen, S D; Afzelius, P; Dam-Larsen, S

    1998-01-01

    The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4....... Furthermore, the fraction of naive CD4 cells increased. These findings have implications for the design of immunotherapy or gene therapy protocols....

  3. Enhancing the function of CD34(+ cells by targeting plasminogen activator inhibitor-1.

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    Sugata Hazra

    Full Text Available Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1, a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO reduced PAI-1 mRNA in diabetic (p<0.01 and non-diabetic (p=0.05 CD34(+ cells. To reduce PAI-1 in human CD34(+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+ cell proliferation and migration in vitro, likely through increased PI3(K activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+ cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

  4. [Knockdown of Puma protects cord blood CD34(+) cells against γ- irradiation].

    Science.gov (United States)

    Zhao, Lei; Zhang, Hong-Yan; Pang, Ya-Kun; Gu, Hai-Hui; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao

    2014-04-01

    Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

  5. Human immunodeficiency virus type 1 restriction by human-rhesus chimeric tripartite motif 5alpha (TRIM 5alpha) in CD34(+) cell-derived macrophages in vitro and in T cells in vivo in severe combined immunodeficient (SCID-hu) mice transplanted with human fetal tissue.

    Science.gov (United States)

    Anderson, Joseph; Akkina, Ramesh

    2008-03-01

    Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutics. The retroviral restriction factor TRIM5alpha (tripartite motif 5alpha protein) has been shown to potently restrict human immunodeficiency virus (HIV)-1 infection in otherwise susceptible cell lines and CD34(+) cell-derived macrophages. A 13-amino acid patch in the C-terminal B30.2 (SPRY) domain of rhesus macaque TRIM5alpha has been shown to be involved in HIV-1 capsid recognition and is critical for viral inhibition. A chimeric human-rhesus TRIM5alpha (TRIM5alpha-HRH) was generated by replacing an 11-amino acid patch in the human isoform with the rhesus 13-amino acid patch. Here we show that lentiviral vector expression of this human-rhesus chimera in HIV-1-permissive MAGI-CXCR4 cells conferred resistance as well as a selective survival advantage on HIV-1 challenge. To apply these findings in a stem cell gene therapy setting, TRIM5alpha-HRH was expressed in CD34(+) cell-derived macrophages in vitro and in SCID-hu mouse-derived thymocytes in vivo. On viral challenge, transgenic macrophages and thymocytes were highly resistant to HIV-1 compared with control cells. Normal development of TRIM5alpha-HRH-expressing macrophages and in vivo-derived T cells was also observed by phenotypic flow cytometric analysis. These results demonstrate the efficacy of TRIM5alpha-HRH in a stem cell setting and its further advancement for use in gene therapy applications.

  6. Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

    Directory of Open Access Journals (Sweden)

    Baumann Gert

    2010-05-01

    Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

  7. [Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34⁺ Cells].

    Science.gov (United States)

    Wu, Jie-Ying; Lu, Yan; Chen, Jin-Song; Wu, Shao-Qing; Tang, Xue-Wei; Li, Yan

    2015-08-01

    cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed. The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A. The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.

  8. Cd117 and Cd34 Staining Patterns in Childhood Benign Mammary Lesions

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    Ayper KAÇAR

    2012-01-01

    Full Text Available Objective: CD117 and CD34 are markers that have both been implied in cancer progression in adult breast lesions. This study was conducted in order to create a retrospective documentation and to analyze the expression patterns of these markers on childhood benign lesions along with a comparison with adult breast lesions’ staining patterns.Material and Method: Nine fibroadenomas, 2 tubular adenomas, 1 mammary hamartoma, 2 gynecomastias, 1 benign phyllodes tumor were retrieved from pathology archives of two reference centers between 2005-2010.Results: CD117 staining was identified in the epithelium of all cases in fibroadenoma/tubular adenoma group and focally positive in 1 mammary hamartoma, 2 gynecomastias, and 1 benign phyllodes tumor. CD117 staining was detected in the stroma of 8 cases. Three fibroadenomas, 1 mammary hamartoma, 2 gynecomastias and 1 benign phyllodes tumor lacked stromal labelling for this marker. All cases were strongly and diffusely positive for CD34 except the benign phyllodes tumor case. This case presented marked loss of stromal CD34 staining when compared to the surrounding stroma. Additionally, pseudoangiomatous stromal hyperplasia was noted in 2 gynecomastias and in the peritumoral stroma of benign phyllodes tumor case.Conclusion: Our study demonstrated that fibroadenoma was the most commonly encountered breast lesion in childhood and that adolescent fibroadenomas showed similar staining patterns for CD117 and CD34 as for adult counterparts. On the other hand, different expression patterns of CD117 and CD34 between adenoma group and the gynecomastias and benign phyllodes tumor group may implicate different mechanisms of development and tumorigenesis among these groups.

  9. Intra-Arterial Immunoselected CD34+ Stem Cells for Acute Ischemic Stroke.

    Science.gov (United States)

    Banerjee, Soma; Bentley, Paul; Hamady, Mohammad; Marley, Stephen; Davis, John; Shlebak, Abdul; Nicholls, Joanna; Williamson, Deborah A; Jensen, Steen L; Gordon, Myrtle; Habib, Nagy; Chataway, Jeremy

    2014-11-01

    Treatment with CD34+ hematopoietic stem/progenitor cells has been shown to improve functional recovery in nonhuman models of ischemic stroke via promotion of angiogenesis and neurogenesis. We aimed to determine the safety and feasibility of treatment with CD34+ cells delivered intra-arterially in patients with acute ischemic stroke. This was the first study in human subjects. We performed a prospective, nonrandomized, open-label, phase I study of autologous, immunoselected CD34+ stem/progenitor cell therapy in patients presenting within 7 days of onset with severe anterior circulation ischemic stroke (National Institutes of Health Stroke Scale [NIHSS] score≥8). CD34+ cells were collected from the bone marrow of the subjects before being delivered by catheter angiography into the ipsilesional middle cerebral artery. Eighty-two patients with severe anterior circulation ischemic stroke were screened, of whom five proceeded to treatment. The common reasons for exclusion were age>80 years (n=19); medical instability (n=17), and significant carotid stenosis (n=13). The procedure was well tolerated in all patients, and no significant treatment-related adverse effects occurred. All patients showed improvements in clinical functional scores (Modified Rankin Score and NIHSS score) and reductions in lesion volume during a 6-month follow-up period. Autologous CD34+ selected stem/progenitor cell therapy delivered intra-arterially into the infarct territory can be achieved safely in patients with acute ischemic stroke. Future studies that address eligibility criteria, dosage, delivery site, and timing and that use surrogate imaging markers of outcome are desirable before larger scale clinical trials. ©AlphaMed Press.

  10. ACE2/Ang-(1-7)/Mas axis stimulates vascular repair-relevant functions of CD34+ cells.

    Science.gov (United States)

    Singh, Neha; Joshi, Shrinidh; Guo, Lirong; Baker, Matthew B; Li, Yan; Castellano, Ronald K; Raizada, Mohan K; Jarajapu, Yagna P R

    2015-11-15

    CD34(+) stem/progenitor cells have been identified as a promising cell population for the autologous cell-based therapies in patients with cardiovascular disease. The counter-regulatory axes of renin angiotensin system, angiotensin converting enzyme (ACE)/Ang II/angiotensin type 1 (AT1) receptor and ACE2/Ang-(1-7)/Mas receptor, play an important role in the cardiovascular repair. This study evaluated the expression and vascular repair-relevant functions of these two pathways in human CD34(+) cells. CD34(+) cells were isolated from peripheral blood mononuclear cells (MNCs), obtained from healthy volunteers. Expression of ACE, ACE2, AT1, and angiotensin type 2 and Mas receptors were determined. Effects of Ang II, Ang-(1-7), Norleu(3)-Ang-(1-7), and ACE2 activators, xanthenone (XNT) and diminazene aceturate (DIZE) on proliferation, migration, and adhesion of CD34(+) cells were evaluated. ACE2 and Mas were relatively highly expressed in CD34(+) cells compared with MNCs. Ang-(1-7) or its analog, Norleu(3)-Ang-(1-7), stimulated proliferation of CD34(+) cells that was associated with decrease in phosphatase and tensin homologue deleted on chromosome 10 levels and was inhibited by triciribin, an AKT inhibitor. Migration of CD34(+) cells was enhanced by Ang-(1-7) or Norleu(3)-Ang-(1-7) that was decreased by a Rho-kinase inhibitor, Y-27632. In the presence of Ang II, XNT or DIZE enhanced proliferation and migration that were blocked by DX-600, an ACE2 inhibitor. Treatment of MNCs with Ang II, before the isolation of CD34(+) cells, attenuated the proliferation and migration to stromal derived factor-1α. This attenuation was reversed by apocynin, an NADPH oxidase inhibitor. Adhesion of MNCs or CD34(+) cells to fibronectin was enhanced by Ang II and was unaffected by Ang-(1-7). This study suggests that ACE2/Ang-(1-7)/Mas pathway stimulates functions of CD34(+) cells that are cardiovascular protective, whereas Ang II attenuates these functions by acting on MNCs. These findings

  11. Smooth muscles and stem cells of embryonic guts express KIT, PDGFRRA, CD34 and many other stem cell antigens: suggestion that GIST arise from smooth muscles and gut stem cells.

    Science.gov (United States)

    Terada, Tadashi

    2013-01-01

    Gastrointestinal stromal tumor (GIST) is believed to original from interstitial cells of (ICC) present in Auerbach's nerve plexus. GIST frequently shows gain-of-function mutations of KIT and PDGFRA. In practical pathology, GIST is diagnosed by positive immunostaining or KIT and/or CD34. The author herein demonstrates that human embryonic gastrointestinal tract smooth muscles (HEGITSM) and human embryonic stem gastrointestinal cells (HEGISC) consistently express KIT, CD34, NCAM, PDGFRA and other stem cell (SC) antigens NSE, synaptophysin, chromogranin, bcl-2, ErbB, and MET throughout the embryonic development of 7-40 gestational week (GW). CK14 was negative. The author examines 42 cases (7-40 GW) of embryonic GI tract (EGI). The HEGISM, HEGIST, and gall bladder smooth muscles (SM) were consistently positive for KIT, CD34, NCAM, PDGFRA, synaptophysin, chromogranin, NSE, bcl-2, ErbB2, and MET in foregut, stomach, GB, midgut, and hindgut throughout the fetal life (7-40 GW). The stem cells (SC) were seen to create the SM, nerves, ICC, and other all structures of GI tract. In adult gastrointestinal walls (n=30), KIT, CD34, PDGFRA, and S100 proteins were expressed in Auerbach's nerve plexus and ICC. The bronchial and vascular SM of embryos did not express these molecules. In GIST, frequent expressions of KIT (100%, 30/30), CD34 (90%, 27/30), and PDGFRA (83%, 25/30) were seen. In general, characteristics of tumors recapitulate their embryonic life. Therefore, it is strongly suggested that GIST may be originated from GI SM and/or GI SC in addition to ICC.

  12. Differences between the CD34+ and CD34- blast compartments in apoptosis resistance in acute myeloid leukemia.

    NARCIS (Netherlands)

    Stijn, van A.; Pol, van der M.A.; Kok, A.; Bontje, PM; Roemen, GM; Beelen, R.H.J.; Ossenkoppele, G.J.; Schuurhuis, G.J.

    2003-01-01

    BACKGROUND AND OBJECTIVES: Altered expression of members of the Bcl-2 family might account for the observed apoptosis resistance to chemotherapy in acute myeloid leukemia (AML). Given the poor prognosis associated with CD34+ expression in AML, we studied the role of spontaneous apoptosis and

  13. The equivalents of human blood and spleen dendritic cell subtypes can be generated in vitro from human CD34+ stem cells in the presence of fms-like tyrosine kinase 3 ligand and thrombopoietin

    OpenAIRE

    Proietto, AI; Mittag, D; Roberts, AW; Sprigg, N; L. Wu

    2012-01-01

    Dendritic cells (DCs) are immune cells specialized to capture, process and present antigen to T cells in order to initiate an appropriate adaptive immune response. The study of mouse DC has revealed a heterogeneous population of cells that differ in their development, surface phenotype and function. The study of human blood and spleen has shown the presence of two subsets of conventional DC including the CD1b/c+ and CD141+CLEC9A+ conventional DC (cDC) and a plasmacytoid DC (pDC) that is CD304...

  14. Expression and clinical significance of GPC3 and CD34 in hepatocellular carcinoma%GPC3和CD34在肝细胞性肝癌中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    畅俊平; 王丽霞

    2013-01-01

    Objective This paper analyzes the GPC3 and CD34 expression in hepatocellular carcinoma,discuss the relationship with diagnosis and treatment of primary liver cancer.Methods In the surgical cases of primary liver cancer,screening 30 specimens cases of cirrhosis of liver cancer.Immunohistochemical markers GPC3 and CD34.Results Cancer tissue show positive in all 30 GPC3 cases,but with different strength,the paracancerous liver cirrhosis area were negative; Cancer organization sinus endothelial shows positive in CD34,all cases show capillarization,different cases show different vascular density,cirrhosis of the liver tissue paracancerous only show positive in stromal vascular,show negative in liver cord.CD34 has high density of positive in cases of GPC3 with strong positive;it has the statistically significant (P<0.05).Conclusion GPC3 and CD34 could use for diagnosis and differential diagnosis of liver cancer,GPC3 may play an important role in the early diagnosis of liver cancer as a carcinoembryonic protein; CD34 shows capillarization of sinusoidal endothelial cancer tissue may related to recurrence and metastasis after interventional treatment.%目的 分析GPC3和CD34在肝癌组织中的表达,探讨与原发性肝癌诊断与治疗的关系.方法 在原发性肝癌手术病例中,筛选肝癌伴有肝硬化的标本30例,免疫组织化学标记GPC3和CD34.结果 33例GPC3癌组织全部阳性,阳性强度不同,癌旁肝硬化区均阴性;CD34癌组织窦内皮阳性,均显示毛细血管化,不同病例显示血管的密度不同,癌旁肝硬化组织只是间质血管阳性,肝索间阴性.GPC3强阳性的病例CD34阳性密度也高,差异有统计学意义(P<0.05).结论 GPC3与CD34可以用于肝癌的诊断与鉴别诊断,GPC3可能作为一种癌胚蛋白在肝癌早期诊断中发挥重要作用;CD34可能与介入治疗后复发与转移有关.

  15. The effect of CD34(+) cell telomere length and hTERT expression on the outcome of autologous CD34(+) cell transplantation in patients with chronic heart failure.

    Science.gov (United States)

    Rozman, Jasmina-Ziva; Perme, Maja Pohar; Jez, Mojca; Malicev, Elvira; Krasna, Metka; Novakovic, Srdjan; Vrtovec, Bojan; Rozman, Primoz

    2017-09-01

    Age-related telomere attrition in stem/progenitor cells may diminish their functional capacity and thereby impair the outcome of cell-based therapies. The aim of the present study was to investigate the effect of CD34(+) cell telomere length and hTERT expression on the clinical outcome of autologous CD34(+) cell transplantation. We studied 43 patients with cardiomyopathy. Their peripheral blood CD34(+) cells were mobilized with granulocyte colony-stimulating factor, enriched by immunoselection and delivered transendocardially. Relative telomere length and expression levels of hTERT were measured using a real-time PCR assay. Immunoselected CD34(+) cells had longer telomere length compared to leukocytes in leukapheresis products (p=0.001). In multivariate analysis, CD34(+) cell telomere length was not associated with the clinical outcome (b=3.306, p=0.540). While hTERT expression was undetectable in all leukapheresis products, 94.4% of the CD34(+) enriched cell products expressed hTERT. Higher CD34(+)hTERT expression was associated with a better clinical outcome on univariate analysis (b=87.911, p=0.047). Our findings demonstrate that CD34(+) cell telomere length may not influence the clinical outcome in cardiomyopathy patients treated with autologous CD34(+) cell transplantation. Larger studies are needed to validate the impact of the CD34(+)hTERT expression on the clinical outcome of autologous CD34(+) cell transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

    Science.gov (United States)

    van de Ven, Rieneke; Reurs, Anneke W; Wijnands, Pepijn G J T B; van Wetering, Sandra; Kruisbeek, Ada M; Hooijberg, Erik; Scheffer, George L; Scheper, Rik J; de Gruijl, Tanja D

    2012-02-01

    Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

  17. Prognostic value of circulating CD34+ cells in myelodysplastic syndromes.

    Science.gov (United States)

    Cesana, Clara; Klersy, Catherine; Brando, Bruno; Nosari, Annamaria; Scarpati, Barbara; Scampini, Linda; Molteni, Alfredo; Nador, Guido; Santoleri, Luca; Formenti, Marta; Valentini, Marina; Mazzone, Antonino; Morra, Enrica; Cairoli, Roberto

    2008-11-01

    We studied circulating (C)CD34(+) cells by flow cytometry in 96 patients with myelodysplastic syndromes (MDS) at diagnosis, and in a subset of 35 cases during follow-up. CCD34(+) counts were stratified within both International Prognostic Scoring System (IPSS) and World Health Organization (WHO) categories. Counts >10/microl were associated with poorer leukemia-free survival, a prognostic value for evolution independent from that of WHO, and a higher progression probability within intermediate-risk IPSS and WHO classes. When serial measurements were performed, counts >10/microl more frequently correlated to evolution. Separating newly diagnosed patients on the basis of 10/microl cut-off of circulating CD34(+) cells retains prognostic utility, especially in intermediate-risk MDS.

  18. CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages.

    Science.gov (United States)

    Matsuoka, S; Ebihara, Y; Xu, M; Ishii, T; Sugiyama, D; Yoshino, H; Ueda, T; Manabe, A; Tanaka, R; Ikeda, Y; Nakahata, T; Tsuji, K

    2001-01-15

    The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin(-)c-Kit(+) long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin(-)c-Kit(+)CD34(-) marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin(-)c-Kit(+) CD34(high) HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin(-)c-Kit(+)CD34(-) cells after 16 weeks, indicating that adult Lin(-)c-Kit(+)CD34(-) HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.

  19. Differential number of CD34+, CD133+ and CD34+/CD133+ cells in peripheral blood of patients with congestive heart failure

    Directory of Open Access Journals (Sweden)

    Fritzenwanger M

    2009-03-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC which are characterised by the simulateous expression of CD34, CD133 and vascular endothelial growth receptor 2 (VEGF 2 are involved in the pathophysiology of congestive heart failure (CHF and their number and function is reduced in CHF. But so far our knowledge about the number of circulating hematopoietic stem/progenitor cells (CPC expressing the early hematopoietic marker CD133 and CD34 in CHF is spares and therefore we determined their number and correlated them with New York Heart Association (NYHA functional class. Methods CD34 and CD133 surface expression was quantified by flow cytometry in the peripheral venous blood of 41 healthy adults and 101 patients with various degrees of CHF. Results CD34+, CD133+ and CD34+/CD133+ cells correlated inversely with age. Both the number of CD34+ and of CD34+/CD133+ cells inversely correlated with NYHA functional class. The number of CD133+ cells was not affected by NYHA class. Furthermore the number of CD133+ cells did not differ between control and CHF patients. Conclusion In CHF the release of CD34+, CD133+ and CD34+/CD133+ cells from the bone marrow seems to be regulated differently. Modulating the releasing process in CHF may be a tool in CHF treatment.

  20. File list: DNS.Bld.10.AllAg.CD34+ [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.10.AllAg.CD34+ hg19 DNase-seq Blood CD34+ SRX062346,SRX062347,SRX027080,SRX...RX026920,SRX026915,SRX026927,SRX026921,SRX062365,SRX214040,SRX040374 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.10.AllAg.CD34+.bed ...

  1. Circulating hematopoietic progenitors and CD34+ cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution

    Directory of Open Access Journals (Sweden)

    Yu JT

    2016-02-01

    Full Text Available Jui-Ting Yu,1,2,* Shao-Bin Cheng,3,* Youngsen Yang,1 Kuang-Hsi Chang,4 Wen-Li Hwang,1 Chieh-Lin Jerry Teng,1,5,6 1Division of Hematology/Medical Oncology, Department of Medicine, Taichung Veterans General Hospital, 2Division of Hematology/Medical Oncology, Tungs' Taichung MetroHarbor Hospital, 3Division of General Surgery, Department of Surgery, 4Department of Medical Research and Education, Taichung Veterans General Hospital, 5Department of Life Science, Tunghai University, 6School of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China *These authors contributed equally to this work Background: Previous studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC and CD34+ cell are positively correlated with CD34+ cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34+ cells required for performing an efficient hematopoietic stem cell (HSC harvest in lymphoma and myeloma patients have not been defined in our institution. Patients and methods: Medical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×106 CD34+ cells/kg and ≥5×106 CD34+ cells/kg, respectively. Results: The minimally required or optimal HSC yield obtained was not influenced by age (≥60 years, sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34+ cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34+ cell and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34+ cell. The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm3 for HPCs and 10/mm3 for CD34+ cells. Furthermore, the cell count cutoff for obtaining

  2. Expression of CD34 and maspin in ameloblastoma from a West African subpopulation.

    Science.gov (United States)

    Udeabor, S E; Adisa, A O; Kolude, B; Barbeck, M; Kirkpatrick, C J; Sader, R A; Ghanaati, S

    2014-08-01

    Ameloblastoma is a locally invasive odontogenic tumor with a high recurrence rate. Its local invasiveness is aided by angiogenesis, which can be correctly estimated by CD34. On the other hand, maspin decreases the local invasive and metastatic capability of cancer cells and functions as an angiogenesis inhibitor. We aim to assess the association between maspin expression and microvessel density in ameloblastoma. Twenty-five formalin-fixed paraffin-embedded (FFPE) blocks of ameloblastoma cases were prepared for antibody processing to CD34 and maspin. Positive immunohistochemical staining was marked by brown cytoplasmic/membrane coloration for CD34 and by nuclear/cytoplasmic coloration for maspin. At the ×40 magnification, we counted blood vessels in two areas of dimension; 300 × 400 μm (area A) and 150 × 200 μm (area B) adjacent to the tumor region to assess relative dispersion of the vessels bordering the tumor. The overall approximate microvessel density (MVD) for area A = 11 (minimum 2, maximum 21) and that for area B = 5 (minimum 1, maximum 10). The MVD in the area A of plexiform ameloblastoma was similar to that of the unicystic, while the hemangiomatous variant had the highest MVD for area A. Maspin positivity was present only in the cytoplasm of ameloblast, stellate reticulum, and the fibrous connective tissue in varying proportions. There was no evidence of the anti-angiogenesis effect of maspin in ameloblastoma from this study. The significance of cytoplasmic localization of maspin in the ameloblasts and stellate reticulum cells needs further investigation.

  3. 免疫磁珠分选白血病KG1a细胞中CD34+CD38-干细胞及其特性研究%Isolation and characteristic of CD34 + CD38-stem cells in leukemia cell line KG1a using magnetic activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    王国征; 李慧; 吴远彬; 李达; 贺艳杰; 周雪云

    2013-01-01

    目的 从白血病KGla细胞中分选CD34+ CD38-干细胞并研究其生物学特性.方法 免疫磁珠法分选CD34+ CD38-细胞,流式细胞术分析细胞表面膜抗原、细胞周期,甲基纤维素培养体系观察其克隆性;以HL60、K562、CD34+ CD38+细胞为对照,甲基偶氮唑蓝法检测柔红霉素对CD34+ CD38-细胞的抑制作用;BALB/c裸鼠皮下接种,观察体内成瘤能力.结果 分选的CD34+ CD38-细胞纯度达95%以上,(69.03 +3.25)%处于Go期,克隆形成率为(38.64±2.68)%,明显抵抗柔红霉素;不同浓度柔红霉素作用后,CD34+ CD38-、CD34+ CD38+、HL60、K562细胞的活性差异有统计学意义(F =961.136,P=0.000);CD34+ CD38-在裸鼠皮下成瘤率显著高于CD34+ CD38+细胞(P<0.05).结论 免疫磁珠法分选白血病干细胞简单易行,分选的细胞符合白血病干细胞生物学特性.%Objective To isolate the CD34+ CD38- stem cells from human acute leukemia cell line KGla and to research its biological characteristics. Methods CD34+ CD38- cells were isolated by magnetic activated cell sorting( MACS) ; the cell surface membrane antigens and cell cycle were analyzed by flow cytometry; its clonality was observed with methyl cellulose system. HL60,K562 and D34+ CD38+ cells were selected as control,and the methyl thiazolyl tetrazolium(MTT) assay was taken to observe the depressant effect of rubidomycin to CD34+ CD38- cells. BALB/c nude mice were inoculated subcutaneous-ly to observe the tumorigenicity. Results The purity of CD34+ CD38- cells were above 95% and(69.03 ± 3. 25) % cells in the G0 phase. The cloning efficiency of CD34+ CD38- cells were( 38. 64 ± 2. 68) % , and the CD34+ CD38- cells were obviously resistant to rubidomycin. There were statistic difference of cytoactive among CD34+ CD38-, CD34+ CD38 + , HL60, and K562 cells under giving the same concentrations of rubidomycin circumstances (F = 961. 136,P= 0.000). The tumorigenesis ability of CD34+ CD38- cells in nude mice was

  4. CD34(low) and SMA(high) represent stromal signature in uterine cervical cancer and are markers for peritumoral stromal remodeling.

    Science.gov (United States)

    Horn, Lars-Christian; Schreiter, Carolin; Canzler, Anika; Leonhardt, Karoline; Einenkel, Jens; Hentschel, Bettina

    2013-12-01

    Peritumoral desmoplastic stromal reaction (DSR) with myofibroblastic phenotype may be of prognostic impact in uterine cervical carcinoma. The present study evaluates the immunostaining (CD34 and smooth muscle actin; SMA) of 97 squamous cell cancers. Staining was scored as low/negative (50%) and DSR as negative/weak and moderate/strong. The staining results were correlated to patient survival. Of the cases, 78.3% showed a decreased of CD34 (SMA staining with more than 50% SMA positive stromal cells. Tumors representing moderate/strong DSR showed a significant decreased CD34 (P=.001) and an increased but not statistically significant SMA staining (P=0.345). Cases with low CD34 and high SMA staining showed reduced 5-year overall survival when compared to cases with high CD34 and low SMA positivity (59.9 vs 81.0%; P=0.025 and 64.6 vs 81.1%; P=0.243). Peritumoral stromal response in cervical carcinoma is immunohistochemically characterized by CD34(low)/SMA(high) and associated reduced overall survival. © 2013.

  5. Effect of Homoharringtonine on Bone Morrow CD34+CD7+ Cells in Chronic Granulocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    LI Yu-feng; DENG Zhi-kui; XUAN Heng-bao; CHEN Bao-an

    2007-01-01

    Objective: To explore the effect of homoharringtonine (HHT) on bone morrow CD34+CD7+ cells in chronic granulocytic leukemia (CGL). Methods: The changes of bone morrow CD34+CD7+ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34+CD7+ cells treated with HHT in vitro were studied. Results: The proportion of CD34+CD7+ cells in CGL (0.145±0.021) was higher than that of normal control (0.052±0.013). The proportion of CD34+CD7+ cells in patients who got cytogenetic responses to HHT (0.072±0.020) decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, (0.137±0.023). the proliferation of CD34+ cells was inhibited and the proportion of CD34+CD7+ cells decreased after cultured with HHT (0.134 in 24 h, 0.126 in 48 h and 0.102 in 72). The apoptosis rate of CD34+CD7+ cells was higher than that in CD34+CD7- cells (35.39%±4.39% versus 24.57%(4.01%, P<0.05) 72 h after culture with HHT. Conclusion: The proportion of CD34+CD7+ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34+CD7+ cells.

  6. Direct Comparison of Wharton's Jelly and Bone Marrow-Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants

    Science.gov (United States)

    van der Garde, Mark; van Pel, Melissa; Millán Rivero, Jose Eduardo; de Graaf-Dijkstra, Alice; Slot, Manon C.; Kleinveld, Yoshiko; Watt, Suzanne M.; Roelofs, Helene

    2015-01-01

    Cotransplantation of CD34+ hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs) enhances HSPC engraftment. For these applications, MSCs are mostly obtained from bone marrow (BM). However, MSCs can also be isolated from the Wharton's jelly (WJ) of the human umbilical cord. This source, regarded to be a waste product, enables a relatively low-cost MSC acquisition without any burden to the donor. In this study, we evaluated the ability of WJ MSCs to enhance HSPC engraftment. First, we compared cultured human WJ MSCs with human BM-derived MSCs (BM MSCs) for in vitro marker expression, immunomodulatory capacity, and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSCs have a more restricted differentiation capacity, both WJ MSCs and BM MSCs expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Most importantly, cotransplantation of either WJ MSCs or BM MSCs with CB CD34+ cells into NOD SCID mice showed similar enhanced recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood, and spleen 6 weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon coincubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although stromal cell-derived factor-1 (SDF-1)-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSCs in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSCs replaced WJ MSCs in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. PMID:26414086

  7. Significance of CD34-and CD34+ Cell Apoptosis and Proliferation in Bone Marrow of Patients with MDS and Their Impact on Survival%骨髓增生异常综合征患者骨髓CD34-和CD34+细胞凋亡与增殖的意义及对生存的影响

    Institute of Scientific and Technical Information of China (English)

    夏冰; 郭青; 赵丹丹; 赵海丰; 韩晓蘋; 王卉; 吴晓雄; 张翼鷟

    2012-01-01

    Alteration in the balance between cell apoptosis and proliferation is one of the pathophysiological mechanisms of the myelodysplastic syndromes (MDS). The question of whether the excessive apoptosis and/ or proliferation predominantly involve the subset of progenitor cells (CD34+ cells) or mature cells (CD34- cells) remains a controversial issue. This study was purposed to analyze the apoptosis and proliferation status of CD34+ and CD34" cells in bone marrow(BM) of patients with MDS, to investigate the pathogenesis of MDS and to determine the relation of apoptosis and proliferation status of CD34+ and CD34" cells with prognosis of MDS. The proprotion of CD34+ cells, the apoptosis and proliferation ratio(A/P) of CD34+ and CD34" cells in BM of 40 patients with MDS, including 20 cases of high-risk MDS and 20 cases of low-risk MDS, and 10 normal persons as control were detected by flow cytometry; the influence of CD34+ and CD34" cell apoptosis and proliferation levels on prognosis of MDS was evaluated by unvariate and multivariate analysis of survival. The results showed that the proportion of CD34+ cells in BM of high-risk MDS patients was significantly higher than that in BM of low-risk MDS patients and in normal BM [(1.92±0.10)% , (1.09±0.04)% , (1.03±0.05)% respectively]. The apoptotic rates (AR) ofbothCD34+ and CD34+ cells were significantly higher in low-risk MDS [(54.75 ±2.18)% ,(80.36 ±1.68)% ] than in high-risk MDS [(24.87 ±2.69)%, (23. 12 ±1.23)%] and in normal BM [(18.51 ±2.74)%, (20.98 ±2.21)%]. When compared between CD34+ cells and CD34- cells in low-risk MDS, a greater AR of CD34- cells was found. However, the higher proliferative rate of CD34+ cells was observed in high-risk MDS. In low-risk MDS, a higher A/P ratio was found in CD34- cells than in CD34+ cells; whereas this ratio was equalized or inverted in high-risk MDS. In addition, the survival and prognosis correlated significantly with AR of CD34+ cells. It is concluded that the early MDS

  8. File list: NoD.Bld.10.AllAg.CD34+ [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.10.AllAg.CD34+ hg19 No description Blood CD34+ SRX031353,SRX088853,SRX01972...9745,SRX014508,SRX088780,SRX088957,SRX088866,SRX019722,SRX019717,SRX041001,SRX019718 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.10.AllAg.CD34+.bed ...

  9. File list: NoD.Bld.05.AllAg.CD34+ [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.05.AllAg.CD34+ hg19 No description Blood CD34+ SRX019720,SRX088853,SRX03135...8866,SRX019717,SRX088780,SRX031425,SRX019745,SRX040992,SRX031453,SRX019718,SRX019722 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.05.AllAg.CD34+.bed ...

  10. Characterization of transcription factor networks involved in umbilical cord blood CD34+ stem cells-derived erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Biaoru Li

    Full Text Available Fetal stem cells isolated from umbilical cord blood (UCB possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1 and 30 TFs were activated by day 56 (Profile-2. Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1 and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34

  11. Interactions of monocyte subpopulations generated from cord blood CD34(+) hematopoietic progenitors with tumor cells: assessment of antitumor potential.

    Science.gov (United States)

    Stec, Malgorzata; Baran, Jaroslaw; Szatanek, Rafal; Mytar, Bozenna; Baj-Krzyworzeka, Monika; Gozdzik, Jolanta; Siedlar, Maciej; Zembala, Marek

    2012-11-01

    Monocytes and their subsets (CD14(++)CD16(+) and CD14(+)CD16(-)) generated from cord blood CD34(+) progenitor cells were used for determination of their capacity to interact with tumor cells in vitro and in vivo. The studies in vitro included adhesion to human umbilical vein endothelial cells, cytotoxicity, production of toxic mediators: reactive oxygen and nitrogen intermediates (ROI and RNI, respectively), and finally their effect on transplantable human tumor growth in nonobese diabetic severe combined immunodeficient mice. The CD14(++)CD16(+) subset exhibited an increased adherence to human umbilical vein endothelial cells and cytotoxicity toward tumor cells in vitro. CD14(+)CD16(-) monocytes showed a higher production of reactive oxygen and nitrogen intermediates after stimulation with tumor cells, and more pronounced inhibition of tumor growth in vivo. The results revealed significant differences in the behavior of CD14(++)CD16(+) and CD14(+)CD16(-) monocyte subsets toward tumor cells, thus providing further evidence that CD34(+) cell-derived monocytes differ in this respect from blood monocytes. The protocol for generation of monocytes with antitumor reactivity described here may be useful to obtain monocytes from CD34(+) progenitor cells of cancer patients. This might offer a basis for a novel approach for various forms of cellular immunotherapy of cancer.

  12. GM-CSF诱导人脐血CD34+造血干细胞CXC细胞趋化因子受体-3表达和配体Γip-10、Mig功能研究

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    CXCR3, know to be predominately expressed on memory/activated T lymphocytes,is a receptor for both γ IP-10 and Mig. We report a novel finding that CXCR3 is also expressed on GM-CSF-stimulated, but not freshly isolated, CD34+ hematopoietic pro genitors from human cord blood. Freshly isolated CD34 + progenitors express low level CXCR3 mRNA, but this expression is highly up-regulated by GM-CSF detected using real time quantitative RT-PCR technique. γ IP-10 and Mig induced GM-CSF stimulated CD34+ progenitor chemotaxis via CXCR3 documented by the fact that anti-CXCR3 mAb blocks γ IP-10 and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colonyforming unit-ganulocyte macrophages. Besides induc tion to chemotaxis, γIP-10 and Mig also induce GM-CSF-stimulated CD34+ progenitor adhesion and aggregation via CXCR3,con firmed by the observation that anti-CXCR3 mAb blocks these functions of γIP-10 and Mig,but not of SDF-1α.γ IP-10 and Mig-in duced integrin (CD49a and CD49b) up-regulation plays crucial role in adhesion of GM-CSF-stimulated CD34+ progenitors. Moreover, γ IP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34+ progenitors. These results indicate that CXCR3-γ IP-10 and -Mig receptor-ligand pairs as well as the effects of GM-CSF on them may be especially important in cytokine/chemokine environment for the physiological and pathophysiological events of differentiation of CD34+ hematopoietic pro genitors into lymphoid and myeloid stem cells, subsequently immune/infl mmatory cells. These processes are including transmigration, relocation,differentiation and maturation of CD34+ hematopoietic progenitors.

  13. The balance of positive and negative effects of TGF-β signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells.

    Science.gov (United States)

    Bai, Hao; Xie, Yin-Liang; Gao, Yong-Xing; Cheng, Tao; Wang, Zack Z

    2013-10-15

    Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34(+)CD31(+)VE-cadherin(+) (VEC(+)) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34(+)CD31(+)VEC(+) cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-β) 1 protein is increased during hPSC differentiation, and that TGF-β signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-β to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34(+)CD31(+)VEC(+) cells. An addition of TGF-β inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34(+)CD31(+)VEC(+) progenitor cells. However, inhibition of TGF-β signaling after mesoderm induction increases CD34(+)CD31(+)VEC(+) progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-β signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

  14. Intracoronary Injection of CD34-Cells in Chronic Ischemic Heart Failure

    DEFF Research Database (Denmark)

    Hansen, Morten; Nyby, Sebastian; Eifer Møller, Jacob;

    2014-01-01

    × 10(6) CD34(+) cells and 14 ± 7 × 10(6) CD133(+) cells). Patients were followed for 7 years and deaths were recorded. Results: During follow-up, 10 patients died (31%). In univariate regression analysis, the total number of BMSCs, CD34(+) cell count and CD133(+) cell count did not significantly...

  15. Interleukin-15 Promotes the Commitment of Cord Blood CD34+ Stem Cells into NK Cells

    Institute of Scientific and Technical Information of China (English)

    张建; 夏青; 孙汭; 田志刚

    2004-01-01

    To explore the effect of rhlL-15 on CB-CD34+ stem cells committing to NK cells, CD34+ stem cells were obtained from cord blood (CB) by magnetic-assisted cell sorting (MACS) method. CD3, CD16 and CD56 molecules expressed on cell surface were detected by flow cytometer. MTF method was used to test the cytotoxicity of NK cells. The results were that stem cell factor (SCF) alone has no effect on CD34+ stem cells. IL-15 stimulated CD34+ stem cells commit to NK cells, and SCF showed strong synergistic effect with IL-15. It was concluded that IL-15 and SCF played different roles during NK cell development, llr15 promoted CD34+ stem cells differentiate to NK cell precursor and SCF improved the effectsof IL-15 on NK cell differentiation.

  16. Evidence that platelet-derived microvesicles may transfer platelet-specific immunoreactive antigens to the surface of endothelial cells and CD34+ hematopoietic stem/ progenitor cells--implication for the pathogenesis of immune thrombocytopenias.

    Directory of Open Access Journals (Sweden)

    Mariusz Z Ratajczak

    2007-03-01

    Full Text Available The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic stem/progenitors may also become affected. Based on our previous work that platelet antigens (e.g., CD41 may be transferred by platelet-derived microvesicles (PMV to the surface of other cells, we asked if platelet derived-antigens, especially those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA 1 a could be also transferred by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells (HUVEC and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the HPA1a antigen is highly expressed on PMV-derived from the HPAla positive platelets and is transferred in PMV-dependent manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive PMV but not by PMV derived from HPAla negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and subsequently exposed to anti-HPA 1 a serum and human NK cells, become subject to elimination by antibody dependent cell cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding" the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that accompany IT (e.g. damage of endothelium, cytopenias.

  17. In vitro effects of imatinib on CD34+ cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase

    Science.gov (United States)

    MENG, FANKAI; ZENG, WEN; HUANG, LIFANG; QIN, SHUANG; MIAO, NINGNING; SUN, HANYING; LI, CHUNRUI

    2014-01-01

    Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34+ cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34+ cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34+ cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase. PMID:24527087

  18. In vitro effects of imatinib on CD34(+) cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase.

    Science.gov (United States)

    Meng, Fankai; Zeng, Wen; Huang, Lifang; Qin, Shuang; Miao, Ningning; Sun, Hanying; Li, Chunrui

    2014-03-01

    Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34(+) cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34(+) cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34(+) cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase.

  19. 急性淋巴细胞白血病CD34+CD38-细胞在NOD/SCID小鼠体内的自我更新与增值能力%Self-renewal and proliferation capability of CD34 + CD38 - cells from acute lymphoblastic leukemia patients transplanted in NOD/SCID mice

    Institute of Scientific and Technical Information of China (English)

    周晓燕; 龙娟; 谭俊杰; 邹琳

    2012-01-01

    Objective To explore the feasibility of establishing mouse model of leukemia by transplantion of human acute lymphoblastic leukemia (ALL) CD34+ CD38- cells into immunodeficient (NOD/SCID) mice, and to study the capability of self-renewal and proliferation in the mice. Methods The CD34+ CD38- cells were isolated from ALL patients and then identified, and the CD34- CD38+ cells were used as control. These cells were transplanted into sublethally irradiated NOD/SCID mice by tail-vein injection of 10 cells per recipient. The changes of peripheral blood were monitored continuously , and histopathological examination of the bone marrow, spleen and liver was performed in each death or dying mouse. Results The number of leukocytes in mouse peripheral blood was increased after inoculation of CD34+ CD38- or CD34- CD38+ cells at 4 weeks, peaked at 8 weeks up to 15 × 109~20 × 109/L, and also increased the number of original and blast lymphocytes. There were mainly increased the number of the original and blast lymphocytes (40% ) in the bone marrow, as well as the leukemic cells infiltrating in the spleen and liver of CD34 ' CD38 ~ mice, which was more obvious than that of the control CD34- CD38+ mice. Conclusions CD34+ CD38- cells from ALL patients can be successfully transplanted into NOD/SCID mice to induce leukemia, indicating that the CD34+ CD38- cells have the capability of self-renewal and proliferation. This model can be used as an important and useful tool for studies on leukemia-initiating cells.%目的 探索急性淋巴细胞白血病(ALL)患者CD34+ CD38-细胞移植到NOD/SCID小鼠体内建立白血病的可行性、自我更新与增殖潜能.方法 分选并鉴定ALL患者骨髓CD34+ CD38-细胞及对照CD34- CD38+细胞后,经尾静脉分别注射104个细胞于亚致死剂量射线照射的NOD/SCID小鼠体内,连续监测小鼠状态以及外周血血象改变,对濒死或死亡小鼠进行骨髓检查、肝脾病理学检查.结果 接种从ALL患者分选的CD

  20. [Extracellular HMGB1 promotes the migration of cord Blood CD34⁺ cells via SDF-1/CXCR-4 axis].

    Science.gov (United States)

    Yang, Lu-Lu; Sun, Zi-Min; Liu, Xin; Zhu, Xiao-Yu; Wang, Xing-Bing; Wang, Jian

    2014-10-01

    This study was aimed to investigate the effect of high mobility group box1(HMGB1) and/or stromal cell derived factor-1(SDF-1) on the migration of cord blood CD34⁺ cells, and to explore whether HMGB1 promotes cord blood CD34⁺ cell migration via SDF-1/CXCR4 axis. Cord blood mononuclear cells were isolated by Ficoll-Paque density centrifugation, CD34⁺ cells were collected by a positive immunoselection procedure (CD34 MicroBeads) according to the manufacturer's instructions, the purity of the isolated CD34⁺ cells was detected by flow cytometry. In vitro chemotaxis assays were performed using Transwell cell chambers to detect cells migration. 1 × 10⁵ cells/well cord blood CD34⁺ cells were added into the upper chambers. Different concentrations of HMGB1 and/or SDF-1 (0, 10, 25, 50, 100, 200 ng/ml) were used to detect the optimal concentrations of HMGB1 and/or SDF-1 for inducing migration of cord blood CD34⁺ cells. Freshly isolated cord blood CD34⁺ cells express CXCR4 (SDF-1 receptor), and HMGB1 receptor TLR-2,TLR-4 and RAGE. To explore which receptors were required for the synergy of HGMB1 and/or SDF-1 on cells migration, the anti-SDF-1, anti-CXCR4 and anti-RAGE antibodies were used to detect the effect of HGMB1 alone or with SDF-1 on cord blood CD34⁺ cells migration. The results showed that the purity of CD34⁺ cells isolated from cord blood mononuclear cells by magnetic cell sorting was 97.40 ± 1.26%, the 25 ng/ml SDF-1 did not induce migration of cord blood CD34⁺ cells, whereas the optimal migration was observed at 100 ng/ml. HMGB1 alone did not induce migration up to 100 ng/ml. The dose test found that the the best synergistic concentrations for cells migration were 100 ng/ml HMGB1 combined with 50 ng/ml SDF-1. The blocking test showed that both the anti-SDF-1 (4 µg/ml) and anti-CXCR4 (5 µg/ml) antibodies could block cell migration induced by HMGB1 or combined with SDF-1, but the cord blood CD34⁺ cells in the presence of anti-RAGE, anti

  1. ELMO1 is upregulated in AML CD34+ stem/progenitor cells, mediates chemotaxis and predicts poor prognosis in normal karyotype AML.

    Directory of Open Access Journals (Sweden)

    Marta E Capala

    Full Text Available Both normal as well leukemic hematopoietic stem cells critically depend on their microenvironment in the bone marrow for processes such as self-renewal, survival and differentiation, although the exact pathways that are involved remain poorly understood. We performed transcriptome analysis on primitive CD34+ acute myeloid leukemia (AML cells (n = 46, their more differentiated CD34- leukemic progeny, and normal CD34+ bone marrow cells (n = 31 and focused on differentially expressed genes involved in adhesion and migration. Thus, Engulfment and Motility protein 1 (ELMO1 was identified amongst the top 50 most differentially expressed genes. ELMO1 is a crucial link in the signaling cascade that leads to activation of RAC GTPases and cytoskeleton rearrangements. We confirmed increased ELMO1 expression at the mRNA and protein level in a panel of AML samples and showed that high ELMO1 expression is an independent negative prognostic factor in normal karyotype (NK AML in three large independent patient cohorts. Downmodulation of ELMO1 in human CB CD34+ cells did not significantly alter expansion, progenitor frequency or differentiation in stromal co-cultures, but did result in a decreased frequency of stem cells in LTC-IC assays. In BCR-ABL-transduced human CB CD34+ cells depletion of ELMO1 resulted in a mild decrease in proliferation, but replating capacity of progenitors was severely impaired. Downregulation of ELMO1 in a panel of primary CD34+ AML cells also resulted in reduced long-term growth in stromal co-cultures in two out of three cases. Pharmacological inhibition of the ELMO1 downstream target RAC resulted in a severely impaired proliferation and survival of leukemic cells. Finally, ELMO1 depletion caused a marked decrease in SDF1-induced chemotaxis of leukemic cells. Taken together, these data show that inhibiting the ELMO1-RAC axis might be an alternative way to target leukemic cells.

  2. Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

    DEFF Research Database (Denmark)

    Ladeby, Rune; Wirenfeldt, Martin; Dalmau, Ishar

    2005-01-01

    following transection of the entorhino-dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion-reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (Brd......U) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early-activated resident microglia, rather than by infiltrating bone marrow-derived cells. The number of CD34(+) microglia peaked at day 3 when 67% of the resident CD11b/Mac-1(+) microglia co-expressed CD34, and all...... CD34(+) cells co-expressed Mac-1, and decreased sharply toward day 5, unlike Mac-1, which was maximally expressed at day 5. Approximately 80% of the CD34(+) cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early...

  3. Levels of circulating CD34+/KDR+ cells do not predict coronary in-stent restenosis.

    Science.gov (United States)

    Haine, Steven E; Van Craenenbroeck, Emeline M; Hoymans, Vicky Y; Miljoen, Hielko P; Vandendriessche, Tom R; Claeys, Marc J; Frederix, Geert; Conraads, Viviane M; Bosmans, Johan M; Vrints, Christiaan J

    2014-01-01

    Angiographic and clinical parameters are poor predictors of in-stent restenosis. Bone marrow-derived CD34(+) cells that coexpress a receptor for vascular endothelial growth factor (kinase insert domain receptor [KDR]) are committed to endothelial lineage. Mobilization and infusion of CD34(+)/KDR(+) cells accelerates re-endothelialization and reduces neointimal thickness in vascular injury models. Bioengineered stents capturing CD34(+) cells also show expedited re-endothelialization. We examined whether circulating CD34(+)/KDR(+) cell counts can be used to predict restenosis in a bare-metal stent (BMS). CD34(+)/KDR(+) cells were counted by flow cytometry in 124 nondiabetic patients before BMS implantation and the relation to in-stent late luminal loss (LLL) was examined by angiography at 6 months (primary end point). Neointima was also quantified as the maximum percentage area stenosis (M%AS) and percentage volume intima hyperplasia (%VIH) on intravascular ultrasonography (secondary end points). Multiple linear regression analysis, taking into account implanted stent length and diameter, revealed no relation between CD34(+)/KDR(+) cell counts and LLL (partial regression coefficient b = 0.11; 95% confidence interval [CI], -0.19-0.42; P = 0.46). Similarly, no relation between CD34(+)/KDR(+) cell counts and M%AS or %VIH could be demonstrated. Moreover, the increase in CD34(+)/KDR(+) cell counts over 6 months was unrelated to LLL (b = -0.15; 95% CI, -0.42-0.12; P = 0.28), M%AS, and %VIH. Although our study does not exclude a pathophysiologic role for CD34(+)/KDR(+) cells in the formation of neointima, cell counts before percutaneous coronary intervention proved to be unrelated to LLL or intravascular ultrasonographically derived restenosis parameters in coronary BMSs at 6 months. Copyright © 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

  4. Clinical significance of detecting lymphatic and blood vessel invasion in stage II colon cancer using markers D2-40 and CD34 in combination.

    Science.gov (United States)

    Lai, Jin-Huo; Zhou, Yong-Jian; Bin, Du; Qiangchen; Wang, Shao-Yuan

    2014-01-01

    This research was conducted to compare differences in colon cancer lymphatic vessel invasion (LVI) with D2-40 antibody labeling and regular HE staining, blood vessel invasion (BVI) with CD34 antibody labeling and HE staining and to assess the possibility of using D2-40-LVI/CD34-BVI in combination for predicting stage II colon cancer prognosis and guiding adjuvant chemotherapy.Anti-D2-40 and anti-CD34 antibodies were applied to tissue samples of 220 cases of stage II colon cancer to label lymphatic vessels and small blood vessels, respectively. LVI and BVI were assessed and multivariate COX regression analysis was performed for associations with colon cancer prognosis. Regular HE staining proved unable to differentiate lymphatic vessels from blood vessels, while D2-40 selectively labeled lymphatic endothelial cell cytosol and CD34 was widely expressed in large and small blood vessels of tumors as well as normal tissues. Compared to regular HE staining, D2-40-labeling for LVI and CD34-labeling for BVI significantly increased positive rate (22.3% vs 10.0% for LVI, and 19.1% vs 9.1% for BVI). Multivariate analysis indicated that TNM stage, pathology tissue type, post-surgery adjuvant chemotherapy, D2-40-LVI, and CD34-BVI were independent factors affecting whole group colon cancer prognosis, while HE staining-BVI, HE staining-LVI were not significantly related. When CD34-BVI/D2-40-LVI were used in combination for detection, the risk of death for patients with two or one positive results was 5.003 times that in the LVI(-)andBVI(-) group (95% CI 2.365 - 9.679). D2-40 antibody LVI labeling and CD34 antibody BVI labeling have higher specificity and accuracy than regular HE staining and can be used as molecular biological indicators for prognosis prediction and guidance of adjuvant chemotherapy for stage II colon cancer.

  5. File list: ALL.Bld.10.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: ALL.Bld.05.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: InP.Bld.05.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. Effect of bone marrow mesenchymal stem cells on the proliferation of bone marrow CD34~+ cells in vitro

    Institute of Scientific and Technical Information of China (English)

    王荣

    2013-01-01

    Objective To investigate the effect on the marrow CD34+ cells by bone marrow mesenchymal stem cells(BMMSC),VarioMACS was used to sort bone marrow CD34+ cells,and then the purity of CD34+ cell was tested by FCM. Marrow mononuclear cells from abortion fetal bone marrow were isolated,and BMMSC were

  20. File list: Unc.Bld.10.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: NoD.Bld.50.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

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  10. File list: NoD.Bld.10.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

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  14. File list: His.Bld.10.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

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  16. File list: Oth.Bld.10.AllAg.CD34_Hematopoietic_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. Isolation and Characterization of CD34+ Blast-Derived Exosomes in Acute Myeloid Leukemia

    Science.gov (United States)

    Hong, Chang Sook; Muller, Laurent; Boyiadzis, Michael; Whiteside, Theresa L.

    2014-01-01

    Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100–1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future. PMID:25093329

  18. Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Chang Sook Hong

    Full Text Available Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab. This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100-1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future.

  19. Genetically engineered blood pharming: generation of HLA-universal platelets derived from CD34+ progenitor cells.

    Science.gov (United States)

    Figueiredo, Constança; Blaszczyk, Rainer

    2014-01-01

    Blood pharming is a recently designed concept to enable in vitro production of blood cells that are safe, effective and readily available. This approach represents an alternative to blood donation and may contribute to overcome the shortage of blood products. However, the high variability of the human leukocyte antigen (HLA) loci remains a major hurdle to the application of off-the-shelf blood products. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a relevant clinical problem. Thus, it would be desirable to generate PLT units devoid of HLA antigens. To reduce the immunogenicity of cell-based therapeutics, we have permanently reduced HLA class I expression using an RNA interference strategy. Furthermore, we demonstrated that the generation of HLA class I-silenced (HLA-universal) PLTs from CD34+ progenitor cells using an shRNA targeting β2-microglobulin transcripts is feasible. CD34+ progenitor cells derived from G-CSF mobilised donors were transduced with a lentiviral vector encoding for the β2-microglobulin-specific shRNA and differentiated into PLTs using a liquid culture system. The functionality of HLA-silenced PLTs and their ability to escape HLA antibody-mediated cytotoxicity were evaluated in vitro and in vivo. Platelet activation in response to ADP and thrombin were assessed in vitro. The immune-evasion capability of HLA-universal megakaryocytes (MKs) and PLTs was tested in lymphocytotoxicity assays using anti-HLA antibodies. To assess the functionality of HLA-universal PLTs in vivo, HLA-silenced MKs were infused into NOD/SCID/IL-2Rγc(-/-) mice with or without anti-HLA antibodies. PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. HLA-universal PLTs demonstrated to be functionally similar to blood-derived PLTs. Lymphocytotoxicity assays showed that HLA-silencing efficiently protects MKs against HLA antibody-mediated complement-dependent cytotoxicity. 80

  20. The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency

    Directory of Open Access Journals (Sweden)

    Miłosz Piotr Kawa

    2017-01-01

    Full Text Available Growth hormone (GH modulates hematopoietic cell homeostasis and is associated with apoptosis control, but with limited mechanistic insights. Aim of the study was to determine whether GH therapeutic supplementation (GH-TS could affect apoptosis of CD34+ cells enriched in hematopoietic progenitor cells of GH deficient (GHD children. CD34+ cells from peripheral blood of 40 GHD children were collected before and in 3rd and 6th month of GH-TS and compared to 60 controls adjusted for bone age, sex, and pubertal development. Next, apoptosis assessment via different molecular techniques was performed. Finally, to comprehensively characterize apoptosis process, global gene expression profile was determined using genome-wide RNA microarray technology. Results showed that GH-TS significantly reduced spontaneous apoptosis in CD34+ cells (p < 0.01 and results obtained using different methods to detect early and late apoptosis in analyzed cells population were consistent. GH-TS was also associated with significant downregulation of several members of TNF-alpha superfamily and other genes associated with apoptosis and stress response. Moreover, the significant overexpression of cyto-protective and cell cycle-associated genes was detected. These findings suggest that recombinant human GH has a direct anti-apoptotic activity in hematopoietic CD34+ cells derived from GHD subjects in course of GH-TS.

  1. The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency

    Science.gov (United States)

    Kawa, Miłosz Piotr; Stecewicz, Iwona; Piecyk, Katarzyna; Paczkowska, Edyta; Rogińska, Dorota; Sobuś, Anna; Łuczkowska, Karolina; Pius-Sadowska, Ewa; Gawrych, Elżbieta; Petriczko, Elżbieta; Walczak, Mieczysław; Machaliński, Bogusław

    2017-01-01

    Growth hormone (GH) modulates hematopoietic cell homeostasis and is associated with apoptosis control, but with limited mechanistic insights. Aim of the study was to determine whether GH therapeutic supplementation (GH-TS) could affect apoptosis of CD34+ cells enriched in hematopoietic progenitor cells of GH deficient (GHD) children. CD34+ cells from peripheral blood of 40 GHD children were collected before and in 3rd and 6th month of GH-TS and compared to 60 controls adjusted for bone age, sex, and pubertal development. Next, apoptosis assessment via different molecular techniques was performed. Finally, to comprehensively characterize apoptosis process, global gene expression profile was determined using genome-wide RNA microarray technology. Results showed that GH-TS significantly reduced spontaneous apoptosis in CD34+ cells (p < 0.01) and results obtained using different methods to detect early and late apoptosis in analyzed cells population were consistent. GH-TS was also associated with significant downregulation of several members of TNF-alpha superfamily and other genes associated with apoptosis and stress response. Moreover, the significant overexpression of cyto-protective and cell cycle-associated genes was detected. These findings suggest that recombinant human GH has a direct anti-apoptotic activity in hematopoietic CD34+ cells derived from GHD subjects in course of GH-TS. PMID:28067847

  2. Thrombopoietin induced proliferation and differentiation of fetal liver CD34+ cells with phenotype change from hemopoiesis to neurogenesis

    Institute of Scientific and Technical Information of China (English)

    Ning Ma; Dongchu Ma; Yi Tao; Yinghui Sun; Di Lin; Huiying Yu; Jinlong Jian; Wei Jia; Boquan Jin

    2008-01-01

    BACKGROUND: Previous studies have reported a neurotrophin-like motif in the N-terminal receptor binding region of the thrombopoietin (TPO) molecule, and have described localization of TPO and TPO receptor in the brain. Therefore, it is believed that TPO may be involved in regulation of neurogenesis.OBJECTIVE: To validate the effect of TPO on trans-differentiation, or differentiation from hematopoietic stem cells (HSCs) to neural stem cells (NSCs).DESIGN, TIME AND SETTING: Comparative studies were performed from March 2004 to April 2007 at the Department of Experimental Medicine, Northern Hospital, and the Department of Immunology, Fourth Military Medical University of Chinese PLA.MATERIALS: Human fetal liver (FL) was obtained from fetuses after water-balloon abortion. Gestational age ranged from 16 to 20 weeks. The study was approved by the Institutional Review Board and Ethics Committee of the Northern Hospital. TPO was kindly provided by Genentech Inc (USA). Iscove's Modified Dulbecco's Medium (IMDM) and neurobasal(tm) medium were purchased from Invitrogen (USA). MACS CD34 multisort kit was purchased from Miltenyi Biotec (Germany). METHODS: CD34+ cells were isolated from human FL mononuclear cells using MACS CD34 multisort kit and cultured at 1 × 105/mL in IMDM, containing TPO for 60 days with weekly changes of half of the medium. After culturing for 30 and 60 days, the TPO-induced cells were resuspended in neurobasal(tm) medium containing 10% fetal brain extracts and plated in an 8-well BIOCOAT(R) poly-D-Lysine Culture Slide and cultured for another 7 days. MAIN OUTCOME MEASURES: Cell number, viability, phenotype and expression of hemopoiesis-related and neurogenesis-related proteins were examined by trypan blue exclusion with hemocytometer, immunoblot, immunocytochemistry and flow cytometry.RESULTS: After 60 days of induction with TPO, the cell number increased by 4.6-fold compared to the initial culture. Although the proportion of the cells expressing the

  3. VEGF、CD34、INS在育龄妇女子宫内膜息肉的表达及意义%Expressions and significances of VEGF, CD34 and insulin in endometrial polyps of women of childbearing age

    Institute of Scientific and Technical Information of China (English)

    孙梅; 马毓梅; 李晓冬; 黄向华

    2011-01-01

    Objective; To explore the expressions of vascular endothelial growth factor (VEGF) , CD34 and insulin in endometrial polyps of women of childbearing age and their relationships with the occurrence of endometrial polyps. Methods: Immunohistochemical SP method was used to detect the expression levels of VEGF, CD34 and insulin in 50 cases with endometrial polyps. Results; In glandular epithelium of endometrial polyps, the expression of VEGF increased significantly (P 0.05) . In endometrial polyps, there was positive correlation between MVD value and VEGF expression (y =0. 813 2), but there was no apparent correlation between expression level of VEGF and insulin expression (γ = -0. 179 7) . Conclusion; VEGF is related to the occurrence of endometrial polyps, which can promote angiogenesis of endometrial polyps. There is no significant difference in the expression level of insulin between endometrial polyps and normal endometrium, it is indicated that there is no correlation between insulin and onset of endometrial polyps at present%目的:探讨育龄妇女EP组织内VEGF、CD34和INS的表达及其与EP发生的关系.方法:免疫组化SP法观察50例EP组织中VEGF、CD34和INS的表达.结果:EP组织腺上皮VEGF表达明显增强(P<0.01),MVD值明显增高,INS表达与正常子宫内膜相比无差异(P>0.05).EP组织MVD值与VEGF表达呈正相关(r=0.813 2),而VEGF的表达强度与INS表达无明显相关关系(r= -0.179 7).结论:VEGF与EP的发生有关,VEGF促进EP组织内的血管生成.EP组织中INS表达与正常子宫内膜无差异,目前未提示INS与EP发病的相关性.

  4. Clinical-scale expansion of CD34(+) cord blood cells amplifies committed progenitors and rapid scid repopulation cells.

    Science.gov (United States)

    Casamayor-Genescà, Alba; Pla, Arnau; Oliver-Vila, Irene; Pujals-Fonts, Noèlia; Marín-Gallén, Sílvia; Caminal, Marta; Pujol-Autonell, Irma; Carrascal, Jorge; Vives-Pi, Marta; Garcia, Joan; Vives, Joaquim

    2017-03-25

    Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34(+) population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34(+) controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγ(null) mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×10(6) CD34(+) cells committed to the granulocytic lineage and 3.9×10(9) neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.

  5. Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells.

    Science.gov (United States)

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke; Debets, Reno

    2012-06-01

    T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.

  6. Effect of CD34 and SMA on diagnosis of phyllodes tumors of breasts and their pathology analysis%CD34与SMa在乳腺叶状肿瘤诊断中的作用及病理形态分析

    Institute of Scientific and Technical Information of China (English)

    王浩; 曹海玲

    2015-01-01

    Objective to evaLuate the pathoLogic significance of the expression of CD34 and SMa in phyLLodes tumors. Methods the immunostaining was done using CD34 and SMa antibody in 10 cases of benign phyLLodes tumors,5 cases of bord-Lines,5 cases of maLignant and 20 cases of fibroadenoma. Results the expression area of CD34 in benign Pt was different from that in Fa,and expression was positiveLy correLated with the maLignant degree of Pt. SMa in Pt was higher than in Fa,and higher as the maLignant degree of Pt. Conclusion CD34 and SMa can be used as vaLuabLe differentiaL makers in Pt and Fa.%目的:观察乳腺叶状肿瘤( Pt)及纤维腺瘤( Fa)CD34、SMa的表达情况并探讨其意义。方法选取Fa患者20例,良性 Pt 患者10例,交界性 Pt 患者5例,恶性 Pt 患者5例进行免疫组化检测 CD34、SMa 的表达情况。结果 CD34在Fa与良性Pt中的表达部位存在差异,其表达强度与Pt的恶性程度呈正比,SMa在Fa中的表达与良性Pt存在强度差异,其表达强度与Pt的恶性程度呈正比。结论免疫组化检测CD34与SMa的表达可做为鉴别 Pt和Fa的有效方法并对Pt的分级有一定帮助。

  7. Expression of Caspase-3 in Cord Blood CD34+ Cells during Culture in vitro

    Institute of Scientific and Technical Information of China (English)

    MAYanping; LIRongping; ZOUPing; XIAOJuan; HUANGShiang; QIAOZhenhua

    2003-01-01

    Objective: To investigate the expression and significance of caspase-3 protein in CD34+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34+ cells during expansion in vitro.

  8. MUTZ-3-derived dendritic cells as an in vitro alternative model to CD34+ progenitor-derived dendritic cells for testing of chemical sensitizers.

    Science.gov (United States)

    Nelissen, Inge; Selderslaghs, Ingrid; Heuvel, Rosette Van Den; Witters, Hilda; Verheyen, Geert R; Schoeters, Greet

    2009-12-01

    The cytokine-dependent CD34(+) human acute myeloid leukaemia cell line MUTZ-3 was used to generate immature dendritic-like cells (MUTZ-3 DC) and their validity as an alternative to primary CD34(+) progenitor-derived DC (CD34-DC) for testing chemical-induced sensitization was assessed. Expression levels of the DC maturation markers HLA-DR, CD86, CD83 and CD11c were studied using flow cytometry after 24 and 48 h exposure to the model compound nickel sulphate (100 and 300 microM). No maturation of MUTZ-3 DC was observed, whereas significantly upregulated expression levels of CD83 and CD86 were noticed in CD34-DC after 24h treatment with 300 microM nickel sulphate compared to control cells. Differential expression of the cytokine genes IL1beta, IL6, IL8, CCL2, CCL3, CCL3L1, CCL4 was analyzed using real-time RT-PCR after 6, 10 and 24h of nickel sulphate exposure. In response to 100 microM nickel sulphate MUTZ-3 DC revealed slightly upregulated mRNA levels after 24h, whereas 300 microM induced transcription of CCL3, CCL3L1 and IL8 significantly after 6 or 10h. These cytokine data correspond to the previously observed effects of 100 microM nickel sulphate in CD34-DC. Our findings underline the stimulatory capacity of nickel sulphate in MUTZ-3 DC with regard to cytokine mRNA induction, but not surface marker expression. Compared to CD34-DC, however, the studied endpoint markers seemed to be less inducible, making the MUTZ-3 DC model in its presented form less suitable for in vitro testing of sensitization. Further assessment of MUTZ-3 DC using other differentiation protocols and an extended set of chemicals will be required to reveal whether this cell line may be a valid alternative model system to primary CD34-DC.

  9. Clinical-scale cultures of cord blood CD34(+) cells to amplify committed progenitors and maintain stem cell activity.

    Science.gov (United States)

    Ivanovic, Zoran; Duchez, Pascale; Chevaleyre, Jean; Vlaski, Marija; Lafarge, Xavier; Dazey, Bernard; Robert-Richard, Elodie; Mazurier, Frédéric; Boiron, Jean-Michel

    2011-01-01

    We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (∼350-fold), CD34(+) cells (∼100-fold), and CFC (∼130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).

  10. Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease

    Directory of Open Access Journals (Sweden)

    Ju Huang

    2017-06-01

    Full Text Available Fabry disease is a rare lysosomal storage disorder (LSD. We designed multiple recombinant lentivirus vectors (LVs and tested their ability to engineer expression of human α-galactosidase A (α-gal A in transduced Fabry patient CD34+ hematopoietic cells. We further investigated the safety and efficacy of a clinically directed vector, LV/AGA, in both ex vivo cell culture studies and animal models. Fabry mice transplanted with LV/AGA-transduced hematopoietic cells demonstrated α-gal A activity increases and lipid reductions in multiple tissues at 6 months after transplantation. Next we found that LV/AGA-transduced Fabry patient CD34+ hematopoietic cells produced even higher levels of α-gal A activity than normal CD34+ hematopoietic cells. We successfully transduced Fabry patient CD34+ hematopoietic cells with “near-clinical grade” LV/AGA in small-scale cultures and then validated a clinically directed scale-up transduction process in a GMP-compliant cell processing facility. LV-transduced Fabry patient CD34+ hematopoietic cells were subsequently infused into NOD/SCID/Fabry (NSF mice; α-gal A activity corrections and lipid reductions were observed in several tissues 12 weeks after the xenotransplantation. Additional toxicology studies employing NSF mice xenotransplanted with the therapeutic cell product demonstrated minimal untoward effects. These data supported our successful clinical trial application (CTA to Health Canada and opening of a “first-in-the-world” gene therapy trial for Fabry disease.

  11. CD34 is required for infiltration of eosinophils into the colon and pathology associated with DSS-induced ulcerative colitis.

    Science.gov (United States)

    Maltby, Steven; Wohlfarth, Carolin; Gold, Matthew; Zbytnuik, Lori; Hughes, Michael R; McNagny, Kelly M

    2010-09-01

    Eosinophil migration into the gut and the release of granular mediators plays a critical role in the pathogenesis of inflammatory bowel diseases, including ulcerative colitis. We recently demonstrated that eosinophil migration into the lung requires cell surface expression of the sialomucin CD34 on mast cells and eosinophils in an asthma model. Based on these findings, we investigated a similar role for CD34 in the migration of eosinophils and other inflammatory cells into the colon as well as explored the effects of CD34 ablation on disease development in a dextran sulfate sodium-induced model of ulcerative colitis. Our findings demonstrate decreased disease severity in dextran sulfate sodium-treated Cd34(-/-) mice, as assessed by weight loss, diarrhea, bleeding, colon shortening and tissue pathology, compared with wild-type controls. CD34 was predominantly expressed on eosinophils within inflamed colon tissues, and Cd34(-/-) animals exhibited drastically reduced colon eosinophil infiltration. Using chimeric animals, we demonstrated that decreased disease pathology resulted from loss of CD34 from bone marrow-derived cells and that eosinophilia in Cd34(-/-)IL5(Tg) animals was sufficient to overcome protection from disease. In addition, we demonstrated a decrease in peripheral blood eosinophil numbers following dextran sulfate sodium treatment. These findings demonstrate that CD34 was expressed on colon-infiltrating eosinophils and played a role in eosinophil migration. Further, our findings suggest CD34 is required for efficient eosinophil migration, but not proliferation or expansion, in the development of ulcerative colitis.

  12. CD34+细胞加速小鼠卒中后的恢复

    Institute of Scientific and Technical Information of China (English)

    理由

    2004-01-01

    日本国立心血管中心的Dr.Akihiko Taguchi报告,给予卒中累及免疫力的小鼠人脐血CD34+细胞,能快速激发缺血区的新血管生成。在这种鼠科动物的模型中,卒中48小时后移植CD34+细胞引起的治疗性血管生成明显加速了功能恢复。Taguchi等指出,CD34+细胞群内循环的内皮祖细胞(EPCs)参与了缺血组织的新血管形成,

  13. Circulating L-selectin levels and endothelial CD34 expression in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Seidelin, J B; Vainer, B; Horn, T

    1998-01-01

    was to compare levels of circulating sL-selectin, expression of the L-selectin ligand CD34 in the affected colon, and inflammatory bowel disease activity. METHODS: Twenty-three patients with UC, 16 patients with CD, and 18 control subjects were included in the study. In blood samples concentrations of serum s......L-selectin were determined by an ELISA technique. In colonoscopically obtained biopsies, CD34 expression was evaluated by immunohistochemical methods using monoclonal CD34 antibodies. Disease activity was determined by a clinical semiquantitative scale. RESULTS: sL-selectin levels were found to be significantly...... neutrophil activation may be the reason for low sL-selectin concentrations during quiescent disease stages, whereas chemokine secretion could explain the increased levels of sL-selectin associated with severe disease activity....

  14. Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment.

    Science.gov (United States)

    Wulf-Goldenberg, Annika; Keil, Marlen; Fichtner, Iduna; Eckert, Klaus

    2012-04-01

    In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34(+) cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation. Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34(+) cord blood stem cell preparations.

  15. Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content.

    Science.gov (United States)

    Voso, M T; Hohaus, S; Moos, M; Pförsich, M; Cremer, F W; Schlenk, R F; Martin, S; Hegenbart, U; Goldschmidt, H; Haas, R

    1999-02-01

    The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non-Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy-1 and Leu-8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR-negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high-dose therapy, a mean number of 4.7x 10(6) CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back-up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.

  16. Autologous CD34~+ and CD133~+ stem cells transplantation in patients with end stage liver disease

    Institute of Scientific and Technical Information of China (English)

    Hosny; Salama; Abdel-Rahman; N; Zekri; Abeer; A; Bahnassy; Eman; Medhat; Hanan; A; Halim; Ola; S; Ahmed; Ghada; Mohamed; Sheren; A; Al; Alim; Ghada; M; Sherif

    2010-01-01

    AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...

  17. Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia.

    Science.gov (United States)

    Sodani, Pietro; Isgrò, Antonella; Gaziev, Javid; Polchi, Paola; Paciaroni, Katia; Marziali, Marco; Simone, Maria Domenica; Roveda, Andrea; Montuoro, Aldo; Alfieri, Cecilia; De Angelis, Gioia; Gallucci, Cristiano; Erer, Buket; Isacchi, Giancarlo; Zinno, Francesco; Adorno, Gaspare; Lanti, Alessandro; Faulkner, Lawrence; Testi, Manuela; Andreani, Marco; Lucarelli, Guido

    2010-02-11

    Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11; 30 mg/m(2) fludarabine from day -17 to -11; 14 mg/kg busulfan starting on day -10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day -5 to -2. Fourteen patients received CD34(+)-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft-selected peripheral blood stem cells CD34(+) and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 x 10(5)/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

  18. Correction of deficient CD34+ cells from peripheral blood after mobilization in a patient with congenital erythropoietic porphyria.

    Science.gov (United States)

    Mazurier, F; Géronimi, F; Lamrissi-Garcia, I; Morel, C; Richard, E; Ged, C; Fontanellas, A; Moreau-Gaudry, F; Morey, M; de Verneuil, H

    2001-03-01

    Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood, and other organs. The onset of most cases occurs in infancy and the main symptoms are cutaneous photosensitivity and hemolysis. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, autografting of genetically modified primitive/stem cells is the only alternative. In the present study, efficient mobilization of peripheral blood primitive CD34(+) cells was performed on a young adult CEP patient. Retroviral transduction of this cell population with the therapeutic human UROS (hUS) gene resulted in both enzymatic and metabolic correction of CD34(+)-derived cells, as demonstrated by the increase in UROS activity and by a 53% drop in porphyrin accumulation. A 10-24% gene transfer efficiency was achieved in the most primitive cells, as demonstrated by the expression of enhanced green fluorescent protein (EGFP) in long-term culture-initiating cells (LTC-IC). Furthermore, gene expression remained stable during in vitro erythroid differentiation. Therefore, these results are promising for the future treatment of CEP patients by gene therapy.

  19. Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34+ cells.

    Science.gov (United States)

    Lee, Eun-Ju; Be, Cheang Ly; Vinson, Andrew R; Riches, Andrew G; Fehr, Friederike; Gardiner, James; Gengenbach, Thomas R; Winkler, David A; Haylock, David

    2015-01-01

    Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche.

  20. Expressão citofotométrica do marcador CD-34 no adenocarcinoma de cólon CD-34 cytophotometric expression in colon adenocarcinoma

    Directory of Open Access Journals (Sweden)

    João Batista Monteiro Tajra

    2008-12-01

    Full Text Available A angiogênese é uma das responsáveis pelo equilíbrio homeostático entre as células. Durante o desenvolvimento do processo de degeneração maligna celular o seu desequilíbrio é considerado um importante marco neoplásico e o CD-34 parece ser um bom marcador de angiogênese. OBJETIVOS - Avaliar qual a expressão citofotométrica do CD-34 no adenocarcinoma de cólon; se apresenta alterações nas diferentes fases evolutivas na classificação modificada de Dukes; e como se expressa no cólon direito e esquerdo. MÉTODOS - Utilizaram-se 19 casos submetidos à técnica imunoistoquímica com anticorpo anti CD-34. Após, as lâminas foram lidas pelo sistema SAMBA com o software Immuno 4, analisando dois índices: marcação e densidade óptica. Os parâmetros foram marcação e expressão do marcador, quer individual quer relacionado à classificação de Dukes e lado. RESULTADOS - A média do índice de marcação foi 66,54 e densidade óptica 43,60. Em relação à classificação de Dukes, 12 do tipo B, tiveram índice de marcação 67,95 e densidade óptica 43,21 e, para os sete do tipo C, índice de marcação 64,12 e densidade óptica 44,27. Não foi possível identificar diferença em relação à classificação de Dukes. Quanto ao lado do tumor, os 11 esquerdos tiveram índice de marcação 72,08 e densidade óptica 46,70, e os oito direitos, índice de marcação 58,93 e densidade óptica 39,44. Em relação ao índice de marcação houve diferença significante, mas quanto à densidade óptica não. CONCLUSÕES - O CD-34 apresentou expressão discreta como marcador de angiogênese; sem diferença entre tipos B e C de Dukes mostrando atividade angiogênica maior à direita do que à esquerda.Angiogenesis is considered to be one of the mechanisms responsible for the homeostatic balance among cells. During cell malignant degeneration, the unbalanced cell proliferation mechanisms demonstrate to be an important factor in cancer evolution

  1. Thrombopoietin mobilizes CD34+ cell subsets into peripheral blood and expands multilineage progenitors in bone marrow of cancer patients with normal hematopoiesis.

    Science.gov (United States)

    Murray, L J; Luens, K M; Estrada, M F; Bruno, E; Hoffman, R; Cohen, R L; Ashby, M A; Vadhan-Raj, S

    1998-03-01

    Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, also mediates biologic effects in vitro on hematopoietic cells more primitive than those committed to the megakaryocyte (MK) lineage. To assess the spectrum of hematopoietic effects of recombinant human (rh)TPO in vivo, we evaluated its proliferative effect on bone marrow (BM) progenitor cells, its maturation effect on BM MKs, and its mobilizing effect on peripheral blood (PB) progenitor cells during a phase I clinical laboratory investigation in which rhTPO was administered to cancer patients with normal hematopoiesis. Twelve patients received a single dose of rhTPO (0.3, 0.6, 1.2, or 2.4 microg/kg of body weight) prior to chemotherapy. BM and PB samples from these patients were analyzed 1 to 2 days before (baseline) and 7 days after rhTPO administration. At higher doses (1.2-2.4 microg/kg), rhTPO produced increased concentrations of primitive CD34+Thy-1+Lin-cells (mean 2.1-fold), CD34+mpl+ cells (mean 5.2-fold), CD34+CD41+CD14- promegakaryoblasts (mean 2.9-fold), and myeloerythroid colony-forming cells (mean threefold) in BM. No significant increases in the frequency of BM colony-forming unit (CFU)-MK were observed. Elevated numbers of both immature (2N-8N) and more mature (64N and 128N) CD41+ MKs were detected in BM, with modal ploidy remaining at 16N. Higher doses of rhTPO (1.2-2.4 microg/kg) also induced increased concentrations of CD34+ cell subsets in PB, including both primitive CD34+Thy-1+Lin- (mean 8.8-fold) and MK lineage-committed CD34+CD41+CD14- cells (mean 14.6-fold) as well as various myeloerythroid colony-forming cells (mean 3.6- to 5.5-fold). These results demonstrate that rhTPO given as a single dose not only promotes proliferation and maturation of cells of the MK lineage, but also expands the pool of BM primitive hematopoietic cells. In addition, rhTPO induces mobilization of hematopoietic progenitors into peripheral circulation. The extent to which such multilineage effects on

  2. Context-Dependent Development of Lymphoid Stroma from Adult CD34+ Adventitial Progenitors

    DEFF Research Database (Denmark)

    Sitnik, Katarzyna Maria; Wendland, Kerstin; Weishaupt, Holger;

    2016-01-01

    ) and thymus that is located within the vascular niche surrounding PDPN-PDGFRβ+/α-Esam-1+ITGA7+ pericytes. CD34+ adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ...

  3. Context-Dependent Development of Lymphoid Stroma from Adult CD34+ Adventitial Progenitors

    Directory of Open Access Journals (Sweden)

    Katarzyna M. Sitnik

    2016-03-01

    Full Text Available Despite the key role of primary and secondary lymphoid organ stroma in immunity, our understanding of the heterogeneity and ontogeny of these cells remains limited. Here, we identify a functionally distinct subset of BP3−PDPN+PDGFRβ+/α+CD34+ stromal adventitial cells in both lymph nodes (LNs and thymus that is located within the vascular niche surrounding PDPN−PDGFRβ+/α−Esam-1+ITGA7+ pericytes. CD34+ adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ re-aggregate grafts, we demonstrate that adult CD34+ adventitial cells are capable of differentiating into multiple lymphoid stroma-like subsets including pericyte-, FRC-, MRC-, and FDC-like cells, the development of which was lymphoid environment-dependent. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34+ adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues.

  4. Intratumoral α-SMA enhances the prognostic potency of CD34 associated with maintenance of microvessel integrity in hepatocellular carcinoma and pancreatic cancer.

    Science.gov (United States)

    Wang, Wen-Quan; Liu, Liang; Xu, Hua-Xiang; Luo, Guo-Pei; Chen, Tao; Wu, Chun-Tao; Xu, Yong-Feng; Xu, Jin; Liu, Chen; Zhang, Bo; Long, Jiang; Tang, Zhao-You; Yu, Xian-Jun

    2013-01-01

    Microvessel density (MVD) as an angiogenesis predictor is inefficient per se in cancer prognosis. We evaluated prognostic values of combining intratumoral alpha-smooth muscle actin (α-SMA)-positive stromal cell density and MVD after curative resection in hypervascular hepatocellular carcinoma (HCC) and hypovascular pancreatic cancer (PC). Tissue microarrays were constructed from tumors of 305 HCC and 57 PC patients who underwent curative resection and analyzed for α-SMA and CD34 expression by immunostaining. Prognostic values of these two proteins and other clinicopathological features were examined. Both low α-SMA density and high MVD-CD34 were associated in HCC with the presence of intrahepatic metastasis and microvascular invasion, and they were related to lymph node involvement and microvascular invasion in PC (pSMA, was an independent prognostic factor for overall survival and recurrence-free survival, the combination of low α-SMA and high CD34 was a predictor of worst prognosis for both types of tumors and had a better power to predict patient death and early recurrence (pSMA-positive cells and vascular endothelial cells overlap, showing major colocalization on vascular walls. Poor microvessel integrity, as indicated by high MVD, together with low perivascular α-SMA-positive cell coverage is associated with early recurrence, unfavorable metastasis, and short survival after tumor resection. This finding highlights the significance of vascular quality in tumor progression, which provides an optimized complement to vascular quantity in prognosis of postoperative patients.

  5. CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor

    DEFF Research Database (Denmark)

    Jinquan, T; Anting, L; Jacobi, H H

    2001-01-01

    phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1...

  6. Negative correlation of CD34+ cells with blood-brain barrier permeability following traumatic brain injury in a rat model.

    Science.gov (United States)

    Jin, Xuelong; Wang, Feifei; Liu, Xingju; Liang, Bin; Chen, Zequn; He, Junfeng; Zhang, Hong; Zhang, Jianning

    2014-11-01

    TBI causes localized cerebral ischemia that, in turn, is accompanied by both changes in BBB permeability and recruitment of CD34(+) cells to the injured tissue. However, it remains unknown whether CD34(+) cell recruitment is linked to BBB permeability. This study is a preliminary investigation into possible correlations between CD34(+) cell recruitment and BBB permeability following TBI in a rat model. Male SD rats were subjected to mild fluid percussion injury. BBB permeability was assessed by measuring extrinsic EB dye extravasation and endogenous EBA expression at days 1, 3, 5, 7, and 12 post injury. The number of CD34(+) cells in the damaged tissue was analyzed by immunohistochemistry at each time point. EB dye extravasation reached a peak at day 3 following TBI, while EBA expression displayed the reverse profile. Accumulation of CD34(+) cells in injured brain tissue was evident at five days post injury. It revealed a negative linear correlation between CD34(+) cell and BBB permeability. The negative linear correlation between CD34(+) cell recruitment and BBB permeability following TBI provides a support for further study of CD34(+) cell transplantation for BBB repair after TBI. © 2014 John Wiley & Sons Ltd.

  7. Sprint interval and sprint continuous training increases circulating CD34+ cells and cardio-respiratory fitness in young healthy women.

    Directory of Open Access Journals (Sweden)

    Emma Harris

    Full Text Available INTRODUCTION: The improvement of vascular health in the exercising limb can be attained by sprint interval training (SIT. However, the effects on systemic vascular function and on circulating angiogenic cells (CACs which may contribute to endothelial repair have not been investigated. Additionally, a comparison between SIT and sprint continuous training (SCT which is less time committing has not been made. METHODS: 12 women (22±2 yrs completed 12 sessions of either SIT (n = 6 or work-matched SCT (n = 6 on 3 days/week. Pre and post-training assessments included brachial artery endothelial function and peripheral blood analysis for CAC number (CD34+/CD34+CD45dim. CAC function was measured by migration and adhesion assays. Cardio-respiratory fitness, carotid arterial stiffness and carotid-radial and brachial-foot pulse wave velocity (PWV were also evaluated. RESULTS: CD34+ CACs increased following training in both groups but CD34+CD45dim did not (Pre CD34+: 40±21/105 leukocytes, Post CD34+: 56±24/105 leukocytes, main time effect p0.05. DISCUSSION: SCT involving little time commitment is comparable to SIT in increasing CD34+ cell number and [Formula: see text]. An increased mobilisation of CD34+ CACs suggests that sprint training may be an effective method to enhance vascular repair.

  8. Tracing CD34+ Stromal Fibroblasts in Palatal Mucosa and Periodontal Granulation Tissue as a Possible Cell Reservoir for Periodontal Regeneration.

    Science.gov (United States)

    Roman, Alexandra; Páll, Emőke; Mihu, Carmen M; Petruţiu, Adrian S; Barbu-Tudoran, Lucian; Câmpian, Radu S; Florea, Adrian; Georgiu, Carmen

    2015-08-01

    The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.

  9. Expression of CD117, CD34 and Ki-67 in gastric stromal tumors and their significances%CD117、CD34、Ki-67在胃间质瘤中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    李鹏; 徐文通

    2013-01-01

    Objective To investigate the clinicopathologic and immunohistochemical features of gastric stromal tumors.Methods A total of 105 cases with gastric stromal tumors were included in the study.The expression of cluster of differentiation 117 (CD117),cluster of differ entiation 34 (CD34),smooth muscle actin (SMA),S-100 protein and Ki-67 in gastric stromal tumors specimens was detected by immunohistochemistry.The clinico-pathologic features of gastric stromal tumors were also analyzed.Results Of 105 gastric stromal tumors specimens,90 were positive for CD117 [85.7 % (90/105)],98 positive for CD34 [93.3 % (98/105)],21 positive for SMA [20.4 % (21/105)],60 positive for Ki-67 [56.1% (60/105)],13 positive for S-100 [12.4 % (13/105)].Conclusion Combined detection of CD117 and CD34 can improve the diagnostic accuracy of gastric stromal tumors.The expression of Ki-67 can be used as a prognostic parameter for gastric stromal tumors as it is correlated with tumor size,risk and mitotic.%目的 探讨胃间质瘤的临床病理和免疫组织化学特征.方法 观察105例胃间质瘤的病理形态特征,并用免疫组织化学EnVision法检测CD117、CD34、SMA、S-100、Ki-67的表达.结果 CD117、CD34、SMA、S-100、Ki-67阳性表达率分别为85.7%(90/105)、93.3%(98/105)、20.4%(21/105)、12.4%(13/105)、56.1%(60/105).结论 联合检测CD117和CD34可以提高胃间质瘤的诊断准确性,Ki-67阳性表达与肿瘤大小、核分裂象、危险程度相关,可作为胃间质瘤的预后指标.

  10. Effect of Interferon-alpha in systemic lupus erthematosus (SLE) serum on the differentiation and maturation of dendritic cells derived from CD34+ hematopoietic precursor cells

    Institute of Scientific and Technical Information of China (English)

    Rong Zhang; Meifen Xing; Weiwen Wang; Xiaofan Yang; Xiaohui Ji

    2009-01-01

    Objective: To study the effect of interferon-alpha IFN-a in the serum of SLE patients on the differentiation and maturation of dendritic cells (DCs) derived from CD34+ hematopoietic precursor cells (HPCs). Methods: Serum samples from SLE patients and normal controls were collected and the concentration of IFN-a detected by ELISA. CD34+HPCs were purified from cord blood by a magnetic cell sorting system (MACS), and cultured to differentiate to DCs. Normal serum, normal serum with exogenous IFN-α, SLE serum with raised levels of IFN-α, or SLE serum with anti-IFN-α neutralizing antibody was added to the culture medium. The phenotype of DCs was analyzed by flow cytometry (FCM) and the capacity of DCs to stimulate allogenic T lymphocyte proliferation was evaluated in a mixed lymphocyte reaction by the Cell Counting Kit-8. Cytokine production was assessed by ELISA. Results: Serum levels of IFN-a were significantly higher in SLE patients than in normal controls and this correlated positively with disease activity. Cultured in SLE serum with raised levels of IFN-α, CD34+HPCs could differentiate into DCs that expressed higher levels of HLA-DR, CD80 and CD86, and showed an enhanced allogenic T-cell stimulatory capacity, while producing lower levels of IL-12 and higher amounts of IL-10 compared with those DCs cultured in normal serum. Conclusion: Increased levels of IFN-a in SLE serum promotes the differentiation and maturation of DCs derived from CD34+ HPCs and could contribute to the pathogenesis of SLE.

  11. IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

    Science.gov (United States)

    Ficara, Francesca; Superchi, Daniela B; Hernández, Raisa Jofra; Mocchetti, Cristina; Carballido-Perrig, Nicole; Andolfi, Grazia; Deola, Sara; Colombo, Augusto; Bordignon, Claudio; Carballido, José M; Roncarolo, Maria Grazia; Aiuti, Alessandro

    2004-12-01

    To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

  12. Expression of S-100, EMA, CD34 and presence of mast cells in eight oral neurofibromas, and a review of 127 cases of the literature

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    Francisco Artur Forte Oliveira

    2013-10-01

    Full Text Available INTRODUCTION: The rarity of oral neurofibromas (ONs generates problems regarding their epidemiological and immunohistochemical characterization. OBJECTIVES: The aim of this study was to evaluate the expression of different markers in ONs and review epidemiologic data reported in the literature. MATERIAL AND METHODS: Clinicopathologic, immunohistochemical (markers S-100, epithelial membrane antigen [EMA], CD34 and histochemical (modified-Ziehl-Neelsen-method studies were performed in eight cases of ON diagnosed in the Department of Pathology and Legal Medicine (DPML, Universidade Federal do Ceará (UFC, Ceará, Brazil, between 1994 and 2010. RESULTS: Oral neurofibromas represented 0.2% of the oral lesions diagnosed by our service in 16 years, and the buccal mucosa was the most frequent oral site (71.4%. Seven (87.5% and 8 (100.0% cases were positive for S-100 and CD34, respectively, and none for EMA. Mast cells were identified in seven cases (87.5%. The literature search indicated that solitary ONs are more common and occur preferentially in females, affecting patients between 30 and 40 years old. The alveolar ridge is the most commonly involved site. CONCLUSION: S-100- and CD34 markers proved to be of great value as a diagnostic tool, unlike EMA staining. Identification of mast cells in most cases suggests their involvement in this tumor pathogenesis. The clinicopathologic data retrieved from the literature enabled the establishment of a more consistent epidemiological profile.

  13. Simultaneous demonstration of mast cells and blood vessels by the combined method CD34--alcian blue-safranin in lip tumors.

    Science.gov (United States)

    Gaje, Puşa; Bocan, Viorica; Cîmpean, Anca Maria; Izvernariu, D A; Streian, Felicia; Raica, M

    2007-01-01

    The aim of the study was to evaluate the mast cell-blood vessel relationship using double staining CD34/AAS. Sections from 14 cases with lip tumors have been stained with Hematoxylin-Eosin. On additional sections from each case, we highlighted blood vessels by immunohistochemistry for CD34 antigen using the method LSAB2-HRP/DAB, followed by alcian blue-safranin stain for mast cells. We quantified the density, distribution and the mast cell types as well as the correlation with the number of blood vessels. All cases have been positive for both staining. We observed a significant correlation between the number of vessels and the mast cells (p = 0.003). In one case, we observed the mast cells stained with safranin (red), the vascular density being less than the mast cells density. Our results confirmed the data from the literature with respect to the large number of mast cells observed in the malignant tumors. The increased vascular density together with the mast cell density suggests a correlation between these two elements in the tumor angiogenesis, possibly though the VEGF secretion. The CD34/AAS stain is a quick and simple method and it allows an optimal correlation between the number of mast cells and blood vessels on the same section. The type of mast cells correlated with microvessel density is a powerful argument towards the involvement of the mast cells in the tumor angiogenesis of the malignances of the lips.

  14. Pigmented hepatocellular adenoma with complete CD34 immunostaining pattern: A diagnostic dilemma

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    Mukul Vij

    2012-01-01

    Full Text Available WHO defines hepatocellular adenoma (HCA as a benign tumor composed of cells closely resembling normal hepatocytes, which are arranged in plates separated by sinusoids. It is more common in women. The present concerns a 41 years female who was found to have a mass lesion in liver on ultrasound while undergoing routine evaluation for dyspepsia. Computed tomography scan of abdomen showed 10 × 8 cm lesion in liver. Extended left hepatectomy was performed. Grossly hepatic cut surface showed circumscribed tumor with dark gray or black color. Microscopy revealed hepatocellular adenoma with abundant Dubin Johnson like pigment deposition. CD34 immunostaining showed complete sinusoidal pattern. We labeled the tumor as pigmented hepatic adenoma with complete CD34 staining pattern. To the best of author′s knowledge only eight cases of pigmented hepatocellular adenoma are described in world literature.

  15. Cd117 and Cd34 Staining Patterns in Childhood Benign Mammary Lesions

    OpenAIRE

    KAÇAR, Ayper; Paker, İrem; AKBIYIK, Fatih; ARIKÖK, Ata Türker; MAMBET, Ervin

    2012-01-01

    Objective: CD117 and CD34 are markers that have both been implied in cancer progression in adult breast lesions. This study was conducted in order to create a retrospective documentation and to analyze the expression patterns of these markers on childhood benign lesions along with a comparison with adult breast lesions’ staining patterns.Material and Method: Nine fibroadenomas, 2 tubular adenomas, 1 mammary hamartoma, 2 gynecomastias, 1 benign phyllodes tumor were retrieved from pathology arc...

  16. Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

    Science.gov (United States)

    Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie

    2003-10-01

    We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

  17. CD34 selected cells for the treatment of poor graft function after allogeneic stem cell transplantation.

    Science.gov (United States)

    Stasia, Alessandra; Ghiso, Anna; Galaverna, Federica; Raiola, Anna Maria; Gualandi, Francesca; Luchetti, Silvia; Pozzi, Sarah; Varaldo, Riccardo; Lamparelli, Teresa; Bregante, Stefania; Van Lint, Maria Teresa; di Grazia, Carmen; Bacigalupo, Andrea

    2014-09-01

    Poor graft function (PGF) is characterized by pancytopenia and a hypoplastic marrow, with complete donor chimerism, usually without severe graft-versus-host disease (GVHD). We report 41 patients with PGF, treated with granulocyte colony-stimulating factor-mobilized CD34 selected cells, at a median interval from transplant of 140 days, without conditioning and without GVHD prophylaxis. Donors were HLA matched siblings (n = 12), unrelated donors (n = 18), or mismatched family members (n = 11). The median number of infused CD34(+) cells was 3.4 × 10(6)/kg. The rate of trilineage recovery was 75%: 83% for HLA matched siblings and 72% for unrelated and mismatched family members (P = .3). The cumulative incidence of acute grade II GVHD was 15%, and no patient developed de novo chronic GVHD. The actuarial 3-year survival is 63%: 76% and 25% for patients with or without trilineage recovery. These data confirm the role of CD34(+) selected cells from the same donor in the treatment of PGF and warrant the request for a second donation also when the donor is unrelated.

  18. Preparation of immunomagnetic nanoparticles and their application in the separation of mouse CD34 + hematopoietic stem cells

    Science.gov (United States)

    Liang, Xin; Xu, Ke; Xu, Jianhe; Chen, Wei; Shen, Hebai; Liu, Jianwen

    2009-06-01

    The magnetic nanoparticles with a diameter of about 60 nm were synthesized by coprecipitation from ferrous and ferric iron solutions and coated with silica. Then the nanoparticles were modified with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS) in order to immobilize anti-CD34 + monoclonal antibodies to the surface of modified magnetic particles. The results of transmission electron microscope (TEM) and Fourier transformed infrared (FT-IR) indicated that the nanoparticles were successfully prepared. Scanning electron microscope (SEM) photo confirmed that the mouse CD34 + cells (cells expressing CD34) were separated by the immunomagnetic nanoparticles. The viability of the separated cells was studied by hematopoietic colony-forming assay, the result of which showed that the target cells still had an ability of proliferation and differentiation. The application of the separated CD34 + cells was in testing the pharmacological effect of three samples isolated from enzyme-digested traditional Chinese medicine Colla corii asini.

  19. Expressions of matrix metalloproteinase-9,IV collagen and CD34 in epithelial ovarian tumor and its significance%基质金属蛋白酶-9、Ⅳ型胶原、CD34在卵巢上皮性肿瘤中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    黄凯清; 柯佩奇; 梁立治; 彭文明; 彭娟; 刘少颜

    2010-01-01

    malignant ovarian carcinomas [benign epithelial ovarian tumors was (17.18±5.64)%,borderline epithelial ovarian tumors was (29.76±7.18)%,well-differentiated malignant epithelial ovarian carcinomas was (57.20±8.55)%,moderately-differentiated malignant epithelial ovarian carcinomas was (71.20±8.48)%, poorly-differentiated malignant epithelial ovarian carcinomas was(90.38±20.03)%](F= 100.072, P < 0.01). The expression of IV collagen in malignant epithelial ovarian carcinomas was different from that in borderline epithelial ovarian tumors and benign epithelial ovarian tumors (F = 11.554,P<0.0l). The expression of MMP-9 was positive correlation with the loss expression of IV collagen and the expression of CD34 (r=0.796,0.802,P< 0.01).Conclusions The positive expression of MMP-9,CD34 and the negative expression of IV collagen are obviously relevant to degree of malignant ovarian carcinomas.The combined testing on expression of MMP-9,CD34, IV collagen on ovarian carcinomas is significant to decide degree of malignant ovarian carcinomas and evaluate future development.

  20. Peripheral blood CD34+ cell count as a predictor of adequacy of hematopoietic stem cell collection for autologous transplantation

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    Combariza, Juan F.

    2016-10-01

    Full Text Available Introduction: In order to carry out an autologous transplantation, hematopoietic stem cells should be mobilized to peripheral blood and later collected by apheresis. The CD34+ cell count is a tool to establish the optimal time to begin the apheresis procedure. Objective: To evaluate the association between peripheral blood CD34+ cell count and the successful collection of hematopoietic stem cells. Materials and methods: A predictive test evaluation study was carried out to establish the usefulness of peripheral blood CD34+ cell count as a predictor of successful stem cell collection in patients that will receive an autologous transplantation. Results: 77 patients were included (median age: 49 years; range: 5-66. The predominant baseline diagnosis was lymphoma (53.2 %. The percentage of patients with successful harvest of hematopoietic stem cells was proportional to the number of CD34+cells in peripheral blood at the end of the mobilization procedure. We propose that more than 15 CD34+cells/μL must be present in order to achieve an adequate collection of hematopoietic stem cells. Conclusion: Peripheral blood CD34+ cell count is a useful tool to predict the successful collection of hematopoietic stem cells.

  1. HEMATOPOIETIC PROGENITOR CELLS AS A PREDICTIVE OF CD34+ ENUMERATION PRIOR TO PERIPHERAL BLOOD STEM CELLS HARVESTING

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    Z. Zulkafli

    2014-09-01

    Full Text Available Background: To date, the CD34+ cell enumeration has relied predominantly on flow cytometry technique. However, flow cytometry is time consuming and operator dependent. The application of the hematopoietic progenitor cells (HPCs channel in Sysmex XE-2100, a fully automated hematology analyzer offers an alternative approach, which is with minimal sample manipulation and less operator dependent. This study evaluates the utility of HPC counts as a predictive of CD34+ enumeration prior to peripheral blood stem cells harvesting. Materials and methods: HPC, CD34+, white blood cell (WBC, reticulocytes (retic, immature platelet fraction (IPF and immature reticulocyte fraction (IRF were determined in 61 samples from 19 patients with hematological malignancies (15 lymphoma and 4 multiple myeloma patients at Hospital Universiti Sains Malaysia (Hospital USM who had received granulocyte-colony stimulating factor (G-CSF and planned for autologous transplantation. Results: CD34+ count showed strong and significant correlation with HPC. The receiver operating characteristics (ROC curve analysis revealed that HPC count > 21.5 x 106 / L can predicts a pre harvest CD34+ count of >20 x 106 / L with sensitivity of 77%, specificity of 64% and area under the curve (AUC of 0.802. Conclusion: We concluded that HPC count can be a useful potential parameter in optimizing timing for CD34+ enumeration prior to leukapheresis.

  2. Generation of induced pluripotent stem cells from peripheral blood CD34+ hematopoietic progenitors of a 31 year old healthy woman

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    Amornrat Tangprasittipap

    2017-04-01

    Full Text Available The MUi019-A human induced pluripotent stem cell line was generated from peripheral blood CD34+ hematopoietic progenitors of a healthy woman using a non-integrative reprogramming method. Episomal vectors carrying reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 and EBNA-1 were delivered using electroporation. The iPSC line can be used as a control in studying disease mechanisms. Furthermore, gene editing approaches can be used to introduce specific mutations into the MUi019-A to model disease while the cell type affected by the disease is inaccessible.

  3. Transient loading of CD34+ hematopoietic progenitor cells with polystyrene nanoparticles

    Science.gov (United States)

    Deville, Sarah; Hadiwikarta, Wahyu Wijaya; Smisdom, Nick; Wathiong, Bart; Ameloot, Marcel; Nelissen, Inge; Hooyberghs, Jef

    2017-01-01

    CD34+ hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon. PMID:28138242

  4. 脐血CD34+细胞体外诱导分化为成熟巨核细胞及产出血小板%In vitro differentiation of umbilical cord blood CD34+ cells into mature megakaryocytes and generation of plateletss

    Institute of Scientific and Technical Information of China (English)

    李昕; 陈方平; 刘竞; 吴新华; 蒋铁斌; 唐雪元

    2009-01-01

    cells into matured cells to form products in vitro.OBJECTIVE: To induce CD34+ cells of umbilical cord blood to differentiate into mature megakaryocytes, and to investigate the mechanism of production of platelets.DESIGN, TIME AND SETTING: This cytology in vitro study was conducted at the Central Laboratory of Xiangya Hospital and Xiangya Third Hospital from 2004 to 2006. MATERIALS: Umbilical cord was collected from healthy full-term pregnant puerperants at the Xiangya Hospital.METHODS: The CD34+ cells were isolated from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in 24-well culture plate at 5x107/L in StemPro-34 serum-free medium, supplemented with L-glutamine, saturated human transferrin, CaCl2, insulin, deionized bovine serum albumin and recombinant human thrombopoietin at 37℃, under 0.05 volume fraction CO2 saturated humidity to be differentiated into megakaryocytes for 14-21 days. Cell medium was absorbed, and centrifuged to obtain supernatant. Samples were centrifuged again, and then supernatant was removed. The remaining was platelet-like particles in cell culture plate. Platelet was isolated from normal platelet-rich plasma.MAIN OUTCOME MEASURES: The following parameters were measured: morphological changes in cultured cells and platelet-like particles in supematant; results of immunohistochemistry; observation results under a microscope; platelet aggregation; CD41 expression.RESULTS: At day 10, silk-like substances were found in megakaryocyte culture medium, with the presence of platelet-sized particles. The production of platelet-sized particles reached a peal at day 16. Cultured cells were strongly positively for platelet-specific antigen GP Ⅱb Ⅲa. Under the optical microscope, mature megakaryocytes were detected, with the presence of some immature megakaryocytes, and platelet-sized particles were found surrounding megakaryocytes. Under the electron microscope, a majority of mature megakaryocytes and a few apoptotic

  5. Vasoreparative dysfunction of CD34+ cells in diabetic individuals involves hypoxic desensitization and impaired autocrine/paracrine mechanisms.

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    Yagna P R Jarajapu

    Full Text Available We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69 and nondiabetic (n = 46 individuals were used to grow endothelial colony forming cells (ECFC, early endothelial progenitor cells (eEPCs and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM. In CM derived from CD34+ cells of diabetic individuals (diabetic-CM, the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM. Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

  6. Vasoreparative dysfunction of CD34+ cells in diabetic individuals involves hypoxic desensitization and impaired autocrine/paracrine mechanisms.

    Science.gov (United States)

    Jarajapu, Yagna P R; Hazra, Sugata; Segal, Mark; Li Calzi, Sergio; LiCalzi, Sergio; Jadhao, Chandra; Jhadao, Chandra; Qian, Kevin; Mitter, Sayak K; Raizada, Mohan K; Boulton, Michael E; Grant, Maria B

    2014-01-01

    We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34+ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

  7. Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody

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    Chun-Li Song

    2015-04-01

    Full Text Available AbstractIntroduction:Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD, with a major limitation of in-stent restenosis (ISR. The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF and anti-CD34 antibody on 316L stainless steel.Methods:Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl-N'-ethylcarbodiimide (EDC and N-hydroxysuccinimide (NHS. Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V10 group. Eight sheets from the D-(H-V10 group were coated with anti-CD34 antibody and termed as the D-(H-V10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody.Results:The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V10 and D-(H-V10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody.Conclusion:Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy.

  8. Misfolded N-CoR is linked to the ectopic reactivation of CD34/Flt3-based stem-cell phenotype in promyelocytic and monocytic acute myeloid leukemia

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    Dawn Sijin Nin

    2015-10-01

    Full Text Available Nuclear receptor co-repressor (N-CoR is the key component of generic co-repressor complex essential for the transcriptional control of genes involved in cellular hemostasis. We have recently reported that N-CoR actively represses Flt3, a key factor of hematopoietic stem cells (HSC self-renewal and growth; and that de-repression of Flt3 by the misfolded N-CoR plays important role in the pathogenesis of promyelocytic and monocytic acute myeloid leukemia (AML. The leukemic cells derived from the promyelocytic and monocytic AML are distinctly characterized by the ectopic reactivation of stem cell phenotypes in relatively committed myeloid compartment. However, the molecular mechanism underlying this phenomenon is not known. Here, we report that N-CoR function is essential for the commitment of primitive hematopoietic cells to the cells of myeloid lineage, and that loss of N-CoR function due to misfolding is linked to the ectopic reactivation of generic stem cell phenotypes in promyelocytic and monocytic AML. Analysis of N-CoR and Flt3 transcripts in mouse hematopoietic cells revealed a positive correlation between N-CoR level and the commitment of myeloid cells and an inverse correlation between N-CoR and Flt3 levels in primitive as well as committed myeloid cells. Enforced N-CoR expression in mouse HSCs inhibited their growth and self-renewal potentials and promoted maturation towards cells of myeloid lineage, suggesting a role of N-CoR in the commitment of cells of myeloid lineage. In contrast to AML cells with natively folded N-CoR, primary and secondary promyelocytic and monocytic AML cells harboring the misfolded N-CoR were highly positive for Flt3 and myeloid antigen based HSC marker CD34. Genetic and therapeutic restoration of N-CoR conformation significantly down-regulated the CD34 levels in monocytic AML cells, suggesting an important role of N-CoR in the suppression of CD34 based hematopoietic stem cell phenotypes. These finding

  9. Mobilisation of hematopoietic CD34+ precursor cells in patients with acute stroke is safe--results of an open-labeled non randomized phase I/II trial.

    Directory of Open Access Journals (Sweden)

    Sandra Boy

    Full Text Available BACKGROUND: Regenerative strategies in the treatment of acute stroke may have great potential. Hematopoietic growth factors mobilize hematopoietic stem cells and may convey neuroprotective effects. We examined the safety, potential functional and structural changes, and CD34(+ cell-mobilization characteristics of G-CSF treatment in patients with acute ischemic stroke. METHODS AND RESULTS: Three cohorts of patients (8, 6, and 6 patients per cohort were treated subcutaneously with 2.5, 5, or 10 µg/kg body weight rhG-CSF for 5 consecutive days within 12 hrs of onset of acute stroke. Standard treatment included i.v. thrombolysis. Safety monitoring consisted of obtaining standardized clinical assessment scores, monitoring of CD34(+ stem cells, blood chemistry, serial neuroradiology, and neuropsychology. Voxel-guided morphometry (VGM enabled an assessment of changes in the patients' structural parenchyma. 20 patients (mean age 55 yrs were enrolled in this study, 5 of whom received routine thrombolytic therapy with r-tPA. G-CSF treatment was discontinued in 4 patients because of unrelated adverse events. Mobilization of CD34(+ cells was observed with no concomitant changes in blood chemistry, except for an increase in the leukocyte count up to 75,500/µl. Neuroradiological and neuropsychological follow-up studies did not disclose any specific G-CSF toxicity. VGM findings indicated substantial atrophy of related hemispheres, a substantial increase in the CSF space, and a localized increase in parenchyma within the ischemic area in 2 patients. CONCLUSIONS: We demonstrate a good safety profile for daily administration of G-CSF when begun within 12 hours after onset of ischemic stroke and, in part in combination with routine i.v. thrombolysis. Additional analyses using VGM and a battery of neuropsychological tests indicated a positive functional and potentially structural effect of G-CSF treatment in some of our patients. TRIAL REGISTRATION: German

  10. Attached segment has higher CD34+ cells and CFU-GM than the main bag after thawing.

    Science.gov (United States)

    Lee, Hye Ryun; Shin, Sue; Yoon, Jong Hyun; Roh, Eun Youn; Song, Eun Young; Han, Kyou Sup; Kim, Byoung Jae

    2015-01-01

    A contiguous segment attached to the cord blood unit (CBU) is required for verifying HLA types, cell viability, and, possibly, potency before transplantation since such a segment is considered to be representative of the CBU. However, little is known regarding the characteristics of contiguous segments in comparison to main bag units due to the difficulty experienced in accessing a large number of cryopreserved CBUs. In this study, we used 245 nonconforming CBUs for allogeneic transplantation. After thawing the cryopreserved CBU, the number of total nucleated cells (TNCs), CD34(+) cells, and CFUs in CB from main bags and segments, as well as cell viability and apoptosis, were examined. The comparative analysis showed that the number of TNCs was significantly higher in CB from main bags, whereas the numbers of CD34(+) cells and CFU-GM were significantly higher in CB from segments. While the cell viability of TNCs in segments was higher, the proportion of apoptotic TNCs was also higher. In contrast, no difference was observed between the proportion of apoptotic CD34(+) cells in main bags and segments. In the correlation analysis, the numbers of TNCs, CD34(+) cells, and CFU-GM in main bags were highly correlated with those in segments, indicating that CB from segments is indeed representative of CB in main bags. Taken together, we conclude that segments have higher CD34(+) cells and CFU-GM and lower TNCs than the main cryopreserved bag, although the two compartments are highly correlated with each other.

  11. Solitary fibrous tumor of the liver expressing CD34 and vimentin: A case report

    Institute of Scientific and Technical Information of China (English)

    Dimitris P Korkolis; George N Zografos; Perikles P Vassilopoulos; Katerina Apostolaki; Chrysanthi Aggeli; George Plataniotis; Emmanuel Gontikakis; Dimitra Volanaki; Maria Sebastiadou; Dimitris Dimitroulopoulos; Dimitris Xinopoulos

    2008-01-01

    A case of a successfully treated solitary fibrous tumor(SFT) of the liver is reported. An 82-year-old femalepresented with left upper abdominal discomfort, afirm mass on palpation, and imaging studies revealeda large tumor, 15 cm in diameter, arising from theleft lobe of the liver. A formal left hepatectomy wasperformed. Microscopic evaluation showed spindleand fibroblast-like cells within the collagenous stroma.Immunohistochemistry disclosed diffuse CD34 andpositive vimentin, supporting the diagnosis of abenign SFT. The patient remained well 21 monthsafter surgery. SFT of the liver is a very rare neoplasmof mesenchymal origin. In most cases it is a benignlesion, although some may have malignant histologicalfeatures and recur locally or metastasize. With lessthan 30 reported cases in the literature, little canbe said regarding its natural history or the benefitsof adjuvant radiochemotherapy. Complete surgicalresection remains the cornerstone of its treatment.

  12. An anti-CD34 antibody-functionalized clinical-grade POSS-PCU nanocomposite polymer for cardiovascular stent coating applications: a preliminary assessment of endothelial progenitor cell capture and hemocompatibility.

    Directory of Open Access Journals (Sweden)

    Aaron Tan

    Full Text Available In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05. However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05, as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05 and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.

  13. The changes of CD34+ cells in C57 mouse bone marrow after irradiation and their roles in dysfunction of hematopoiesis%辐射小鼠骨髓CD34+细胞的变化及其意义

    Institute of Scientific and Technical Information of China (English)

    马增春; 高月; 刘永学; 谭洪玲; 张立; 陶来宝; 陈鹏

    2001-01-01

    Objective To observe the changes of CD34+ cells in C57 mouse bone marrow after irradiation and investigate the role of apoptosis in radiation-induced dysfunction of hematopoiesis. MethodsFlow cytometric enumeration of CD34+ hematopietic stem and progenitor cells by double fluorescent labeling apoptosis detection by Annexin V-FITC kit,and cell cycle detection by PI labeling were carried out. Results  ①Compared with the normal group,the percentage of CD34+ cells in bone marrow nucleated cells decreased at least for 14 days after irradiation,and the changes were related with irradiation doses.②At 6 h after irradiation,the largest amount of apoptopic cells could be detected.③Bone marrow cell cycle was perturbed after 5.5 Gy irradiation. Conclusion The percentage of CD34+ hematopietic stem and progenitor cells in C57 mouse bone marrow decreased after irradiation,and apoptosis might be responsible for the changes of the bone marrow cells.%目的 研究γ射线照射后C57小鼠骨髓中CD34+细胞的数量变化规律及其意义。方法 流式细胞仪测定CD34+细胞在骨髓有核细胞中的比例;Annexin V-FITC试剂盒检测骨髓细胞的凋亡;细胞固定后PI染色测定细胞周期。结果 ①CD34+细胞在骨髓有核细胞中的比例随照射剂量的加大而降低,在5.5 Gy照射后14 d内小鼠CD34+细胞的减少表现为持续性;②小鼠照射后6 h骨髓细胞凋亡率最高,以5.5 Gy照射组最为明显;③5.5 Gy照射后小鼠骨髓细胞周期紊乱。结论 γ射线损伤骨髓中的干祖细胞,造成骨髓中干祖细胞的数量减少,其途径之一是诱导骨髓细胞凋亡。

  14. Human embryos in the original position?

    Science.gov (United States)

    DiSilvestro, Russell

    2005-06-01

    Two different discussions in John Rawls' A Theory of Justice lead naturally to a rather conservative position on the moral status of the human embryo. When discussing paternalism, he claims that the parties in the original position would seek to protect themselves in case they end up as incapacitated or undeveloped human beings when the veil of ignorance is lifted. Since human embryos are examples of such beings, the parties in the original position would seek to protect themselves from their embryonic stages onward. When discussing the basis of equality, Rawls claims that the parties in the original position would guarantee basic rights for all those with the capacity to take part in this original position. To guarantee the basic rights of infants and young children, he goes on to interpret this capacity as a "potentiality that is ordinarily realized in due course." Since human embryos have this potentiality, they too should have basic rights.

  15. Mast Cell-activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor cells

    Directory of Open Access Journals (Sweden)

    Zoulfia eAllakhverdi

    2013-12-01

    Full Text Available Background: Shortly after allergen exposure, the number of bone marrow and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates bone marrow to release these effector cells in increased numbers. We hypothesize that mast cells may play a predominant role in this process. Objective: To examine the effect of IgE-activated mast cells on bone marrow mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors. Methods: Primary mast cells were derived from CD34+ precursors and activated with IgE/anti-IgE. Bone marrow mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL1/TNF or IgE/anti-IgE activated mast cells in Transwell system. Results: Bone marrow mesenchymal stromal cells produce low level of TSLP under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated mast cells. The latter also triggers BM-MSCs production of G-CSF, and GM-CSF while inhibiting SDF-1. Mast cell-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes. Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated mast cells trigger bone marrow mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of mast cells should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases.

  16. Tube-Like Structures with Co-Expression of D2-40 and CD34: Newly Formed Vasculatures?

    Directory of Open Access Journals (Sweden)

    Bin Jiang, Jeffrey Mason, Anahid Jewett, William CS Cho, Yan-gao Man

    2012-01-01

    Full Text Available Background: A great number of in vitro and in vivo studies have suggested that many pathways or factors can stimulate angiogenesis and lymphangiogenesis, which facilitate tumor progression and metastasis. However, the morphological and immunohistochemical profile of newly formed vasculatures has not been elucidated, making it difficult to differentiate them from the pre-existing ones, and to identify their unique molecular profiles for diagnosis and therapeutic interventions.Experimental findings: As cytokeratin (CK-19 is a well-recognized stem cell marker and CK-19-positive cells are frequently detected in the peripheral blood of patients with metastatic cancer, our recent studies have assessed the involvement of CK-19 in the formation of new vasculatures in primary colorectal cancer (CRC tissues. Our studies showed that a subset of lymph node-positive cases harbored some isolated normal epithelial structures with distinct CK-19 immunostaining within an otherwise CK-19-negative background. These structures are exclusively located within or adjacent to lymphoid follicles and are often surrounded by tube-like structures expressing lymphatic endothelial marker D2-40. Similar structures are more frequently seen at the junctions between pre-invasive and invasive CRC with the following features: (1. they consist of a single layer of endothelial cells that express both D2-40 and CD34, (2. their endothelial walls are often incomplete with disseminated cells protruding into the adjacent stroma, and (3. they are exclusively associated with disseminated CK-19-positive cellsHypothesis: Based on these findings, we propose that these tube-like structures represent newly formed vasculatures, which are derived by the convergence of aberrant lymphocyte infiltration and tumor stem cells. Because of their close physical proximity, tumor stem cells within the epithelial and stromal components contribute equally and coordinately to the morphogenesis of new

  17. Cytokines in different combination plus total saponins of panax ginseng for inducing the differentiation of CD34+ cells cultured in vitro%不同组合细胞因子与人参总皂苷对体外培养CD34+细胞分化的诱导效应

    Institute of Scientific and Technical Information of China (English)

    王建伟; 张华; 姜蓉; 王亚平

    2007-01-01

    定向诱导分化为红系血细胞的影响.③人参总皂苷对CD34+细胞形成红系祖细胞能力的影响.结果:①各因子组合中以Flt-3配基+血小板生成素+干细胞因子+白细胞介素3+红细胞生成素细胞因子组合诱导CD34+细胞分化为红系血细胞的能力最强,2周时CD71+细胞比例为(61.20±5.31)%.②人参总皂苷20 mg/L是液体培养诱导CD34+细胞向红系分化的最佳浓度,与干细胞因子+白细胞介素3+红细胞生成素协同作用CD34+细胞2周后,CD71+细胞比例从(48.39±5.07)%增加到(56.20±1.40)%.③人参总皂苷10~50 mg/L均能提高CD34+细胞形成早期红系祖细胞、晚期红系祖细胞的集落产率.结论:含有Flt-3配基+血小板生成素+干细胞因子+白细胞介素3+红细胞生成素的细胞因子组合,可诱导CD34+细胞定向生成大量的红系细胞;人参总皂苷能促进CD34+细胞定向诱导分化为红系血细胞.%BACKGROUND: Hematopoietic growth factors (HGFs) can be involved in different hematopoietic cells and different phase of differentiation. Erythropoietin (EPO) can promote he proliferation and differentiation of erythroid progenitor cells in vivo and in vitro, Interleukin-3 (IL-3) appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. IL-3 acts to enhance the effect on the proliferation and differentiation of various hematopoietic cells.OBJECTIVE: To investigate the effect of HGFs and total saponins of panax ginseng (TSPG) on erythropoietic differentiation of purified CD34+ cells from human bone marrow.DESIGN: An observational comparative experiment.SETTING: Chongqing Medical University.MATERIALS: This experiment was performed in the Department of Histology and Embryology, Institute of Basic Medical Research and Key Laboratory of Biochemistry and Molecular Pharmacology in Chongqing Medical University from July 2002 to January 2004. Human bone marrow was collected from extracted ribs of

  18. Effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34 + hematopoietic cells in bone marrow of asthmatic mice

    Institute of Scientific and Technical Information of China (English)

    毛辉; 殷凯生; 王曾礼; 李富宇; 张希龙; 刘春涛; 雷松

    2004-01-01

    Background Corticosteroids remain the most effective therapy available for asthma. They have widespread effects on asthmatic airway inflammation. However, little is known about the effects of corticosteroids on the production of bone marrow inflammatory cells in asthma. This study observed the effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34 + hematopoietic cells, so as to explore the possible effectiveness of a bone marrow-targeted anti-inflammatory strategy.Methods Balb/c mice were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. For two consecutive weeks, asthmatic mice were challenged with OVA while being given either prednisone, montelukast, prednisone plus montelukast, or sterile saline solution. The mice were killed 24 hours after the last challenge with OVA, and bronchoalveolar lavage fluid (BALF),peripheral blood, and bone marrow were collected. Eosinophils in peripheral blood and BALF, and nucleated cells in BALF, peripheral blood, and bone marrow were counted. The percentages of CD34+cells, CD4 + T lymphocytes and CD8 + T lymphocytes among nucleated cells in peripheral blood and bone marrow were counted by flow cytometry. Immunocytochemistry and in situ hybridization were employed to detect expression of CD34 and interleukin (IL)-5Rαx mRNA (CD34 + IL-5Rα mRNA+ cells)among bone marrow hematopoietic cells.Results Compared with the sterile saline solution group, the number of eosinophils in BALF and peripheral blood, CD34 + cells in peripheral blood and bone marrow, and CD34 + IL-5Rc mRNA+ cells in bone marrow of mice from the prednisone and prednisone plus montelukast groups were significantly lower (P<0.01). The number of eosinophils in BALF from the montelukast group was also significantly lower (P<0.05).Conclusions The results suggest that, in this asthmatic mouse model, prednisone probably inhibits proliferation, differentiation, and migration of CD34 + cells in bone marrow, blocks

  19. Positive selection on the human genome.

    Science.gov (United States)

    Vallender, Eric J; Lahn, Bruce T

    2004-10-01

    Positive selection has undoubtedly played a critical role in the evolution of Homo sapiens. Of the many phenotypic traits that define our species--notably the enormous brain, advanced cognitive abilities, complex vocal organs, bipedalism and opposable thumbs--most (if not all) are likely the product of strong positive selection. Many other aspects of human biology not necessarily related to the 'branding' of our species, such as host-pathogen interactions, reproduction, dietary adaptation and physical appearance, have also been the substrate of varying levels of positive selection. Comparative genetics/genomics studies in recent years have uncovered a growing list of genes that might have experienced positive selection during the evolution of human and/or primates. These genes offer valuable inroads into understanding the biological processes specific to humans, and the evolutionary forces that gave rise to them. Here, we present a comprehensive review of these genes, and their implications for human evolution.

  20. Differentiation of CD34+ hematopoietic stem cells into hepatocyte-like cells in vivo%CD34+造血干细胞移植后体内向肝样细胞分化

    Institute of Scientific and Technical Information of China (English)

    范烨; 张峰; 李相成; 常在; 王科; 钱晓峰; 王学浩

    2007-01-01

    目的 观察人脐血CD34+造血干细胞移植后在体内向肝样细胞分化的情况.方法 CD34+造血干细胞分离自足月妊娠产妇脐血,实验动物采用6~8周的非肥胖糖尿病/重症联合免疫缺陷(nonobese diabetic/severe combined immunodeficiency disease,NOD/SCID)小鼠,20 μl CCl4腹腔注射建立小鼠急性肝损伤模型,其中部分小鼠24 h后通过尾静脉注射入肝细胞生长因子(hepatocyte growth factor,HGF)的裸DNA质粒,建模48 h后将CD34+造血干细胞通过尾静脉注入小鼠体内,观察各组死亡率、肝功能恢复情况,并通过RT-PCR、免疫组化、FISH等手段检测小鼠肝组织内人源性的、分泌白蛋白的肝样细胞.结果 实验各组之间肝功能恢复无明显差异.各组存活率相近.肝组织石蜡切片显示,同时注射HGF质粒及CD34+造血干细胞的实验组,其肝组织损伤程度最轻,单独注射HGF质粒或CD34+造血干细胞的两个实验组结果相近,对照组最重.在注射CD34+造血干细胞的两组小鼠肝组织中,通过RT-PCR、免疫组化、FISH等方法均可检测到人源性的肝样细胞,联合应用HGF的实验组中,此种分化细胞数量更多,分布更广.另外与此同时可观察到融合细胞的存在.结论 脐血中CD34+的造血干细胞可以分化成肝样细胞,HGF可以促进这一分化进程.

  1. Opposing roles for CD34 in B16 melanoma tumor growth alter early stage vasculature and late stage immune cell infiltration.

    Directory of Open Access Journals (Sweden)

    Steven Maltby

    Full Text Available Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/- mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/- mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/- mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.

  2. Expression and significance of caspase-3 in CD34+ cord blood cells%Caspase-3在脐血CD34+造血干/祖细胞中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    马艳萍; 邹萍; 肖娟; 黄士昂

    2002-01-01

    目的:探讨脐血CD34+ 干/祖细胞在不同细胞因子支持下的体外扩增过程中Caspase-3表达及意义.方法:采用RT-PCR、Wester blot和流式细胞仪分析技术测定脐血CD34+ 细胞在体外扩增过程中的生物学特性及Caspase-3的表达.结果:Caspase-3 mRNA在新鲜分离的脐血CD34+ 细胞中低水平表达,在细胞因子支持下体外培养3 d,扩增的CD34+ 细胞中Caspase-3 mRNA和蛋白质表达上调,但在该两种细胞中仅能检测到分子量为32 000的无活性酶原形式的Caspase-3,随着体外培养时间的延长,在IL-3、IL-6和GM-CSF组合条件下,Caspase-3被激活,可检测到分子量为20 000的裂解片段.结论:虽然造血干细胞的凋亡是个复杂的过程,但在脐血CD34+ 干/祖细胞体外扩增过程中,Caspase-3参与了凋亡事件并发挥着重要的作用.

  3. Fluctuations of CD34 cells number in blood of cancer patients during final year of life

    Directory of Open Access Journals (Sweden)

    Alexey Shoutko

    2012-12-01

    Full Text Available The purpose of the work was the comparative study of dynamic relations between the fluctuation of survival rate and presence of hematopoietic progenitor cells in circulating blood of oncologic patients in the final months of their life. The registered associativity turbulence in the content of CD34 cells in a blood of the advanced oncologic patients and turbulence of probability of their death leads to the assumption that the reason for life’s cessation may be the abnormal morphogenic activity of young cells produced in the bone marrow toward the tumor tissues of the host, as well as a lack of such activity toward the critical normal tissues. Thus, the cytotoxic, especially long-term, therapy of patients with advanced cancer may be timely even harmful for them and compromise the ultimate result of treatment.The regimes of cytotoxic therapy of patients in cases of premature exhaustion of haematopoietic resource should be optimized based on individual periodicity of blood saturation with progenitor cells. The development of reliable and simple methods for clinical monitoring of quickly changing prognosis of patients status seems to become important aim of coming investigation. The data might be useful for optimization of regimes of cytotoxic therapy of individual patients in emergency.

  4. Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.

    Science.gov (United States)

    Yang, H; Acker, J P; Cabuhat, M; Letcher, B; Larratt, L; McGann, L E

    2005-05-01

    In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells, and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417, respectively) or platelets (P=0.88 and 0.126, respectively). However, the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001, respectively) and platelets (P=0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007, respectively) but not to platelets (P=0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

  5. Paclitaxel-Fe3O4 nanoparticles inhibit growth of CD138–  CD34– tumor stem-like cells in multiple myeloma-bearing mice

    Directory of Open Access Journals (Sweden)

    Yang C

    2013-04-01

    Full Text Available Cuiping Yang,1,3,* Jing Wang,2,* Dengyu Chen,1,* Junsong Chen,1 Fei Xiong,4 Hongyi Zhang,1 Yunxia Zhang,2 Ning Gu,4 Jun Dou11Department of Pathogenic Biology and Immunology, Medical School, 2Department of Gynecology and Obstetrics, Zhongda Hospital, Southeast University, Nanjing, 3Department of Pathogenic Biology and Immunology, School of Basic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, 4School of Biological Science and Medical Engineering, Southeast University, Nanjing, People’s Republic of China*These authors contributed equally to this workBackground: There is growing evidence that CD138– CD34– cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138– CD34– tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs on CD138– CD34– tumor stem cells in multiple myeloma-bearing mice.Methods: CD138– CD34– cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated.Results: CD138– CD34– cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 106 CD138– CD34– tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P <  0.005, and the groups that received nanoparticles alone (P < 0.005 or paclitaxel alone (P < 0.05. In addition

  6. Bole of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34(+) progenitor cells

    NARCIS (Netherlands)

    Kamps, AWA; Smit, JW; Vellenga, E

    1999-01-01

    The present study focused on whether it is possible to expand monocytic cells from CD34(+) progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6. It was demonstrated that CD34(+) cells differentiate without expans

  7. Igf1r+/CD34+ immature ICC are putative adult progenitor cells, identified ultrastructurally as fibroblast-like ICC in Ws/Ws rat colon

    DEFF Research Database (Denmark)

    Wang, X Y; Albertí, E; White, E J

    2009-01-01

    by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r(+)/CD34(+) cells were found in Ws...

  8. miR-92a Corrects CD34+ Cell Dysfunction in Diabetes by Modulating Core Circadian Genes Involved in Progenitor Differentiation.

    Science.gov (United States)

    Bhatwadekar, Ashay D; Yan, Yuanqing; Stepps, Valerie; Hazra, Sugata; Korah, Maria; Bartelmez, Stephen; Chaqour, Brahim; Grant, Maria B

    2015-12-01

    Autologous CD34(+) cells are widely used for vascular repair; however, in individuals with diabetes and microvascular disease these cells are dysfunctional. In this study, we examine expression of the clock genes Clock, Bmal, Per1, Per2, Cry1, and Cry2 in CD34(+) cells of diabetic and nondiabetic origin and determine the small encoding RNA (miRNA) profile of these cells. The degree of diabetic retinopathy (DR) was assessed. As CD34(+) cells acquired mature endothelial markers, they exhibit robust oscillations of clock genes. siRNA treatment of CD34(+) cells revealed Per2 as the only clock gene necessary to maintain the undifferentiated state of CD34(+) cells. Twenty-five miRNAs targeting clock genes were identified. Three of the miRNAs (miR-18b, miR-16, and miR-34c) were found only in diabetic progenitors. The expression of the Per2-regulatory miRNA, miR-92a, was markedly reduced in CD34(+) cells from individuals with DR compared with control subjects and patients with diabetes with no DR. Restoration of miR-92a levels in CD34(+) cells from patients with diabetes with DR reduced the inflammatory phenotype of these cells and the diabetes-induced propensity toward myeloid differentiation. Our studies suggest that restoring levels of miR-92a could enhance the usefulness of CD34(+) cells in autologous cell therapy.

  9. Removal of inhibitory substances with recombinant fibronectin-CH-296 plates enhances the retroviral transduction efficiency of CD34(+)CD38(-) bone marrow cells.

    Science.gov (United States)

    Chono, H; Yoshioka, H; Ueno, M; Kato, I

    2001-09-01

    In retroviral gene transduction, the efficiency of viral infection was reduced by the proteoglycans and some other materials secreted by the producer lines. In order to remove these inhibitors we have developed the rFN-CH-296-facilitated protocol. Because the rFN-CH-296 molecule has strong ability to bind a retroviral vector, rFN-CH-296 bound plates are utilized to wash out the unbound putative inhibitors present in a virus supernatant. The gene transduction efficiencies of human CD34(+)CD38(-) BMCs with a GALV-pseudotyped vector and the rFN-CH-296-facilitated protocol were compared with the protocol without a coating plate with CH-296, the mean gene transduction efficiencies being found to be 95.5 and 71.1%, respectively.

  10. Bullous allergic drug eruption with presence of myeloperoxidase and reorganization of the dermal vessels observed by using CD34 and collagen IV antibodies

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu-Velez

    2011-01-01

    Full Text Available Context: Bullous allergic reactions are inflammatory skin disorders, presenting usually as a result of some type of reaction to medication. Case Report: A 67-year-old female was evaluated for the presence of diffuse patches of erythema, microvesiculation, vesicles, crusts, and oozing of sudden appearance on the extremities and on the rest of the body after taking sulfamethoxazole in combination with trimethoprim. Skin biopsies for hematoxylin and eosin examination, as well as for direct immunofluorescence and immunohistochemistry analysis were performed. H&E staining demonstrated classic features. Direct immunofluorescence revealed strong deposits of fibrinogen in the vessels of the skin. The immunohistochemistry stain showed strong positivity of myeloperoxidase within the blister cavity. The distribution of the vessels around the inflammatory process were noticed by using antibodies to CD34 as well to collagen IV. Conclusions: sulfamethoxazole is catalysed by CYP2C9 and/or myeloperoxidase. Thus, myeloperoxidase appears to be of importance in this disorder.

  11. Cocultivation of umbilical cord blood CD34+ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Gang Xie; Jin-Fu Wang; Ying Xiang; Li-Yan Qiu; Bing-Bing Jia; Li-Juan Wang; Guo-Zhong Wang; Guo-Ping Huang

    2006-01-01

    AIM: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.METHODS: UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity,and long-term culture-initiating culture (LTC-IC) output.Finally, the severe-combined immunodeficient (SCID)mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis.RESULTS: There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells,CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID)mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION: The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.

  12. Toll-like receptors 2 and 4 mediate the capacity of mesenchymal stromal cells to support the proliferation and differentiation of CD34{sup +} cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xingbing, E-mail: wangxingbing91@hotmail.com [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Cheng, Qiansong; Li, Lailing; Wang, Jian; Xia, Liang [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Xu, Xiucai [The Center Laboratory of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Sun, Zimin [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China)

    2012-02-01

    Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1-10 by reverse transcription-polymerase chain reaction. TLR1-6, but not TLR7-10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 and TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34{sup +} hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34{sup +} cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM{sub 3}CSK{sub 4}) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM{sub 3}CSK{sub 4} and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.

  13. Human language reveals a universal positivity bias.

    Science.gov (United States)

    Dodds, Peter Sheridan; Clark, Eric M; Desu, Suma; Frank, Morgan R; Reagan, Andrew J; Williams, Jake Ryland; Mitchell, Lewis; Harris, Kameron Decker; Kloumann, Isabel M; Bagrow, James P; Megerdoomian, Karine; McMahon, Matthew T; Tivnan, Brian F; Danforth, Christopher M

    2015-02-24

    Using human evaluation of 100,000 words spread across 24 corpora in 10 languages diverse in origin and culture, we present evidence of a deep imprint of human sociality in language, observing that (i) the words of natural human language possess a universal positivity bias, (ii) the estimated emotional content of words is consistent between languages under translation, and (iii) this positivity bias is strongly independent of frequency of word use. Alongside these general regularities, we describe interlanguage variations in the emotional spectrum of languages that allow us to rank corpora. We also show how our word evaluations can be used to construct physical-like instruments for both real-time and offline measurement of the emotional content of large-scale texts.

  14. Expressão citofotométrica dos marcadores tumorais Ki-67 e CD34 no adenocarcinoma de estômago Citophotometric expression of tumoral markers: Ki67 and CD34 in stomach adenocarcinoma

    Directory of Open Access Journals (Sweden)

    José Augusto Menezes Freitas de Campos

    2007-09-01

    Full Text Available RACIONAL: O câncer de estômago ocupa o terceiro lugar das neoplasias malignas em todo o mundo e é a segunda causa de morte por câncer. No entanto, sua incidência está em declínio nos últimos anos, principalmente em paises como Estados Unidos, Inglaterra e Austrália, assim como onde a sua incidência sempre foi alta - Japão, China, Coréia e Leste da Ásia -, com estimativa de 780 casos anuais por 100 mil habitantes. OBEJTIVOS: Descrição da expressão citofotométrica dos marcadores Ki-67 e CD34 e comparação entre a expressão deles no adenocarcinoma de estômago. MÉTODOS: Foram selecionados inicialmente 60 blocos com espécime de adenocarcinoma gástrico coletados nos serviços de patologia do Hospital do Gama - Brasília, DF e Hospital Dom Orione - Araguaina,TO. A imunoistoquímica com os marcadores Ki-67 e CD34 foi feita no Laboratório de Citologia e Histopalogia Ltda - CITOLAB, Curitiba, PR. Foram aproveitados do total 35 blocos para o estudo histológico e imunoistoquímico que foram analisados pelo sistema de citometria de imagem computarizado SAMBA 4000 no Instituto de Pesquisas Médicas do Hospital Universitário Evangélico de Curitiba. Foram estudados o índice de marcagem e a densidade óptica para ambos os marcadores analisados separadamente e depois comparados. RESULTADOS: Das 35 lâminas, 15 (42,85% marcaram o Ki-67; 26 (74,28% o CD34 e 12 lâminas (34,28% marcaram com os dois marcadores. Quando se comparou o índice de marcagem entre Ki-67 e CD34, observou-se que existe diferença significativa (PBACKGROUND: Stomach cancer is rated as the third malignant neoplasm in the world, being the second most frequent cause of death. However, its incidence has been declining in the last few years in countries like the United States, England and Australia, and in countries where its incidence has always been high - Japan, China, Korea and in the Eastern part of Asia, which has an estimated 780 cases per year per 100

  15. Expressão citofotométrica do marcador CD34 no carcinoma epidermóide de esôfago Citophotometric expression of CD34 in squamous cell carcinoma of the esophagus

    Directory of Open Access Journals (Sweden)

    Olímpia Alves Teixeira Lima

    2007-12-01

    Full Text Available RACIONAL: O câncer de esôfago está entre as seis neoplasias malignas mais comuns do mundo. Devido à sua grande agressividade clínica, o subtipo carcinoma epidermóide constitui um dos tumores de pior prognóstico, com alto índice de morbi-mortalidade. Marcadores de biologia molecular tem sido apontados como forte coadjuvante no diagnóstico e graduação de tumores. A angiogênese, evento essencial para a progressão tumoral, pode ser estudada pelo marcador CD34. OBJETIVO: Determinar por citofotometria, usando o sistema SAMBA 4000, a expressão do marcador CD34 no carcinoma epidermóide de esôfago e, correlacioná-los com dados clínico-patológicos (idade, sexo, grau de diferenciação do tumor, estadio, tamanho, localização, profundidade e acometimento de linfonodos. MÉTODOS: Avaliaram-se 29 amostras teciduais de carcinoma epidermóide de esôfago utilizando-se coloração imunoistoquímica com marcador anti-CD34. A quantificação da expressão deste marcador foi realizada por citometria de imagem, pelo sistema SAMBA 4000 nas variáveis índice de marcagem e densidade óptica. A correlação entre subgrupos e análise estatística dos resultados foi realizada com o programa SPSS. RESULTADOS: A expressão média do marcador CD34 foi de 73,40% + 15,20 no índice de marcagem e 56,10 + 23,54 na densidae óptica. O CD34 não apresenta correlação estatisticamente significativa com as características clínico-histopatológicas estudadas (idade, sexo, grau de diferenciação do tumor, estadio, tamanho, localização, profundidade e acometimento de linfonodos. CONCLUSÃO: O marcador CD34 apresenta expressão no carcinoma epidermóide de esôfago, com maior valor no índice de marcagem em relação à densidade óptica. Ele4 não apresenta correlação com as características clínico-histopatológicas estudadas.BACKGROUND: Esophagus carcinoma is rated among the six more common malignant neoplasias in the world. Due to its aggressive

  16. 体外诱导骨髓CD34+细胞生成血管内皮细胞的方法%Induction of CD34+ cell into endothelial cell in vitro

    Institute of Scientific and Technical Information of China (English)

    李清刚; 张育; 王质刚; 徐秀红; 张玉海; 李哲先

    2002-01-01

    目的体外分离狗骨髓CD34+细胞,建立诱导分化血管内皮细胞的方法,并进行生物学鉴定.方法利用免疫磁珠法分离狗骨髓CD34+细胞,在体外经血管内皮细胞生长因子(VEGF)、内皮细胞生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)诱导后,观察细胞生长状况,以光镜、电镜进行形态学鉴定,应用免疫组化方法检测冯维靳布兰德因子(Von Willebrandfactor,vWF)表达.结果光镜下细胞单层融合生长,呈铺路石样形态,单个核位于中央,细胞群体倍增时间为35 h.电镜下可见到Weibel-Palade小体,在细胞胞浆vWF染色阳性.结论采用免疫磁珠法可从骨髓获得高纯度的CD34+细胞,在体外诱导分化成血管内皮细胞.

  17. Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group

    Science.gov (United States)

    Braulke, Friederike; Platzbecker, Uwe; Müller-Thomas, Catharina; Götze, Katharina; Germing, Ulrich; Brümmendorf, Tim H.; Nolte, Florian; Hofmann, Wolf-Karsten; Giagounidis, Aristoteles A. N.; Lübbert, Michael; Greenberg, Peter L.; Bennett, John M.; Solé, Francesc; Mallo, Mar; Slovak, Marilyn L.; Ohyashiki, Kazuma; Le Beau, Michelle M.; Tüchler, Heinz; Pfeilstöcker, Michael; Nösslinger, Thomas; Hildebrandt, Barbara; Shirneshan, Katayoon; Aul, Carlo; Stauder, Reinhard; Sperr, Wolfgang R.; Valent, Peter; Fonatsch, Christa; Trümper, Lorenz; Haase, Detlef; Schanz, Julie

    2015-01-01

    International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%–20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34+) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34+ peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34+ blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913). PMID:25344522

  18. Thyrotropin regulates IL-6 expression in CD34+ fibrocytes: clear delineation of its cAMP-independent actions.

    Directory of Open Access Journals (Sweden)

    Nupur Raychaudhuri

    Full Text Available IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD. In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO. Recently, CD34(+ fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+ orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR, the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(- orbital fibroblasts but reverts when CD34(+ fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt and NF-κB (-78 to -62 nt binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO.

  19. Actual or ideal body weight to calculate CD34+ cell dose in patients undergoing autologous hematopoietic SCT for myeloma?

    Science.gov (United States)

    Singh, V; Krishnamurthy, J; Duffey, S; Meagher, R; Villa, M; Monreal, J; Evens, A; Frankfurt, O; Altman, J; Gordon, L; Tallman, M; Williams, S; Winter, J; Singhal, S; Mehta, J

    2009-02-01

    CD34+ cell dose calculations are usually based on actual body weight (ABW). We have shown that ideal body weight (IBW) may provide a better basis for this in a small population of patients with hematologic malignancies. This was studied further in 514 myeloma autografts. The CD34+ cell doses (10(6)/kg) by IBW and ABW were 1.37-39.36 (median 6.03) and 1.15-29.67 (median 4.84), respectively. IBW-based cell doses correlated slightly better with engraftment than ABW-based doses (higher r(2)): 0.5 x 10(9)/l neutrophils 0.83 versus 0.82, 1.0 x 10(9)/l neutrophils 0.78 versus 0.77, 20 x 10(9)/l platelets 0.54 versus 0.53 and 50 x 10(9)/l platelets 0.57 versus 0.55. When outliers (hematologic recovery in 16 days) were excluded, the findings were similar: 0.5 x 10(9)/l neutrophils 0.85 versus 0.84, 1.0 x 10(9)/l neutrophils 0.85 versus 0.84, 20 x 10(9)/l platelets 0.86 versus 0.85 and 50 x 10(9)/l platelets 0.85 versus 0.84. CD34+ cell doses based on IBW as well as ABW significantly affected engraftment when analyzed separately as continuous variables. However, when analyzed together, only the dose based on IBW retained significance. We conclude that calculation of CD34+ cell numbers for autotransplantation should be based on IBW.

  20. Stem and Progenitor Cell Expansion in Co-culture of Mobilized CD34 + Cells and Osteopetrotic Mouse Stroma

    Institute of Scientific and Technical Information of China (English)

    Na LI; Shahin Rafii; JF Stoltz; Malcolm A.S. Moore; Pierre Feugier; Deog-Yeon JO; Jae Hung Shieh; Karen L. MacKenzie; JF Lesesve; V Latger-Cannard; D Bensoussan; Ronald G Crystal

    2005-01-01

    @@ 1 Introduction Culture systems capable of expanding and/or maintaining hematopoietic stem cells will not only facilitate our understanding of stem cell biology, but also broaden clinical applications. Among various in vitro hematopoietic culture systems, co-cultures of marrow or CD34+ cells with an adherent stromal layer that can produce cytokines and extracellular matrix components most effectively supports long-term hematopoiesis ( LTC ), mimicking the bone marrow micro-environment.

  1. Immunohistochemical Comparison of the Expression of CD34 and CD105 in Odontogenic Keratocyst and Dentigerous Cyst

    Science.gov (United States)

    Jamshidi, Shokoofeh; Zargaran, Massoumeh; Roshanaei, Ghodratollah; Hadadi, Fatemeh; Dehghani| Nazhvani, Ali

    2017-01-01

    Statement of the Problem: Odontogenic keratocyst (OKC) is a developmental odontogenic cyst with specific histopathological features, high recurrence rate, and aggressive clinical behavior. Angiogenesis might be considered as an important factor for the growth, expansion, and distribution of this lesion. Purpose: The aim of the present study was to determine the mean vascular densities (MVD) of OKCs and dentigerous cysts to evaluate their relationship with the biologic behavior of these lesions. Materials and Method: In this cross-sectional analytical study, angiogenesis was assessed in OKC and dentigerous cyst by measuring the MVD. Immunohistochemistry was carried out using CD34 and CD105. The results were analyzed with independent samples t-test. The data were analyzed, setting p value at 0.05. Results: The MVDs with the use of CD34 and CD105 markers were significantly higher in OKC compared to dentigerous cyst (p< 0.05). In addition, MVDs obtained by CD105 in dentigerous cysts and OKC were significantly less than those based on CD34 (p< 0.05). Conclusion: Based on the results of the present study, it can be suggested that angiogenesis might be one of the possible mechanisms involved in higher aggressive biologic behavior and greater recurrence rate of OKC compared to dentigerous cysts.

  2. 凋亡抑制因子survivin在CD+34细胞上的表达及意义

    Institute of Scientific and Technical Information of China (English)

    韩晓雁; 蔡真

    2005-01-01

    @@ Survivin是近年来发现的一种凋亡蛋白抑制因子,是凋亡抑制因子(IAPs)家族的成员之一,主要通过抑制caspase 3和caspase 7的活性而阻断细胞凋亡过程.目前已知的人类IAP蛋白有7种,分别为:XIAP、cIAP-1、cIAP-2、NAIP、survivin、Apollon和livin[1].Survivin常表达于增殖旺盛的细胞,如胚胎细胞和癌细胞[2],但目前研究发现survivin不是肿瘤特异性的,它在正常人的造血干/祖细胞(CD+34细胞)上也有表达.Fukuda等检测IAPs家族各成员在CD+34细胞上的表达,发现除survivin表达外,XIAP、cIAP-1、cIAP-2和Apollon在新鲜分离的CD+34细胞上均有表达,livin和NAIP未发现表达,经细胞因子TPO、SCF(干细胞因子)和FL(flt3配体)刺激后只有survivin-mRNA表达上调[3].

  3. Index of CD34+ Cells and Mononuclear Cells in the Bone Marrow of Spinal Cord Injury Patients of Different Age Groups: A Comparative Analysis

    Directory of Open Access Journals (Sweden)

    Vidyasagar Devaprasad Dedeepiya

    2012-01-01

    Full Text Available Introduction. Recent evidence of safety and efficacy of Bone Marrow Mononuclear Cells (BMMNC in spinal cord injury makes the Bone Marrow (BM CD34+ percentage and the BMMNC count gain significance. The indices of BM that change with body mass index and aging in general population have been reported but seldom in Spinal Cord Injury (SCI victims, whose parameters of relevance differ from general population. Herein, we report the indices of BMMNC in SCI victims. Materials and Methods. BMMNCs of 332 SCI patients were isolated under GMP protocols. Cell count by Trypan blue method and CD34+ cells by flow cytometry were documented and analysed across ages and gender. Results. The average BMMNC per ml in the age groups 0–20, 21–40, 41–60, and 61–80 years were 4.71, 4.03, 3.67, and 3.02 million and the CD34+ were 1.05%, 1.04%, 0.94%, and 0.93% respectively. The decline in CD34+ was sharp between 20–40 and 40–60 age groups. Females of reproductive age group had lesser CD34+. Conclusion. The BMMNC and CD34+ percentages decline with aging in SCI victims. Their lower values in females during reproductive age should be analysed for relevance to hormonal influence. This study offers reference values of BMMNC and CD34+ of SCI victims for successful clinical application.

  4. [Association of CD34 cell surface antigen expression with cytomorphological characteristics of acute promyelocytic leukemia blasts and clinical characteristics of patients: one center experience].

    Science.gov (United States)

    Ostojić, Alen; Pazur, Marina; Siftar, Zoran; Paro, Mirjana Mariana Kardum; Jelić-Puskarić, Biljana; Gredelj-Simec, Njetocka; Radić-Kristo, Delfa; Kardum-Skelin, Ika; Vrhovac, Radovan; Jaksić, Branimir

    2011-09-01

    The aims of the study were to investigate the association between cytomorphology and immunophenotypic expression of CD34 cell surface antigen of blasts and their relationship with clinical and laboratory characteristics of patients with acute promyelocytic leukemia (APL). Sixteen consecutive patients (male 69% and female 31%) diagnosed with APL at Department of Hematology, Merkur University Hospital between August 1998 and December 2010 were included in the study. The mean age of patients was 43.9 (range: 18-78, SD 14.9). The patients' clinical and laboratory features, cytomorphological characteristics of APL-blasts and their immunophenotype determined by flow cytometry were analyzed. Patients were divided into two groups, CD34- and CD34+, and were then compared according to clinical and laboratory characteristics. There was no difference according to age, sex or white blood cell count between two groups. The mean value of hypogranular/agranular APL-blasts was markedly higher in CD34+ group than CD34- group (34%, range 9-60, SD 24.4 vs. 11.5%, range 0-38, SD 13.7), with borderline statistical significance (P=0.055). CD34- patients had significantly better overall survival than CD34+ ones (P=0.02). Patients without Auer rods detected in APL-blasts had higher CD34 expression (69.4% +/- 33.8) compared to patients with detected Auer rods (7.3% +/- 24.8), but statistical significance was not reached (p=0.053). Our results are consistent with the results of other published studies and point to the fact that higher CD34 expression and lower cytoplasmic granularity of APL-blasts are factors that seem to define a specific subgroup of APL patients. Together with other diagnostic tools currently available, they could be of value in planning treatment of APL patients.

  5. [Expression of adhesion molecules on CD34+ cells of BM and PB stem cell samples during high-dose chemotherapy combined with transplantation of autologous PB stem cells].

    Science.gov (United States)

    Liu, Peng; Han, Xiao-Hong; Shi, Yuan-Kai; He, Xiao-Hui; Yang, Cheng; Ai, Bin

    2004-12-01

    This study was aimed to investigate the expressions of adhesion molecules such as CD54, CD49d and CD62L by CD34(+) cells sampled from different stages of bone marrow (BM) and peripheral blood (PB) before/after G-CSF mobilization and after transplantation through the direct labeling with three colour-immunofluorescence and flow cytometry, and to explore the differences in expression of adhesion molecules on CD34(+) cells from different origins and their clinical significance. Mononuclear cells collected from BM and PB before mobilization, after collection of stem cells and hematopoietic recostruction of BM at the end of transplantation were marked with CD54-FITC, CD49d-FITC and CD62L-FITC separately, as well as CD34-PE and CD45PerCE. 3-color fluorescene analysis was carried out by FACS. The expression differences of CD34(+) and adhesion molecules between BM and APBSC were compared. The results showed that expression differences of CD54, CD49d and cd62Lon CD34(+) cells belore mobilization, after collection and reconstraction of transplantation were not statiscally significant, the difference of CD54, CD49d and CD62L on CD34(+) between 1st and 2nd collections of hematopoietic stem cells also were not statiscally significant. In the collected APBSC, the expression level of CD34(+) CD49d(+) was significantly lower than those in BM before mobilization (P = 0.001). It is concluded that the method of chemotherapy combined with G-CSF mobilization can down-regulate CD49d expression in BM CD34(+) cells, thus can mobilize and move theirs into peripheral blood. After the reconstitution by transplantation, the expression of CD49d on CD34(+) cells tends to normal, the clinical significance needs to be elucidated by accumulation of much more cases.

  6. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

    Directory of Open Access Journals (Sweden)

    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  7. Nestin-positive progenitor cells isolated from human fetal pancreas have phenotypic markers identical to mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ling Zhang; Tian-Pei Hong; Jiang Hu; Yi-Nan Liu; Yong-Hua Wu; Ling-Song Li

    2005-01-01

    AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro.METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescenceactivated cell sorting (FACS) and radioimmunoassay (RIA)were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 timesin vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cellmarkers; (3) FACS analysis revealed that CD44, CD90and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreaticduodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intracellular insulin content was detected by RIA after the induction culture.CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulinproducing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.

  8. Effects of hyperbaric oxygen therapy in the management of chronic wounds and its correlation with CD34 ± endothelial progenitor cells%高压氧治疗下肢慢性创面愈合与外周血内皮祖细胞的相关性

    Institute of Scientific and Technical Information of China (English)

    马辕华; 雷永红; 周敏; 李雪; 赵宏宇

    2011-01-01

    Objective To evaluate the effects of hyperbaric oxygen(HBO)therapy in the management of chronic wound and observe the correlation between wound healing and CD34 + endothelial progenitor cells(EPCs).Methods A total of 119 patients with chronic wound in lower extremities lasting >3 months were recruited for this randomized,single-center,placebo-controlled clinical trial.The changes of CD34 + average count before and after HBO therapy were detected by flow cytometry(FACS).There were 97 patients on long-term HBO therapy and in 22 patients on hyperbaric air therapy as control group.The CD34/Scal-1 + and CD34/CXCR4 dual-positive populations of gated cell were determined respectively by FACs.The outcomes of two groups were compared.Treatment was administered within a single-place hyperbaric chamber for 90-main daily(session duration 120 main)for 5 days a week for 4 weeks (20 treatment sessions).Results The wound size decreased at the4-week end point(62.7% +22.3% in the HBO group vs 34.4% +20.6% in the control group,P <0.05).Mter 10 episodes of HBO therapies for chronic non-healing wound,the peripheral CD34 + EPCs average count rose from 0.24% + 0.03% at pretreatment to 1.32% ±0.05% while the number was 1.75% + 0.17% after 20 episodes of HBO(P < 0.05)Both were significantly different from that of the patients at pre-treatment.However the overall circulating white cell count was not significantly elevated.The CD34/Scal-1 + and CD34/CXCR4 dual-positive populations of gated cell in HBO group were 5.8 and 5.2 folds than those at pre-treatment respectively.The number of EPCs was positively correlated with wound healing in lower extremities(correlation coefficient 0.84 ; P < 0.01).Conclusion Adjunctive treatment of HBO facilitates the healing of chronic non-healing wound in selected patients through the mobilization of EPCs.%目的 探讨高压氧(HBO)对中国人下肢创伤后小腿部慢性创面愈合的影响,以及创面愈合与外周循环中CD

  9. 半叶马尾藻多糖对辐射损伤小鼠骨髓CD+34细胞和SCF mRNA表达的影响%Effect of Sargassum hemiphyllum polysaccharides on the mRNA expression of CD+34 cells and SCF in irradiated mice

    Institute of Scientific and Technical Information of China (English)

    孟庆勇; 刘志辉; 郑辉

    2005-01-01

    腹腔注射10~40mg·kg-1·d-1半叶马尾藻多糖[Sargassum hemiphyllum (Turner)C.Agardh poly-saccharides,SHP],7d后,小鼠经5Grγ射线照射,观察SHP对辐射损伤小鼠骨髓CD+34细胞占骨髓有核细胞的百分率、CD34基因表达和SCF(stem cell factor,干细胞因子)mRNA基因表达的变化,探讨SHP抗辐射损伤保护小鼠造血功能的机理.用流式细胞术测定CD+34细胞含量,通过RT-PCR检测CD+34和SCF mRNA基因表达.结果显示SHP能显著提高辐射小鼠CD+34细胞的百分率,提高CD+34和SCFmRNA基因的表达.增加CD+34细胞数、促进CD+34和SCF mRNA基因表达可能是该海藻多糖抗辐射、保护小鼠造血功能的重要机理之一.

  10. Properties of monocytes generated from haematopoietic CD34(+) stem cells from bone marrow of colon cancer patients.

    Science.gov (United States)

    Stec, Malgorzata; Baran, Jarosław; Szatanek, Rafał; Mytar, Bożenna; Lenart, Marzena; Czupryna, Antoni; Szczepanik, Antoni; Siedlar, Maciej; Zembala, Marek

    2013-04-01

    Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34(+) stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34(+) haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14(+) monocytes were found. They consisted of two subpopulations: CD14(++)CD16(+) and CD14(+)CD16(-), at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14(+) monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3(+) T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu369-377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8(+) T lymphocytes (CTL). The CD14(++)CD16(+) subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34(+) stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.

  11. Direct visualization of neo-vessel formation following peripheral injection of bone marrow derived CD34+ cells in experimental myocardial damage.

    Science.gov (United States)

    Ciulla, M M; Ferrero, S; Gianelli, U; Paliotti, R; Magrini, F; Braidotti, P

    2007-01-01

    The definitive fate of peripherally injected PKH26 labelled bone marrow mononuclear cells expressing the CD34+ antigen following experimental myocardial cryodamage in rats (n=10) has been examined by direct visualization on photoconverted light and electron microscopy images. One week after the injection in each rat of about 150,000 CD34+ cells early stage PKH26+ vascular structures were localized in the infarcted areas, suggesting that a potential benefit of this therapeutic approach consists in the regeneration of the vasculature.

  12. Impact of mobilized blood progenitor cell quality determined by the CFU-GM/CD34+ ratio on rapid engraftment after blood stem cell transplantation.

    Science.gov (United States)

    Fu, S Q; Abboud, C N; Brennan, J K; Ifthikharuddin, J J; Nichols, D; Liesveld, J L

    2002-01-01

    To find a parameter to predict the quality of collected mobilized CD34+ blood as hemopoietic reconstituting cells, the ratio of CFU-GM to CD34+ cells was examined. One hundred six consecutive patients who underwent blood stem cell transplantation at the University of Rochester from 01/01/99 to 12/31/99 were examined retrospectively for the number of days to reach an absolute neutrophil count of 500 or 1000 cells/microl and an absolute platelet count of 20,000 or 50,000 cells/microl without transfusion support as measures of engraftment. Linear regression analyses were conducted to determine factors influencing engraftment. The number of CD34+ cells/kg and CFU-GM/kg correlated highly with the number of nucleated blood cells/kg. In this population, in which 90% of patients received >2 x 10(6) CD34+ cells/kg, neither the number of CD34+ cells/kg nor the number of CFU-GM/kg correlated with the time to engraftment as judged by neutrophil or platelet levels. In contrast, the lower the ratio of CFU-GM to CD34+ cells, the more rapid the reconstitution of platelets to 20,000/microl (P = 0.03) and 50,000/microl (P = 0.02). Thus, a lower ratio of the CFU-GM/CD34+ appended to reflect a greater number of hematopoietic reconstituting cells in the blood cell collection. The CFU-GM/CD34+ ratio is an apparent predictor of earlier platelet engraftment, suggesting that the ratio reflects the engraftment potential of mobilized donor progenitor cells.

  13. Low CD34 dose is associated with poor survival after reduced-intensity conditioning allogeneic transplantation for acute myeloid leukemia and myelodysplastic syndrome.

    Science.gov (United States)

    Törlén, Johan; Ringdén, Olle; Le Rademacher, Jennifer; Batiwalla, Minoo; Chen, Junfang; Erkers, Tom; Ho, Vincent; Kebriaei, Partow; Keever-Taylor, Carolyn; Kindwall-Keller, Tamila; Lazarus, Hillard M; Laughlin, Mary J; Lill, Michael; O'Brien, Tracey; Perales, Miguel-Angel; Rocha, Vanderson; Savani, Bipin N; Szwajcer, David; Valcarcel, David; Eapen, Mary

    2014-09-01

    Reduced-intensity conditioning/nonmyeloablative conditioning regimens are increasingly used in allogeneic hematopoietic cell transplantation (HCT). Reports have shown CD34(+) dose to be important for transplantation outcome using myeloablative conditioning. The role of CD34(+) dose of peripheral blood progenitor cells (PBPC) has not been previously analyzed in a large population undergoing reduced-intensity conditioning/nonmyeloablative HCT. We studied 1054 patients, ages 45 to 75 years, with acute myeloid leukemia or myelodysplastic syndrome who underwent transplantation between 2002 and 2011. Results of multivariate analysis showed that PBPC from HLA-matched siblings containing after myeloablative HCT, CD34(+) dose did not affect relapse or graft-versus-host disease with either donor type. An upper cell dose limit was not associated with adverse outcomes. These data suggest that PBPC CD34(+) doses >4 × 10(6) CD34(+)/kg and >6 × 10(6) CD34(+)/kg are optimal for HLA-matched sibling and unrelated donor HCT, respectively.

  14. Analysis of CD34+ cell collection using two mobilization regimens for newly diagnosed multiple myeloma patients reveals the separate impact of mobilization and collection variables.

    Science.gov (United States)

    Abuabdou, Ahmed; Rosenbaum, Eric R; Usmani, Saad Zafar; Barlogie, Bart; Cottler-Fox, Michele

    2014-10-01

    Mobilization regimens for CD34+ cells have generally been judged successful based on the number of cells collected without evaluating mobilization separately from collection. Using retrospective data for patients who collected CD34+ cells on Total Therapy protocols 3a/3b (VTD-PACE) and Total Therapy 4/5 using a novel regimen that added low dose melphalan to VTD-PACE (MVTD-PACE), we analyzed mobilization and collection variables separately. A significant difference favoring MVDT-PACE was found in mean CD34+ cells/µL on day 2 of collection and in mean ratio of CD34+ cells/µL on day 2 to day 1. However, because apheresis variables and growth factor dose during collection were manipulated to optimize individual collections, the two regimens were not significantly different when the mean total CD34+ cells ×10(6) /kg collected was compared. Thus, when evaluating a chemotherapy regimen or new growth factor for mobilization, it is important to realize that total CD34+ cells collected is dependent on both mobilization and collection variables.

  15. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    Science.gov (United States)

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

  16. Timing of Peripheral Blood Stem Cell Yield: Comparison of Alternative Methods with the Classic Method for CD34+ Cell Determination

    Directory of Open Access Journals (Sweden)

    I. Fatorova

    2014-01-01

    Full Text Available Hematopoietic stem cells (HSCs, still represent a certain mystery in biology, have a unique property of dividing into equal cells and repopulating the hematopoietic tissue. This potential enables their use in transplantation treatments. The quality of the HSC grafts for transplantation is evaluated by flow cytometric determination of the CD34+ cells, which enables optimal timing of the first apheresis and the acquisition of maximal yield of the peripheral blood stem cells (PBSCs. To identify a more efficient method for evaluating CD34+ cells, we compared the following alternative methods with the reference method: hematopoietic progenitor cells (HPC enumeration (using the Sysmex XE-2100 analyser, detection of CD133+ cells, and quantification of aldehyde dehydrogenase activity in the PBSCs. 266 aphereses (84 patients were evaluated. In the preapheretic blood, the new methods produced data that were in agreement with the reference method. The ROC curves have shown that for the first-day apheresis target, the optimal predictive cut-off value was 0.032 cells/mL for the HPC method (sensitivity 73.4%, specificity 69.3%. HPC method exhibited a definite practical superiority as compared to other methods tested. HPC enumeration could serve as a supplementary method for the optimal timing of the first apheresis; it is simple, rapid, and cheap.

  17. 外周血CD_(34)~+细胞检测对外周血干细胞采集结果及时机选择的意义%Significance of peripheral CD_(34)~+ cell count on the harvest of mobilized peripheral hematopoietic stem cells

    Institute of Scientific and Technical Information of China (English)

    唐暐; 王苓; 赵维莅; 沈志祥; 胡炯

    2010-01-01

    Objective Autologous hematopoietic stem cell transplantation (Auto-HSCT) has been widely used in hematological malignancies.To mobilize and harvest sufficient number of peripheral CD_(34)~+ cells is one of key issues for auto-HSCT. Peripheral CD_(34)~+ cell numeration has been used as an indicator for apheresis while we mostly rely on the peripheral WBC or MNC count. In this study, we try to evaluate the association of peripheral CD_(34)~+ count to the CD_(34)~+ cells number in the apheresis product and to find out a potential threshold. Methods From Jan 2007 to Dec 2009, a total of 57 apherosis for auto-HSCT were analysed. All patients were mobilized by cyclophophamide (CTX) plus G-CSF(5-10μg/kg) regimen. The apheresis were performed with COBE SPECTRA VERSION 6 and CD_(34)~+ count of both peripheral and apheresis products were analysed by flow cytometry. Results The median number of MNC in apheresis products was 4.6(0.3-10.5)×10~8/kg with median CD_(34)~+ cells at 2.4(0.16-34.9)×10~6/kg. The peripheral CD_(34)~+ count was the only parameter associated with the MNC and CD_(34)~+ cell numbers in the apheresis products while the WBC number was irrelevant to the results of apheresis. Our data showed that when the peripheral CD_(34)~+ count reach 15/μl, the efficacy of a single apheresis significantly improved with 81 % and 60 % reached 1 and 2×10~6 CD_(34)~+ cells/kg respectively and the total number of MNC and CD_(34)~+ cells were significantly superior to apheresis with peripheral CD_(34)~+ cells <15/μl, thus indicated that CD_(34)~+ ≥15 /μl can be used as the threshold for apheresis. Furthermore, the ROC analysis demonstrated that CD_(34)~+ cells ≥25(26.5-28.6) /μl is the best indicator level for a successful single apheresis. Conclusion Our study clearly showed that peripheral CD_(34)~+ cell count is a key indicator of apherosis. CD_(34)~+ cells at 15/μl can be used as the threshold to start apheresis in the clinical setting.%目的 研究外周血CD

  18. A novel use of growth factors, CD34 positive cells, and fibrin for fingertip injury: Description of a case

    Directory of Open Access Journals (Sweden)

    Francesco Romano

    2016-01-01

    Full Text Available Traumatic soft tissue injuries of the finger represent a frequent hand injury challenging hand surgeons. We report a case involving a 30-year-old man with a dorsal index finger soft tissue wound failing conservative treatment. The novel use of applied fibrin membranes and concentrated growth factors yielded complete resolution of the injury in 6 months without need for skin grafting.

  19. Changes in the number of circulating CD34+cells after eccentric exercise of the elbow flexors in relation to muscle damage

    Institute of Scientific and Technical Information of China (English)

    Ho Seong Lee; Makii Muthalib; Takayuki Akimoto; Kazunori Nosaka

    2015-01-01

    Background:It has been reported that strenuous exercise increases the number of bone marrow-derived progenitor cells such as CD34+cells in the blood, but no previous studies have investigated the changes in circulating CD34+cells following resistance exercise. This study tested the hypothesis that the number of CD34+cells in the blood would increase after eccentric exercise of the elbow flexors, but decrease in recovery, and the magnitude of the changes would be dependent on the magnitude of muscle damage. Methods:Nine men (28.0 ± 6.6 years) performed exercises consisting of 10 sets of six maximal voluntary eccentric contractions of the elbow flexors with their non-dominant arm. Six of them performed the same exercise with the same arm 4 weeks later. Changes in indirect markers of muscle damage were measured before, within 10 min after, and at 24, 48, 72, and 96 h after eccentric exercise. Differential leukocyte counts (total leukocytes, neutrophils, lymphocytes, monocytes) and CD34+cells in the blood were measured before, immediately after, and at 2, 24, 48, 72, and 96 h following the exercises. Results:After eccentric exercise, significant ( p<0.05) decreases in maximal voluntary isometric contraction torque and increases in delayed onset muscle soreness and plasma creatine kinase activity were observed. However, no significant changes in leukocytes and CD34+cells were evident. The changes in muscle damage markers were significantly ( p<0.05) smaller following the second exercise session as compared with the first exercise session, but the changes in leukocytes and CD34+cells were not significantly different between sessions. Conclusion:These results did not support the hypothesis, and showed that eccentric exercise-induced muscle damage to the elbow flexors did not influence the number of circulating CD34+cells.

  20. Allogeneic T cells induce rapid CD34+ cell differentiation into CD11c+CD86+ cells with direct and indirect antigen-presenting function

    Science.gov (United States)

    Abbasian, Javaneh; Mahmud, Dolores; Mahmud, Nadim; Chunduri, Sandeep; Araki, Hiroto; Reddy, Pavan; Hoffman, Ronald; Arpinati, Mario; Ferrara, James L. M.; Rondelli, Damiano

    2006-01-01

    Dendritic cells (DCs) derive from CD34+ cells or monocytes and stimulate alloimmune responses in transplantation. We hypothesized that the interaction between CD34+ cells and allogeneic T cells would influence the function of hematopoietic stem cells (HSCs). Cord blood (CB) CD34+ cells proliferated briskly in response to allogeneic, but not autologous, T cells when mixed with irradiated T cells for 6 days in vitro. This proliferation was significantly inhibited by an anti-HLA class II monoclonal antibody (mAb), by an anti-TNFα mAb, or by CTLA4-Ig. Allogeneic T cells induced the differentiation of CD34+ progenitors into cells with the morphology of dendritic monocytic precursors and characterized by the expression of HLA-DR, CD86, CD40, CD14, and CD11c, due to an endogenous release of TNFα. Cotransplantation of CD34+ cells with allogeneic T cells into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice resulted in a greater engraftment of myeloid CD1c+ dendritic cells compared with cotransplantation with autologous T cells. In vitro, CD34+ cell-derived antigen-presenting cells (APCs) were functionally capable of both direct and indirect presentation of alloantigens. Based on these findings, we hypothesize that in HSC transplantation the initial cross talk between allogeneic T cells and CD34+ cells may result in the increased generation of APCs that can present host alloantigens and possibly contribute to the development of graft-versus-host disease. (Blood. 2006;108:203-208) PMID:16478883

  1. Comparative Analysis of Telomerase Activity in CD117+CD34+ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yuan Li; Shan-Shan Lu; Ting Xu; Hong-Qi Zhang; Hua Li

    2015-01-01

    Background:This study characterized the cardiac telocyte (TC) population both in vivo and in vitro,and investigated its telomerase activity related to mitosis.Methods:Using transmission electron microscopy and a phase contrast microscope,the typical morphological features of cardiac TCs were observed;by targeting the cell surface proteins CD 1 17 and CD34,CD 117+CD34+ cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture.Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8.Under this conditioned medium,the process of cell division was captured,and the telomerase activity ofCD 117+CD34+ cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs),cardiac fibroblasts (CFBs),cardiomyocytes (CMs).Results:Cardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms).In addition,64% of the primary cultured cardiac TCs were composed of CD 117+CD34+ cardiac TCs;which was verified by immunofluorescence.In a live cell imaging system,CD 117+CD34+ cardiac TCs were observed to enter into cell division in a short time,followed by an significant invagination forming across the middle of the cell body.Using a real-time quantitative telomeric-repeat amplification assay,the telomerase concentration in CD117+CD34+ cardiac TCs was obviously lower than in BMSCs and CFBs,and significantly higher than in CMs.Conclusions:Cardiac TCs represent a unique cell population and CD117+CD34+ cardiac TCs have relative low telomerase activity that differs from BMSCs,CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis.

  2. Comparative Analysis of Telomerase Activity in CD117+CD34+ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

    Science.gov (United States)

    Li, Yuan-Yuan; Lu, Shan-Shan; Xu, Ting; Zhang, Hong-Qi; Li, Hua

    2015-01-01

    Background: This study characterized the cardiac telocyte (TC) population both in vivo and in vitro, and investigated its telomerase activity related to mitosis. Methods: Using transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD117 and CD34, CD117+CD34+ cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117+CD34+ cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs), cardiac fibroblasts (CFBs), cardiomyocytes (CMs). Results: Cardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms). In addition, 64% of the primary cultured cardiac TCs were composed of CD117+CD34+ cardiac TCs; which was verified by immunofluorescence. In a live cell imaging system, CD117+CD34+ cardiac TCs were observed to enter into cell division in a short time, followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117+CD34+ cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs. Conclusions: Cardiac TCs represent a unique cell population and CD117+CD34+ cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis. PMID:26168836

  3. Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34(+)CD38(-) stem and progenitor cells in chronic myeloid leukemia.

    Science.gov (United States)

    Dolinska, Monika; Piccini, Alexandre; Wong, Wan Man; Gelali, Eleni; Johansson, Anne-Sofie; Klang, Johannis; Xiao, Pingnan; Yektaei-Karin, Elham; Strömberg, Ulla Olsson; Mustjoki, Satu; Stenke, Leif; Ekblom, Marja; Qian, Hong

    2017-08-19

    Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34pos Hematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction

    Directory of Open Access Journals (Sweden)

    Mohsen Moslem

    2015-01-01

    Full Text Available Mesenchymal stem/stromal cells (MSCs represent a promising cell source for research and therapeutic applications, but their restricted ex vivo propagation capabilities limit putative applications. Substantial self-renewing of stem cells can be achieved by reprogramming cells into induced pluripotent stem cells (iPSCs that can be easily expanded as undifferentiated cells even in mass culture. Here, we investigated a differentiation protocol enabling the generation and selection of human iPSC-derived MSCs exhibiting relevant surface marker expression profiles (CD105 and CD73 and functional characteristics. We generated such iPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs. All such iPSC-MSCs exhibited the characteristics of normal bone marrow-derived (BM MSCs. In direct comparison to BM-MSCs our iPSC-MSCs exhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passages. Considering functional capabilities, iPSC-MSCs provided supportive feeder layer for CD34+ hematopoietic stem cells’ self-renewal and colony forming capacities. Furthermore, iPSC-MSCs gained immunomodulatory function to suppress CD4+ cell proliferation, reduce proinflammatory cytokines in mixed lymphocyte reaction, and increase regulatory CD4+/CD69+/CD25+ T-lymphocyte population. In conclusion, we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source or reprogramming factor composition and we suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeutic approaches.

  5. Comparison of reendothelialization and neointimal formation with stents coated with antibodies against endoglin and CD34 in a porcine model

    Directory of Open Access Journals (Sweden)

    Cui S

    2015-04-01

    Full Text Available Song Cui,1* Xian-Tao Song,1 Chao Ding,2* Li-Jun Meng,3 Shu-Zheng Lv,1 Kefeng Li4,5 1The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Department of Cardiology, Anzhen Hospital Affiliated to Capital Medical University, Beijing, People’s Republic of China; 2Department of Cardiology, Huimin People’s Hospital, Binzhou, People’s Republic of China, 3Department of Cardiology, Binzhou Central Hospital, Binzhou, People’s Republic of China; 4School of Medicine, University of California, San Diego, CA, USA; 5Tianjin SunnyPeak Biotech Co, Ltd, Tianjin, People’s Republic of China *These authors contributed equally to this work Abstract: Anti-CD34 coated stents are the only commercialized antibody-coated stents currently used for coronary artery diseases with various limitations. Endoglin plays important roles in the proliferation of endothelial cells and vascular remodeling and could be an ideal target surface molecule. The objective of this study was to investigate the efficacy of stents coated with anti-endoglin antibodies (ENDs in terms of endothelial recovery and the reduction of neointimal formation. The performance of ENDs was evaluated by comparing with stents coated with anti-CD34 antibodies (CD34s, sirolimus-eluting stents (SESs, and bare metal stents (BMSs. Stents were randomly assigned and placed in the coronary arteries of juvenile pigs. Histomorphometric analysis and scanning electron microscopy were performed after stent implantation. Our results showed at 14 days after stent implantation, the neointima area and percent area stenosis in ENDs and CD34s were remarkably decreased compared with those in BMSs and SESs (P<0.05. Moreover, the percentage of reendothelialization was significantly higher in ENDs and CD34s than that in SESs or BMSs at both 7 and 14 days (P<0.05. There was no difference in the neointima area, percent area stenosis, and percentage of reendothelialization in ENDs compared with CD34s. The artery

  6. Expressions of fibroblast growth factor-1, mitogen activated protein kinase-1 and CD34 in the lesions of patients with psoriasis vulgaris and their significance%银屑病患者成纤维细胞生长因子1、丝裂原活化蛋白激酶1、CD34的表达及意义

    Institute of Scientific and Technical Information of China (English)

    焦健; 张春红; 吕世峰

    2013-01-01

    Objective To estimate the relationship of fibroblast growth factor-1 (FGF-1),mitogen activated protein kinase-1 (MAPK-1) and CD34 expressions with clinicopathological findings in lesions of psoriasis vulgaris.Methods Skin specimens were collected from 30 patients with psoriasis vulgaris,whose general information and clinical findings were recorded.The protein expressions of FGF-1,MAPK-1 and CD34 in these specimens were detected by tissue chip technology and immunohistochemistry,and the mRNA expression of FGF-1 by reverse transcription-PCR.The correlations of FGF-1 protein expression with patients' gender,age,clinical stage,psoriasis area and severity index (PASI) score,angiogenesis and abnormal epidermal proliferation were statistically analyzed.Results The protein and mRNA expressions of FGF-1 were significantly higher in the lesions than in the control skin (both P < 0.05).The protein expressions of CD34 and MAPK-1 were significantly increased,and positively associated with the protein expression of FGF-1 in psoriatic lesions.The up-regulation of FGF-1 protein was unrelated to the age or gender of patients,but associated with clinical stage and PASI score.The proportion of patients with increased FGF-1 expression was significantly larger in patients at active stage and with high PSAI score than in those at quiescent stage and with low PSAI score (P < 0.05).Conclusions In psoriasis vulgaris,FGF-1 may participate in the abnormal epidermal proliferation and vascular dysplasia in dermal papilla,and the upregulation of FGF-1 appears to be associated with the progression of disease and aggravation of lesions.%目的 探讨寻常性银屑病皮损成纤维细胞生长因子1(FGF-1)、丝裂原活化蛋白激酶1(MAPK-1)、CD34与临床病理的相关性.方法 收集30例寻常性银屑病患者的皮损,记录患者的一般情况和临床表现.用组织芯片技术和免疫组化法检测银屑病皮损中FGF-1、MAPK-1、CD34蛋白的表达,反转录PCR(RT-PCR)

  7. A neutralizing anti-TGF-beta1 antibody promotes proliferation of CD34+Thy-1+ peripheral blood progenitors and increases the number of transduced progenitors.

    Science.gov (United States)

    Imbert, A M; Bagnis, C; Galindo, R; Chabannon, C; Mannoni, P

    1998-05-01

    The subset of blood cells that expresses both CD34 and Thy1 (CD90) cell surface molecules is enriched in hematopoietic stem cell activity and can be obtained from the peripheral blood of cancer patients after mobilization by chemotherapy and granulocyte colony-stimulating factor (G-CSF). Because transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of hematopoietic progenitor proliferation and differentiation, in this study we analyzed the impact of neutralizing TGF-beta1 activity during culture and retroviral transduction of CD34+Thy1+ cells. When purified CD34+Thy1+ cells were cultured in the presence of a neutralizing antibody against TGF-beta1, the percentage of cycling cells, proliferation, and absolute number of clonogenic progenitors were increased in comparison to the cultures performed without the addition of antibody. Antibody-mediated neutralization of TGF-beta1 during retroviral transduction performed by coculture of CD34+Thy1+ cells with a MFG-S-nlsLacZ retroviral vector-producing cell line did not affect the percentage of transduced progenitors as assessed by direct X-Gal staining of colonies in clonogenic assays. However, due to the better expansion of CD34+Thy1+ cells in the presence of anti-TGF-beta1, the absolute number of transduced progenitors recovered at the end of the culture was increased.

  8. NUP98/11p15 translocations affect CD34+ cells in myeloid and T lymphoid leukemias.

    Science.gov (United States)

    Crescenzi, Barbara; Nofrini, Valeria; Barba, Gianluca; Matteucci, Caterina; Di Giacomo, Danika; Gorello, Paolo; Beverloo, Berna; Vitale, Antonella; Wlodarska, Iwona; Vandenberghe, Peter; La Starza, Roberta; Mecucci, Cristina

    2015-07-01

    We assessed lineage involvement by NUP98 translocations in myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML), and T-cell acute lymphoblastic leukaemia (T-ALL). Single cell analysis by FICTION (Fluorescence Immunophenotype and Interphase Cytogenetics as a Tool for Investigation of Neoplasms) showed that, despite diverse partners, i.e. NSD1, DDX10, RAP1GDS1, and LNP1, NUP98 translocations always affected a CD34+/CD133+ hematopoietic precursor. Interestingly the abnormal clone included myelomonocytes, erythroid cells, B- and T- lymphocytes in MDS/AML and only CD7+/CD3+ cells in T-ALL. The NUP98-RAP1GDS1 affected different hematopoietic lineages in AML and T-ALL. Additional specific genomic events, were identified, namely FLT3 and CEBPA mutations in MDS/AML, and NOTCH1 mutations and MYB duplication in T-ALL.

  9. Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors

    Science.gov (United States)

    Poggi, Marjorie; Canault, Matthias; Favier, Marie; Turro, Ernest; Saultier, Paul; Ghalloussi, Dorsaf; Baccini, Veronique; Vidal, Lea; Mezzapesa, Anna; Chelghoum, Nadjim; Mohand-Oumoussa, Badreddine; Falaise, Céline; Favier, Rémi; Ouwehand, Willem H.; Fiore, Mathieu; Peiretti, Franck; Morange, Pierre Emmanuel; Saut, Noémie; Bernot, Denis; Greinacher, Andreas; BioResource, NIHR; Nurden, Alan T.; Nurden, Paquita; Freson, Kathleen; Trégouët, David-Alexandre; Raslova, Hana; Alessi, Marie-Christine

    2017-01-01

    Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels. PMID:27663637

  10. Transcriptional activity of telomerase complex in CD34- stem cells of cord blood in dependence of preparation time.

    Directory of Open Access Journals (Sweden)

    M Bojdys-Szyndlar

    2009-12-01

    Full Text Available The aim of the study was to determine whether the expression of telomerase subunits encoding genes changes during the process of cord blood preparation. It should establish if the commonly accepted 24 hours time interval in stem cells kriopreservation procedure significantly influences their immortalization and so decreases the "quality" of cord blood stem cells. Investigation includes 69 women. Spontaneous labour was the inclusion condition. The material was collected at birth after clamping of umbilical cord by direct vasopuncture. CD34- cells were extracted from cord blood (MACS, Miltenyi Biotec; Bisley, Surrey, UK. The expression profile of telomerase activators and inhibitors encoding genes was determined using HG_U133A oligonucleotide microarray (Affymetrix. We used a real-time quantitative RT-PCR assay to quantify the telomerase TERT, hTR and TP1 subunits mRNA copy numbers in CD34- cells in 0, 6, 12 and 24 hours after cord blood collection. We observed significant decrease of numbers of copies of TERTA+B mRNA within the successive hours of observation. Significant decrease of numbers of TERTA mRNA copies was confirmed after 24 hours. However, we observed significant increase of numbers of copies of TERTB mRNA after 6 hours of observation. Similar level was maintained during another 6h. The significantly lower number of copies of TERTB mRNA was observed after 24h. We also observed significant increase of number of copies of TERT mRNA after 6 hours. Number of copies of TERT mRNA significantly decreased after another 6h, remaining, however, on a higher then initial one. The significant lower number of copies of TERT mRNA was observed 24h after delivery. The possible explanation of those results is discussed in the paper.

  11. Dominant unit CD34+ cell dose predicts engraftment after double-unit cord blood transplantation and is influenced by bank practice.

    Science.gov (United States)

    Purtill, Duncan; Smith, Katherine; Devlin, Sean; Meagher, Richard; Tonon, Joann; Lubin, Marissa; Ponce, Doris M; Giralt, Sergio; Kernan, Nancy A; Scaradavou, Andromachi; Stevens, Cladd E; Barker, Juliet N

    2014-11-06

    We investigated the unit characteristics associated with engraftment after double-unit cord blood (CB) transplantation (dCBT) and whether these could be reliably identified during unit selection. Cumulative incidence of neutrophil engraftment in 129 myeloablative dCBT recipients was 95% (95% confidence interval: 90-98%). When precryopreservation characteristics were analyzed, the dominant unit CD34(+) cell dose was the only characteristic independently associated with engraftment (hazard ratio, 1.43; P = .002). When postthaw characteristics were also included, only dominant unit infused viable CD34(+) cell dose independently predicted engraftment (hazard ratio, 1.95; P banks were more likely to have low recovery (P banks and units with cryovolumes other than 24.5 to 26.0 mL were more likely to have poor postthaw viability. Precryopreservation CD34(+) cell dose and banking practices should be incorporated into CB unit selection.

  12. Producción de eritrocitos a partir de células CD34⁺ de sangre de cordón umbilical

    OpenAIRE

    Labbrozzi, Juan Pablo

    2016-01-01

    El objetivo de este estudio es la generación ex-vivo de eritrocitos a partir de la maduración y diferenciación de células CD34+ purificadas de sangre de cordón umbilical. El desarrollo de la terapia celular y la generación de diferentes tipos celulares a partir de células madre ha abierto el campo a la producción de diferentes componentes sanguíneos, entre ellos los eritrocitos, a partir de células madre hematopoyéticas (células CD34+). Existen diferentes fuentes de células CD34+, siendo el c...

  13. Differential cytogenomics and miRNA signature of the Acute Myeloid Leukaemia Kasumi-1 cell line CD34+38- compartment.

    Science.gov (United States)

    Pedranzini, Laura; Mottadelli, Federica; Ronzoni, Simona; Rossella, Franca; Ferracin, Manuela; Magnani, Ivana; Roversi, Gaia; Colapietro, Patrizia; Negrini, Massimo; Pelicci, Pier Giuseppe; Larizza, Lidia

    2010-10-01

    The t(8;21) Acute Myeloid Leukaemia (AML) Kasumi-1 cell line with N822K KIT mutation, is a model system for leukemogenesis. As AML initiating cells reside in the CD34(+)CD38(-) fraction, we addressed the refined cytogenomic characterization and miRNA expression of Kasumi-1 cell line and its FACS-sorted subpopulations focussing on this compartment. By conventional cytogenetics, Spectral-Karyotyping and array-CGH the cytogenomic profile of Kasumi-1 cells evidenced only subtle regions differentially represented in CD34(+)CD38(-) cells. Expression profiling by a miRNA platform showed a set of miRNA differentially expressed in paired subpopulations and the signature of miR-584 and miR-182 upregulation in the CD34(+)CD38(-) fraction. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  14. Inhibition of CD34+ cell migration by matrix metalloproteinase-2 during acute myocardial ischemia, counteracted by ischemic preconditioning [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Dominika Lukovic

    2017-02-01

    Full Text Available Background. Mobilization of bone marrow-origin CD34+ cells was investigated 3 days (3d after acute myocardial infarction (AMI with/without ischemic preconditioning (IP in relation to stromal-derived factor-1 (SDF-1α/ chemokine receptor type 4 (CXCR4 axis, to search for possible mechanisms behind insufficient cardiac repair in the first days post-AMI. Methods. Closed-chest reperfused AMI was performed by percutaneous balloon occlusion of the mid-left anterior descending (LAD coronary artery for 90min, followed by reperfusion in pigs. Animals were randomized to receive either IP initiated by 3x5min cycles of re-occlusion/re-flow prior to AMI (n=6 or control AMI (n=12. Blood samples were collected at baseline, 3d post-AMI, and at 1-month follow-up to analyse chemokines and mobilized CD34+ cells. To investigate the effect of acute hypoxia, SDF-1α and matrix metalloproteinase (MMP-2 in vitro were assessed, and a migration assay of CD34+ cells toward cardiomyocytes was performed. Results. Reperfused AMI induced significant mobilisation of CD34+ cells (baseline: 260±75 vs. 3d: 668±180; P<0.001 and secretion of MMP-2 (baseline: 291.83±53.40 vs. 3d: 369.64±72.89; P=0.011 into plasma, without affecting the SDF-1α concentration. IP led to the inhibition of MMP-2 (IP: 165.67±47.99 vs. AMI: 369.64±72.89; P=0.004 3d post-AMI, accompanied by increased release of SDF-1α (baseline: 23.80±12.36 vs. 3d: 45.29±11.31; P=0.05 and CXCR4 (baseline: 0.59±0.16 vs. 3d: 2.06±1.42; P=0.034, with a parallel higher level of mobilisation of CD34+ cells (IP: 881±126 vs. AMI: 668±180; P=0.026, compared to non-conditioned AMI. In vitro, CD34+ cell migration toward cardiomyocytes was enhanced by SDF-1α, which was completely abolished by 90min hypoxia and co-incubation with MMP-2. Conclusions. Non-conditioned AMI induces MMP-2 release, hampering the ischemia-induced increase in SDF-1α and CXCR4 by cleaving the SDF-1α/CXCR4 axis, with diminished mobilization of

  15. CyFlow Space流式细胞仪CD34+细胞绝对计数分析%Research on absolute enumeration of CD34+ cells detected by CyFlow Space flow cytometry

    Institute of Scientific and Technical Information of China (English)

    杨玉琮; 程小丽; 陈葳; 尤元义; 李旭

    2011-01-01

    目的 比较在Partec CyFlow Space流式细胞仪上采用不同设门方法计数CD34+造血干细胞结果的差异.方法 采集经骨髓动员的患者外周静脉血标本30例,在分别采用同型对照设门法和国际血液治疗与移植工程协会(ISHAGE)推荐设门法设置流式细胞仪后进行标本测定.结果 两种设门法标本计数结果具有相关性(r=0.990),但ISHAGE设门法的计数结果低于同型对照设门法(P<0.05).回收试验显示,ISHAGE设门法计数不同细胞浓度标本的预期值与回收值的相关系数为0.998 9;对3种细胞浓度重复检测的变异系数为2.2%~3.8%.结论 两种设门法计数CD34+细胞的相关性较好,但ISHAGE设门法更能反映造血干细胞的特点,具有良好的准确度和精密度,且无需同型对照抗体,节约试剂,更适合临床常规使用.%Objective To compare the difference of CD34+ cells counts detected by Partec CyFlow Space flow cytometry using two different gating strategies. Methods 30 peripheral blood samples were collected from patients after being treated by bone marrow mobilization,and measured by using ISHAGE gating strategy, recommended by the International Society for Hernatotherapy and Graft Engineering,and isotype gating strategy. Results There was fine correlation between the results detected by the two methods (r=0. 990) ,but the detection result of ISHAGF gating was lower than that of isotype gating strategy (P<O. 05). Recovery test indicated that the correlation coefficient between expected and tested value was 0. 9989 , with the coefficient of variation was 2. 2% to 3. 8% when three samples with different cell concentration were detected by using ISHAGF gating strategy. Conclusion There could be fine correlation between the results detected by the two gating strategies,but ISHAGF gating strategy is more accurate, with lower cost, and more suitable for clinical laboratory application.

  16. Effects of different delivery routes of CD34+ stem cells on cardiac function in the ischemic cardiomyopathy of rats%不同CD34+干细胞移植途径对缺血性心肌病大鼠心功能影响的研究

    Institute of Scientific and Technical Information of China (English)

    张瑞宏; 王岩; 李为民; 李悦; 赵翠萍; 井玲

    2008-01-01

    目的 探讨同种异体CD34+干细胞不同移植途径对心肌梗死后心力衰竭大鼠心功能的影响,同时评价干细胞移植的有效途径和安全性.方法 Wistar雄性大鼠64只被随机分成3组:干细胞移植组(30只);急性心肌梗死模型组(20只);假手术组(14只).移植组和模型组结扎左冠状动脉(冠脉)前降支造成缺血性心肌病模型;假手术组只开胸,不结扎冠脉.另选同种异体大鼠30只,将粒细胞集落刺激因子(G-CSF)动员的外周血CD34+干细胞经免疫磁珠法分离纯化后制成干细胞悬液,在冠脉结扎后7 d分别经股静脉和心外膜注入到移植组大鼠体内,模型组和假手术组注入等量磷酸盐缓冲液(PBS).于制模前、干细胞移植术后1、2和4周分别行心脏超声检查,术后4周进行血流动力学测定.结果 与模型组比较,心肌梗死后4周移植组左室射血分数(LVEF)、左室短轴缩短率(ΔFS)、左室收缩期末压(LVESP)和左室内压最大上升速率(+dp/dt max)明显提高(P均<0.01),左室收缩期末直径(LVESD)、左室舒张期末压(LVEDP)、左室内压最大下降速率(-dp/dt max)和左室等容舒张时间常数(Tc)均明显减小(P均<0.01);股静脉移植和心外膜移植两种途径对心功能的影响差异无统计学意义(P均>0.05).结论 经股静脉和心外膜两种途径移植外周血干细胞是安全可行的,二者均能有效地改善心功能,有助于梗死心肌的修复.%Objective To compare the effects of CD34-positive stem cells delivered by different routes on cardiac funetion in rats with ischemic cardiomyopathy,and to evaluate the efficacy and safety of stem cell transplantation. Methods Sixty-four male Wistar rats were randomized into cell infusion group(n=30),acute myocardial infarction(AMI)model group(n=20)and sham operation group(n=14).AMI model was reproduced by ligation of left anterior descending coronary artery. CD34+ stem cells mobilized with granulocyte

  17. 肿瘤坏死因子α和白细胞介素4对脐血CD34+造血干细胞来源的树突状细胞体外培养体系的影响%Influence of tumor necrosis factor-alpha and interleukin-4 on the in vitro culture system for dendritic cells derived from cord blood CD34+hematopoietic precu rsor cells

    Institute of Scientific and Technical Information of China (English)

    顾镭; 邢美芬; 季晓辉; 孙志达; 杨晓帆; 王慧娟; 李明顺

    2008-01-01

    瘤坏死因子aoh、白细胞介素448 h加入诱导培养的树突状细胞各表型表达相对较佳,其细胞表达CD80,CD86均较其他组高,尤以CD86表达为著,并具有激发异体T细胞增殖能力.结论:CD34+造血干细胞经过合适的培养体系能够诱导分化为功能性树突状细胞,以GM-CSF与肿瘤坏死因子α O h加入、白细胞介素4 48 h加入的GM-CSF+肿瘤坏死因子α+白细胞介素4方案更为可取.%BACKGROUND: Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.OBJECTIVE: To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND SETTING: Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS: The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.METHODS: CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured

  18. Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative Function of CD34+ CACs in Diabetic Retinopathy

    Science.gov (United States)

    Duan, Yaqian; Moldovan, Leni; Miller, Rehae C.; Beli, Eleni; Salazar, Tatiana; Hazra, Sugata; Al-Sabah, Jude; Chalam, KV; Raghunandan, Sneha; Vyas, Ruchi; Parsons-Wingerter, Patricia; Oudit, Gavin Y.; Grant, Maria B.

    2016-01-01

    Purpose: In diabetes, the impaired vasoreparative function of Circulating Angiogenic Cells (CACs) is believed to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the protective arm of renin-angiotensin system (RAS) ACE2 Angiotensin-(1-7) Mas plays an important role in restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with different stages of retinopathy. Methods: Study subjects (n43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene expressions in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by migration ability toward CXCL12 using the QCM 5M 96-well chemotaxis cell migration assay. Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls, suggestive of compensation (p0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also increased in diabetics without DR, while was reduced in NPDR compared to controls (p0.0002), indicating a possible loss of compensation of the protective RAS at this stage of DR. The presence of even mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects with Angiotensin-(1-7), migratory ability to a chemoattractant CXCL12 was restored (p0.0008). By VESGEN analysis, an increase in small vessel density was observed in NPDR subjects when compared with the controls. Conclusions: These data suggest the protective RAS axis within diabetic CACs may help maintain their vasoreparative potential

  19. Platelet-rich plasma diminishes calvarial bone repair associated with alterations in collagen matrix composition and elevated CD34+ cell prevalence.

    Science.gov (United States)

    Giovanini, Allan Fernando; Deliberador, Tatiana Miranda; Gonzaga, Carla Castiglia; de Oliveira Filho, Marco Antonio; Göhringer, Isabella; Kuczera, Juliane; Zielak, João Cesar; de Andrade Urban, Cícero

    2010-06-01

    The interaction between platelets and both type I and III collagens plays an important role in modulating platelet adhesion and aggregation, also contributing to the chemotaxis of CD34+ cells. The interaction with type III collagen can maintain high levels of collagen and alter the biology of bone repair when the PRP is used. The aim of this study was to evaluate the effect of platelet-rich plasma (PRP) and autograft on the presence of type III and type I collagens, the ratio between them, as well as the presence of CD34+ progenitor cells, while comparing these results by means of a histomorphometric analysis of the bone tissue. Four bone defects (8.0mm in diameter and 2.0mm in depth) were produced on the calvarium of 23 rabbits. The surgical defects were treated with either autogenous bone grafts, autogenous bone grafts with PRP and PRP alone. Animals were euthanized at 2, 4 or 6 weeks post-surgery. Histological, histomorphometric and immunohistochemical analyses were performed to assess repair time, as well as the expression of type I and III collagens, and number of progenitor CD34+ cells. Data were analyzed using the ANOVA and Student-Newman-Keuls test (alpha=5%). An enlarged granulation and medullary tissue areas in the PRP groups were observed. The use of PRP in this study hindered bone deposition, also enhanced type III to type I collagen ratio and the chemotaxis of CD34+ progenitor cells, similarly to a thrombogenic effect.

  20. Diagnostic value of immunohistochemical staining of GP73, GPC3, DCP, CD34, CD31, and reticulin staining in hepatocellular carcinoma.

    Science.gov (United States)

    Yao, Shuzhe; Zhang, Jianping; Chen, Haiyan; Sheng, Yan; Zhang, Xiaoying; Liu, Zhiyan; Zhang, Cuijuan

    2013-09-01

    It has been reported that Golgi protein-73 (GP73), glypican-3 (GPC3), and des-γ-carboxy prothrombin (DCP) could serve as serum markers for the early detection of hepatocellular carcinoma (HCC). This study aimed to evaluate a panel of immunostaining markers (including GP73, GPC3, DCP, CD34, and CD31) as well as reticulin staining to distinguish HCC from the mimickers. Our results revealed that CD34 immunostaining and reticulin staining were highly sensitive for the diagnosis of HCC. A special immunoreaction pattern of GP73--a diffuse coarse-block pattern in a perinuclear region or a concentrated cluster-like or cord-like pattern in a certain part of the cytoplasm--was observed in HCC cells, in contrast to the cytoplasmic fine-granular pattern in surrounding non-tumor cells and non-malignant nodules. This coarse-block pattern correlated significantly with less differentiated HCC. In comparison, GPC3 displayed a good advantage in diagnosing well-differentiated HCC. In our study, DCP and CD31 showed little diagnostic value for HCC as an immunostaining marker. When GP73, GPC3, and CD34 were combined, the specificity improved to 96.6%. Our findings demonstrate for the first time that the immunohistochemical panel of GP73, GPC3, and CD34 as well as reticulin staining is highly specific for the pathological diagnosis of HCC.

  1. Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34+ Stem Cells

    Directory of Open Access Journals (Sweden)

    Mehdi Azad

    2013-07-01

    : Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters.

  2. 胚胎干细胞体外诱导分化为CD34+造血前体细胞

    Institute of Scientific and Technical Information of China (English)

    周其锋; 何志旭; 冯炼强; 黄绍良; 李树浓

    2002-01-01

    [目的]胚胎干细胞(embryonic stem cell,ES)体外诱导分化,制备CD34+造血前体细胞.[方法]胚胎干细胞在含甲基纤维素培养液中分化形成胚胎体(embryoid body,EB),然后加入造血刺激因子诱导产生CD34+造血前体细胞,间接免疫荧光及流式细胞仪检测分化结果.[结果]胚胎干细胞分化第9~15天均有CD34+细胞,第13天比例最高,可达17.36%.[结论]胚胎干细胞分化成胚胎体,在造血刺激因子的存在下,可大量获得CD34+造血前体细胞.

  3. Imunossupressão com ciclofosfamida e fludarabina, com suporte de células tronco hematopoéticas autólogas CD-34+, para tratamento de esclerose sistêmica severa Immunosuppression with cyclophosphamide and fudarabine, with support of autologous CD-34+ stem cells, in the treatment of severe scleroderma

    Directory of Open Access Journals (Sweden)

    Carlos Alberto von Mühlen

    2004-04-01

    , including scleroderma. In this paper the authors present a patient with the diffuse form of scleroderma who received immunosuppression with cyclophosphamide 0.5 g/m² and fludarabine followed by infusion of autologous CD-34+ cells without T lymphocyte purging. This patient was followed up with cutaneous score measures, imaging and laboratory tests, as well as a well-being questionnaire. After transplantation there were major positive responses on skin and gastrointestinal tract, with cessation of diarrhea and malabsorption syndrome. These clinical improvements did not last past 6 months. We observed transitory stem cell transfusion reaction and localized herpes zoster. The patient died 12 months after the procedure due to aspirative pneumonia and gastrointestinal complications. The immunosuppressive regimen, and not the autologous stem cell transplantation, could have been responsible for the observed transitory patient improvement.

  4. Expression of platelet-bound stromal-cell derived factor-1 (SDF-1) and number of CD34(+) progenitor cells in patients with congestive heart failure.

    Science.gov (United States)

    Jorbenadze, Rezo; Schleicher, Erwin; Bigalke, Boris; Stellos, Konstantinos; Gawaz, Meinrad

    2014-01-01

    Platelet-bound stromal cell-derived factor-1 (SDF-1) plays a crucial role in attachment of circulating CD34(+) progenitor cells to the vascular wall, facilitating tissue healing after injury. However there is no evidence about expression of platelet-bound SDF-1 in patients with congestive heart failure (CHF). The aim of our study was to evaluate expression of platelet-bound SDF-1 and number of CD34(+) progenitor cells in patients with CHF. Forty-eight patients with idiopathic dilated cardiomyopathy (DCM) and 61 patients with ischaemic cardiomyopathy (ICM) were consecutively enrolled into the study. Blood taken from 109 consecutive patients was studied for surface expression of platelet-bound SDF-1 and number of CD34(+) progenitor cells by flow cytometry. The highest expression of platelet-bound SDF-1 was observed in patients with severe impairment of left ventricular systolic function compared with patients with mild or moderate impairment of left ventricular systolic function (mild vs. moderate vs. severe impairment of left ventricular systolic function: MFI ± SD: 35.6 ± 34 vs. 101.45 ± 73 vs. 124.86 ± 86.7, Kruskal-Wallis p SDF-1 number of CD34(+) progenitor cells was the highest in severe impairment of left ventricular systolic function (mild vs. moderate vs. severe impairment of left ventricular systolic function: mean ± SD: 260.4 ± 177.5 vs. 580.7 ± 340.5 vs. 640.82 ± 370.6, Kruskal-Wallis p SDF-1 expression was associated with number of circulating CD34(+) progenitor cells (r = 0.454, p SDF-1 and number of CD34(+) cells were higher in patients with DCM compared with patients with ICM (p SDF-1 and CD34(+) progenitor cells are especially increased in patients with severe impairment of left ventricular systolic function in CHF.

  5. Negative association of donor age with CD34+ cell dose in mixture allografts of G-CSF-primed bone marrow and G-CSF-mobilized peripheral blood harvests

    Institute of Scientific and Technical Information of China (English)

    Li Yan; Chang Yingjun; Xu Lanping; Zhang Xiaohui; Huang Xiaojun

    2014-01-01

    Background The effects of donor characteristics on CD34+ cell dose remain controversial.Recently,we developed a novel haploidentical transplant protocol,in which mixture allografts of granulocyte colony-stimulating factor (G-CSF)-primed bone marrow (G-BM) and G-CSF-mobilized peripheral blood (G-PB) were used.The aim of this study was to investigate the effects of donor characteristics on CD34+ cell dose in mixture allografts of G-BM and G-PB.Methods A total of 162 healthy adult donors,who underwent bone marrow harvest and peripheral blood collection between January 2009 and November 2010 in Peking University People's Hospital,were prospectively investigated.G-CSF was administered subcutaneously at a dose of 5 μg/kg once a day for 5-6 consecutive days.Bone marrow and peripheral blood stem cells were harvested on the fourth day and fifth day,respectively.A final total CD34+ cell dose less than 2× 106 cells/kg recipient body weight was considered a poor mobilization.Results Of the 162 donors,31 (19.1%) did not attain this threshold.The obtained median CD34+ cell doses in bone marrow,peripheral blood,and mixture allografts were 0.83×106/kg,2.40×106/kg,and 3.47×106/kg,respectively.Multiple regression analysis showed that donor age had a significant negative effect on CD34+ cell dose in either G-BM,or G-PB,or mixture allografts of G-BM and G-PB.And a 1-year increase in age was associated with a 5.6% decrease in the odds of achieving mobilization cutoff.No significant correlation was found for donor gender,body mass index (BMI),and weight.Conclusion Donor age is the only factor among the four parameters,including age,gender,weight,and BMI,that influence CD34+ cell dose in mixture allografts of G-BM and G-PB,and younger donors should be chosen to obtain sufficient CD34+ cells for transplantation.

  6. Combination of low O(2) concentration and mesenchymal stromal cells during culture of cord blood CD34(+) cells improves the maintenance and proliferative capacity of hematopoietic stem cells.

    Science.gov (United States)

    Hammoud, Mohammad; Vlaski, Marija; Duchez, Pascale; Chevaleyre, Jean; Lafarge, Xavier; Boiron, Jean-Michel; Praloran, Vincent; Brunet De La Grange, Philippe; Ivanovic, Zoran

    2012-06-01

    The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O(2) concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co-culture of cord blood CD34(+) cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O(2) and assessed the impact on total cells, CD34(+) cells, committed progenitors (colony-forming cells-CFC) and stem cells activity (pre-CFC and Scid repopulating cells-SRC). Not surprisingly, the expansion of total cells, CD34(+) cells, and CFC was higher in co-culture and at 20% O(2) compared to simple culture and low O(2) concentrations, respectively. However, co-culture at low O(2) concentrations provided CD34(+) cell and CFC amplification similar to classical culture at 20% O(2) . Interestingly, low O(2) concentrations ensured a better pre-CFC and SRC preservation/expansion in co-culture. Indeed, SRC activity in co-culture at 1.5% O(2) was higher than in freshly isolated CD34(+) cells. Interleukin-6 production by MSC at physiologically low O(2) concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co-culture and low O(2) concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool.

  7. Positive Darwinian selection in human population: A review

    Institute of Scientific and Technical Information of China (English)

    WU DongDong; ZHANG YaPing

    2008-01-01

    This paper reviews a large number of genes under positive Darwinian selection in modern human populations, such as brain development genes, immunity genes, reproductive related genes, percep-tion receptors. The research on the evolutionary property of these genes will provide important insight into human evolution and disease mechanisms. With the increase of population genetics and com-parative genomics data, more and more evidences indicate that positive Darwinian selection plays an indispensable role in the origin and evolution of human beings. This paper will also summarize the methods to detect positive selection, analyze the interference factors faced and make suggestions for further research on positive selection.

  8. Effects of SDF-1 on the proliferation and homing-related function of UCB CD34 + cells%基质衍生因子-1对脐带血CD34+细胞增殖及归巢相关功能影响的研究

    Institute of Scientific and Technical Information of China (English)

    段相会; 李桥川

    2013-01-01

    目的 探讨基质衍生因子-1(SDF-1)对脐带血(UCB) CD34+细胞增殖及归巢相关功能影响.方法 采用流式细胞仪测定、造血祖细胞培养、结晶紫染色测定、Transwell实验等方法.结果 ①SDF-1在体外能通过抑制UCB CD34+细胞凋亡增强细胞生存能力;②与其他造血因子协同SDF-1可在一定程度上促进粒系祖细胞增殖;③SDF-1能提高CD34+细胞的黏附能力和迁移能力.结论 SDF-1能够应用于UCB CD34+细胞体外培养,增强UCB CD34+细胞的归巢相关功能.

  9. Expression of CD105 and CD34 receptors controls BMP-induced in vitro mineralization of mouse adipose-derived stem cells but does not predict their in vivo bone-forming potential.

    Science.gov (United States)

    Madhu, Vedavathi; Kilanski, Allison; Reghu, Nikitha; Dighe, Abhijit S; Cui, Quanjun

    2015-05-01

    Adipose-derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose-derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone-forming potential. Four purified populations of mouse ADSCs: CD105(+) CD34(+), CD105(-) CD34(-), CD105(+) CD34(-) and CD105(-) CD34(+) were obtained using fluorescence-activated cell sorting (FACS) and their BMP-responsiveness was determined in vitro. CD105(+) CD34(-) population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP-responsive phenotype and larger cell size while expression of CD34 correlated with low BMP-responsive phenotype and smaller cell size. CD105(+) CD34(-) population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105(+) CD34(-) ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP-responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  10. Concurrent hypermulticolor monitoring of CD31, CD34, CD45 and CD146 endothelial progenitor cell markers for acute myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Yumi [College of Pharmacy, Seoul National University, 1 Gwanak-Ro, Gwanak Gu, Seoul 151-742 (Korea, Republic of); Nam, Myung Hyun [Department of Laboratory Medicine, Korea University Ansan Hospital, Korea University College of Medicine (Korea, Republic of); Hyuk, Song Woo [Cardiology College of Medicine, Korea University (Korea, Republic of); Yoon, Soo Young [College of Medicine, Korea University, Seoul (Korea, Republic of); Song, Joon Myong, E-mail: jmsong@snu.ac.kr [College of Pharmacy, Seoul National University, 1 Gwanak-Ro, Gwanak Gu, Seoul 151-742 (Korea, Republic of)

    2015-01-01

    Highlights: • We observe EPCs and HPCs in patient for AMI diagnosis. • We detect two EPC subtypes using quantum dot and AOTF. • Quantum dot has narrower emission wavelength range than fluorescence dye. • AOTF provide smaller spectral interference than bandpass filters. • Quantum dot and AOTF are suitable for detecting large number of molecular markers concurrently. - Abstract: The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(−/dim), CD146(−) as early outgrowth EPCs and CD31(+), CD34(+), CD45(−/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(−/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(

  11. Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells.

    Directory of Open Access Journals (Sweden)

    Paola Guglielmelli

    Full Text Available Myeloproliferative neoplasms (MPN are chronic myeloid cancers thought to arise at the level of CD34+ hematopoietic stem/progenitor cells. They include essential thrombocythemia (ET, polycythemia vera (PV and primary myelofibrosis (PMF. All can progress to acute leukemia, but PMF carries the worst prognosis. Increasing evidences indicate that deregulation of microRNAs (miRNAs might plays an important role in hematologic malignancies, including MPN. To attain deeper knowledge of short RNAs (sRNAs expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, we sequenced with Illumina HiSeq2000 technology CD34+ cells from healthy subjects and PMF patients. We detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs, in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543 were deregulated also in PMF granulocytes. Moreover, 3'-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. We thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease.

  12. Proportion of CD34 + CD38- Cell Population in Bone Marrow of Patients with De Novo AML as Prognostic Factor of Complete Rimission at First Course of Induction Chemotherapy%初诊急性髓系白血病骨髓CD34+CD38-细胞群比例是初次诱导缓解率的预后因素

    Institute of Scientific and Technical Information of China (English)

    张翠萍; 魏辉; 王慧君; 怀磊; 何侃; 陈一瑞; 林冬; 王建祥

    2011-01-01

    为了研究非M3急性髓系白血病(AML non-M3) CD34+ CD38 -细胞群及其G0期比例与临床和实验室特征的关系,使用流式细胞仪检测40例初治AML non-M3骨髓单个核细胞CD34和CD38的表达,测定各群细胞的细胞周期,并分析CD34+ CD38 -细胞群及其G0期比例与临床实验室特征及初次诱导缓解率的关系.结果显示,CD34+ CD38 -细胞群及其G0期的比例与染色体核型、初诊WBC计数及FLT3/ITD阳性均无明显相关性,但与诱导治疗后骨髓中幼稚细胞比例相关.诱导停疗第7天骨髓中有幼稚细胞患者CD34+ CD38-细胞比例为(12.47±26.26)%,诱导停疗第7天无幼稚细胞患者CD34+ CD38 -细胞比例为(2.62±7.20)% (p =0.031).诱导停疗第1天骨髓中可见幼稚细胞患者CD34+细胞群比例为(17.40±21.20)%,而诱导停疗第1天无幼稚细胞患者该比例为(5.64±6.96)%(p=0.00l).CR组患者治疗前CD34+ CD38 -细胞群的比例为(2.51±9.72)%,明显低于非CR组患者(24.92 +27.04%) (p =0.001).而在AMI non-M2b患者中,CR组患者治疗前CD34+ CD38 -细胞群的比例为(1.60±4.82)%,较非CR组患者更为降低(p <0.001).单因素分析显示,诱导化疗后是否取得CR与年龄(p=0.022)、CD34+ CD38 -细胞群比例(p=0.008)、诱导停疗第7天骨髓幼稚细胞比例(p=0.011)相关.多因素分析显示,仅有CD34+ CD38 -细胞群的比例与是否获得CR有相关趋势(p =0.052).结论:初治AML患者CD34+CD38 -细胞群的比例是AML初次诱导缓解率的预后因素.%This study was to investigate the relationship between the CD34* CD38 cell population and its proportion in Go phase of de novo AML non-M3 at diagnosis and the clinical and experimental characteristics. The flow cytometry was used to detect the expression of the cell surface antigen CD34 and CD38 in the bone marrow mononuclear cells (MNC) of the AML non-M, at diagnosis and investigate the cell cycle of the subpopulations, and then the relationships between the

  13. Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder after high-dose immunosuppressive therapy and autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases.

    Science.gov (United States)

    Nash, Richard A; Dansey, Roger; Storek, Jan; Georges, George E; Bowen, James D; Holmberg, Leona A; Kraft, George H; Mayes, Maureen D; McDonagh, Kevin T; Chen, Chien-Shing; Dipersio, John; Lemaistre, C Fred; Pavletic, Steven; Sullivan, Keith M; Sunderhaus, Julie; Furst, Daniel E; McSweeney, Peter A

    2003-09-01

    High-dose immunosuppressive therapy followed by autologous hematopoietic stem cell transplantation (HSCT) is currently being evaluated for the control of severe autoimmune diseases. The addition of antithymocyte globulin (ATG) to high-dose chemoradiotherapy in the high-dose immunosuppressive therapy regimen and CD34 selection of the autologous graft may induce a higher degree of immunosuppression compared with conventional autologous HSCT for malignant diseases. Patients may be at higher risk of transplant-related complications secondary to the immunosuppressed state, including Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorder (PTLD), but this is an unusual complication after autologous HSCT. Fifty-six patients (median age, 42 years; range, 23-61 years) with either multiple sclerosis (n = 26) or systemic sclerosis (n = 30) have been treated. The median follow-up has been 24 months (range, 2-60 months). Two patients (multiple sclerosis, n = 1; systemic sclerosis, n = 1) had significant reactivations of herpesvirus infections early after HSCT and then developed aggressive EBV-PTLD and died on days +53 and +64. Multiorgan clonal B-cell infiltrates that were EBV positive by molecular studies or immunohistology were identified at both autopsies. Both patients had positive screening skin tests for equine ATG (Atgam) and had been converted to rabbit ATG (Thymoglobulin) from the first dose. Of the other 54 patients, 2 of whom had partial courses of rabbit ATG because of a reaction to the intravenous infusion of equine ATG, only 1 patient had a significant clinical reactivation of a herpesvirus infection (herpes simplex virus 2) early after HSCT, and none developed EBV-PTLD. The T-cell count in the peripheral blood on day 28 was 0/microL in all 4 patients who received rabbit ATG; this was significantly less than in patients who received equine ATG (median, 174/microL; P =.001; Mann-Whitney ranked sum test). Although the numbers are limited

  14. Predictive factors for poor peripheral blood stem cell mobilization and peak CD34(+) cell count to guide pre-emptive or immediate rescue mobilization.

    Science.gov (United States)

    Sancho, Juan-Manuel; Morgades, Mireia; Grifols, Joan-Ramon; Juncà, Jordi; Guardia, Ramon; Vives, Susana; Ferrà, Christelle; Batlle, Monsterrat; Ester, Anna; Gallardo, David; Millà, Fuensanta; Feliu, Evarist; Ribera, Josep-Maria

    2012-08-01

    Failure in mobilization of peripheral blood (PB) stem cells is a frequent reason for not performing hematopoietic stem cell transplantation (HSCT). Early identification of poor mobilizers could avoid repeated attempts at mobilization, with the administration of pre-emptive rescue mobilization. Data from the first mobilization schedule of 397 patients referred consecutively for autologous HSCT between 2000 and 2010 were collected. Poor mobilization was defined as the collection of 13.8/μL was enough to ensure ≥ 2 × 10(6) CD34(+)cells/kg, with high sensitivity (90%) and specificity (91%). The prevalence of poor mobilization was high, being associated with disease type, therapy with purine analogs and multiple chemotherapy regimens. The threshold of CD34(+) cell count in PB identified poor mobilizers, in whom the administration of immediate or pre-emptive plerixafor could be useful to avoid a second mobilization.

  15. P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART.

    Directory of Open Access Journals (Sweden)

    Inna Menkova-Garnier

    2016-04-01

    Full Text Available Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an in vitro system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway, survival (IL7R-IL7 axis and death (Fas, P2X7, CD39/CD73. P2X7 expression was abnormally strong and there was no CD73 mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that in vitro inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs.

  16. Granulocyte colony-stimulating factor mobilizes peripheral blood CD34+ cells in patients with type 2 diabetes mellitus%粒细胞集落刺激因子动员2型糖尿病患者外周血CD34+细胞的变化

    Institute of Scientific and Technical Information of China (English)

    窦立冬; 崔晓兰; 陈建河; 王立梅; 王意忠

    2011-01-01

    背景:与正常人比较,2型糖尿病患者外周血CD34+细胞含量已有明显下降.目的:分析2型糖尿病患者外周血动员后CD34+细胞比例的变化.方法:将234例2型糖尿病患者按病程分为5组(5~<10年,10~15年和≥15年),予以粒细胞集落刺激因子动员.动员5 d后,使用流式细胞仪检测外周血CD34+细胞的含量,并利用Person简单相关及多元回归方法分析其与病程、血脂、尿酸的关系.结果与结论:糖尿病者外周血动员后CD34+细胞水平与三酰甘油相关(r=-0.202,P=0.002),与载脂蛋白B相关(r=-0.276,P=0.000),与尿酸相关(r=-0.297,P=0.000).经统计分析发现,糖尿病者外周血动员后CD34+细胞数随糖尿病病程进展而逐渐下降.%AbstractBACKGROUND: The number of mobilized peripheral blood CD34+ cells significantly decreases in type 2 diabetes mellitus patients compared with normal subjects.OBJECTIVE: To investigate the changes of mobilized peripheral blood CD34+ cells in type 2 diabetes mellitus patients. METHODS: A total of 234 patients with type 2 diabetes mellitus were divided into five categories according to disease duration, newly diagnosed type 2 diabetes (≤1 year) and diabetes duration (1-5, 5-10, 10-15, ≥15 years). CD34+ cells were mobilized with granulocyte colony-stimulating factor. Five days later, the number of peripheral blood CD34+ cells was detected with flow cytometry. Its relationship with disease duration, blood lipids and uric acid was analyzed by person-related analysis and multiple regression analysis.RESULTS AND CONCLUSION: The number of mobilized peripheral blood CD34+ cells was associated with the triacylglycerol (r=-0.202, P=0.002), the apoprotein-B (r=-0.276, P=0.000), and the uric acid (r=-0.297, P=0.000). Statistical analysis found that, the number of mobilized peripheral blood CD34+ cells decreased with development of diabetes mellitus.

  17. Inhibition of CK2{alpha} and PI3K/Akt synergistically induces apoptosis of CD34+CD38- leukaemia cells while sparing haematopoietic stem cells.

    Science.gov (United States)

    Cheong, June-Won; Min, Yoo Hong; Eom, Ju In; Kim, Soo Jeong; Jeung, Hoi Kyung; Kim, Jin Seok

    2010-11-01

    The CD34(+)CD38(-) leukaemia cell population contains leukaemia stem cells (LSCs) responsible for treatment failure in acute myeloid leukaemia (AML) and, thus, novel therapies are required to eradicate LSCs without harming healthy haematopoietic stem cells (HSC). The present study evaluated the effects of co-treatment with LY294002 (a PI3K/Akt inhibitor) and apigenin (a CK2 inhibitor) (LY/Api) at subtoxic concentrations on leukaemia cell lines and primary AML cells. LY/Api synergistically induced apoptosis in leukaemia cells, especially CD34(+)CD38(-) leukaemia cells. However, these effects were negligible in HSCs. LY/Api-induced apoptosis was accompanied by activation of caspase cascades and disruption of mitochondrial membrane potential. Caspase inhibitor or Akt overexpression abrogated this synergistic induction in apoptosis by LY/Api. LY/Api also led to remarkable down-regulation of anti-apoptotic proteins including Bcl-xL and NF-κB in CD34(+)CD38(-) leukaemia cells, but not in healthy hematopoietic stem cells. Inhibition of both CK2 and PI3K/Akt pathways may be a promising LSCs-targeted therapeutic strategy for AML.

  18. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, increases the number of circulating CD34⁺CXCR4⁺ cells in patients with type 2 diabetes.

    Science.gov (United States)

    Aso, Yoshimasa; Jojima, T; Iijima, T; Suzuki, K; Terasawa, T; Fukushima, M; Momobayashi, A; Hara, K; Takebayashi, K; Kasai, K; Inukai, T

    2015-12-01

    We investigated the effects of sitagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, on the number of circulating CD34(+)CXCR4(+)cells, a candidate for endothelial progenitor cells (EPCs), plasma levels of stromal cell-derived factor (SDF)-1α, a ligand for CXCR4 receptor and a substrate for DPP-4, and plasma levels of interferon-inducible protein (IP)-10, for a substrate for DPP-4, in patients with type 2 diabetes. We studied 30 consecutive patients with type 2 diabetes who had poor glycemic control despite treatment with metformin and/or sulfonylurea. Thirty diabetic patients were randomized in a 2:1 ratio into a sitagliptin (50 mg/day) treatment group or an active placebo group (glimepiride 1 mg/day) for 12 weeks. Both groups showed similar improvements in glycemic control. The number of circulating CD34(+)CXCR4(+) cells was increased from 30.5 (20.0, 47.0)/10(6) cells at baseline to 55.5 (31.5, 80.5)/10(6) cells at 12 weeks of treatment with 50 mg/day sitagliptin (P = 0.0014), while showing no significant changes in patients treated with glimepiride. Plasma levels of SDF-1α and IP-10, both physiological substrates of endogenous DPP-4 and chemokines, were significantly decreased at 12 weeks of sitagliptin treatment. In conclusion, treatment with sitagliptin increased the number of circulating CD34(+)CXCR4(+) cells by approximately 2-fold in patients with type 2 diabetes.

  19. Fusion of dendritic cells and CD34+CD38- acute myeloid leukemia (AML) cells potentiates targeting AML-initiating cells by specific CTL induction.

    Science.gov (United States)

    Lei, Zhang; Zhang, Gui-Mei; Hong, Mei; Feng, Zuo-Hua; Huang, Bo

    2009-05-01

    Distinct leukemia-initiating cells (L-ICs) represent a critical target for therapeutic intervention of acute myeloid leukemia (AML). A potential strategy to eradicate L-ICs is to generate L-IC-specific cytotoxic T lymphocytes (CTLs). However, owing to rarity and immortality of L-ICs, it is difficult for antigen-presenting cells to capture L-ICs for specific antigen presentation. Here, we report a novel approach by fusing allogeneic dendritic cells (DCs) and CD34CD38 AML progenitor cells, through which specific CTLs were effectively induced, leading to the cytolysis to AML-initiating cells. Fusion of either DC/CD34CD38 AML cell or DC/CD34 AML cell could effectively induce the proliferation and activation of CTLs. However, only the former CTLs could effectively attack AML progenitor cells, and result in the unkilled progenitor/initiating cells losing the abilities of active proliferation in vitro and engraftment in NOD-SCID mice. These findings suggest that AML progenitor/initiating cell-specific CTLs may be generated based on allogeneic DC/progenitor cell fusion strategy; the induced CTLs may potentially eradicate AML by targeting L-ICs directly or indirectly.

  20. Efficacy of single dose pegfilgrastim in enhancing the mobilization of CD34+ peripheral blood stem cells in aggressive lymphoma patients treated with cisplatin-aracytin-containing regimens.

    Science.gov (United States)

    Nosari, A; Cairoli, R; Ciapanna, D; Gargantini, L; Intropido, L; Baraté, C; Scarpati, B; Santoleri, L; Nador, G; Pezzetti, L; Morra, E

    2006-09-01

    Systematic data on the ability of pegfilgrastim to mobilize stem cells after chemotherapy are scarce. We evaluated the efficacy of a single 6 mg dose of pegfilgrastim for mobilizing peripheral blood stem cells (PBSC) in aggressive lymphoma patients. Between July 2004 and October 2005, 17 aggressive non-Hodgkin's lymphoma and 11 poor-risk Hodgkin's lymphoma were treated with cycles containing cisplatin-aracytin. At the end of chemotherapy, the patients received 6 mg of pegfilgrastim. Duration of grade 4 neutropenia, adverse events, time to neutrophil recovery, peak and harvest of CD34+ cells were recorded. Twenty-seven out of 28 patients harvested a median of 17.3 x 10(6)/CD34+ cells (range 2.5-28.9) after a median of 9 days (range 8-12 days), with a single apheresis procedure in 25 cases. All patients had grade 3-4 neutropenia, median duration 3 days. The only adverse event was mild bone pain. To date, 13 patients have been autografted with a median of 15.4 x 10(6) CD34+ pegfilgrastim-mobilized cells per kg (range 2.5-28.9) with rapid and sustained engraftment. Mobilization, harvesting and autografting of pegfilgrastim-mobilized PBC can be successfully achieved in pretreated patients with aggressive lymphoma.

  1. Correlation between survival and number of mobilized CD34+ cells in patients with multiple myeloma or Waldenström macroglobulinemia.

    Science.gov (United States)

    Kakihana, Kazuhiko; Ohashi, Kazuteru; Akiyama, Hideki; Sakamaki, Hisashi

    2010-12-01

    High-dose chemotherapy followed by autologous stem cell transplantation is the established treatment for symptomatic multiple myeloma (MM) or Waldenström macroglobulinemia (WM). We retrospectively analyzed the impact of mobilized CD34+ cell number on clinical outcomes in patients with MM or WM who underwent autologous stem cell transplantation in our hospital from 1997 to 2007. A total of 39 patients were identified. All patients received peripheral stem cell support after a conditioning regimen. We defined patients with collection of a large number (≥ 8 × 10(6)/kg) of CD34+ cells as super mobilizers (SM), and all others as normal mobilizers (NM). Although hematological engraftment was earlier in the SM group, overall survival did not differ significantly between groups (P = 0.392). Likewise, no significant differences were seen in progression-free survival (P = 0.201) or survival after relapse (P = 0.330). In conclusion, our retrospective study could not find any correlation between survival and number of mobilized CD34+ cells, in contrast to previously reported results.

  2. IL-7 activates the phosphatidylinositol 3-kinase/AKT pathway in normal human thymocytes but not normal human B cell precursors.

    Science.gov (United States)

    Johnson, Sonja E; Shah, Nisha; Bajer, Anna A; LeBien, Tucker W

    2008-06-15

    IL-7 signaling culminates in different biological outcomes in distinct lymphoid populations, but knowledge of the biochemical signaling pathways in normal lymphoid populations is incomplete. We analyzed CD127/IL-7Ralpha expression and function in normal (nontransformed) human thymocytes, and human CD19(+) B-lineage cells purified from xenogeneic cord blood stem cell/MS-5 murine stromal cell cultures, to further clarify the role of IL-7 in human B cell development. IL-7 stimulation of CD34(+) immature thymocytes led to phosphorylation (p-) of STAT5, ERK1/2, AKT, and glycogen synthase kinase-3 beta, and increased AKT enzymatic activity. In contrast, IL-7 stimulation of CD34(-) thymocytes (that included CD4(+)/CD8(+) double-positive, and CD4(+) and CD8(+) single-positive cells) only induced p-STAT5. IL-7 stimulation of CD19(+) cells led to robust induction of p-STAT5, but minimal induction of p-ERK1/2 and p-glycogen synthase kinase-3 beta. However, CD19(+) cells expressed endogenous p-ERK1/2, and when rested for several hours following removal from MS-5 underwent de-phosphorylation of ERK1/2. IL-7 stimulation of rested CD19(+) cells resulted in robust induction of p-ERK1/2, but no induction of AKT enzymatic activity. The use of a specific JAK3 antagonist demonstrated that all IL-7 signaling pathways in CD34(+) thymocytes and CD19(+) B-lineage cells were JAK3-dependent. We conclude that human CD34(+) thymocytes and CD19(+) B-lineage cells exhibit similarities in activation of STAT5 and ERK1/2, but differences in activation of the PI3K/AKT pathway. The different induction of PI3K/AKT may at least partially explain the different requirements for IL-7 during human T and B cell development.

  3. The loss of CD34+ cells in peripheral hematopoietic stem cell products cryopreserved by non-controlled rate freezing and stored at -80 °C after overnight storage.

    Science.gov (United States)

    Donmez, Ayhan; Yilmaz, Fergun; Soyer, Nur; Cagirgan, Seckin; Arik, Bahar; Tombuloglu, Murat

    2014-10-01

    Although peripheral blood stem cell (PBSC) products cryopreserved by non-controlled rate freezing and stored at -80 °C after overnight storage are used frequently, data regarding the rate of loss of CD34+ cells in these products are limited. In this prospective study, CD34+ cells were counted at three (fresh, post-overnight and post-thaw) points in 83 PBSC products from 41 patients by flow cytometry. Compared to fresh products, the mean losses of post-overnight and post-thaw total CD34+ cells are 16.3% and 38.4% (p = 0.02), and the mean losses of post-overnight and post-thaw viable CD34+ cells are 16.5% and 48.5%, respectively (p < 0.001). The numbers of fresh viable, post-thaw total and post-thaw viable CD34+ cells were inversely correlated with the durations of neutrophil and platelet engraftment. Our results indicate that the mean loss of post-thaw total and viable CD34+ cells is approximately 20% higher than that observed in standard cryopreservation methods. In addition, fresh viable, post-thaw total and especially post-thaw viable CD34+ cell levels are valuable predictors of both neutrophil and platelet engraftments. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Mobilization and collection of CD34+ cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses

    Directory of Open Access Journals (Sweden)

    Kátia Aparecida de Brito Eid

    2015-06-01

    Full Text Available Introduction: The use of peripheral hematopoietic progenitor cells (HPCs is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G- CSF for mobilization is a single daily dose of 10 µg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures. Objective: The aim of this study was to compare a fractionated dose of 15 µg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight. Methods: Patients were divided into two groups: Group 10 - patients who received a single daily dose of 10 µg G-CSF/kg body weight and Group 15 - patients who received a fractioned dose of 15 µg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved. Results: Group 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3% for Group 10 and 36 (24.7% for Group 15. For Group 10, a median of three (range: 1-7 leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59 were collected whereas for Group 15 the corresponding values were one (range: 1-3 and 5.29 × 106 cells/kg body weight (±4.95. A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001. Conclusions: To collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 µg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed.

  5. Effects of lentiviral vector-mediated RNAi on expression of CCR5 in CD34 + cells in peripheral blood of mice%慢病毒载体介导的RNAi对小鼠外周血CD34+细胞CCR5表达的影响

    Institute of Scientific and Technical Information of China (English)

    姜兆磊; 朱家全; 鲍春荣; 丁芳宝; 汤敏; 梅举

    2011-01-01

    目的 构建含趋化因子受体5(CCR5) -短发夹状RNA(shRNA)的重组慢病毒,观察其对小鼠外周血CD34+造血干细胞(CD34+细胞)CCR5mRNA表达的影响.方法 使用Ambion RNAi靶序列及参考相关文献设计并确定RNA干扰(RNAi)靶序列.将靶序列转入克隆载体pBShH1扩增,再转入含增强型绿色荧光蛋白(EGFP)的FG12慢病毒载体,酶切、测序鉴定.包装CCR5-shRNA慢病毒并测定滴度,转染小鼠外周血CD34+细胞,检测转染效率.将CCR5-shRNA慢病毒转染的CD34+细胞回输小鼠体内,1周后采用Real-Time PCR检测细胞CCR5 mRNA的表达.结果 相关鉴定结果显示:目的片段插入了FG12载体,并带入pBSHH1上的H1 RNA polymeraseⅢ启动子,CCR5-shRNA慢病毒载体构建完成;病毒感染滴度为5×107 TU/mL;CCR5-shRNA慢病毒对小鼠外周血CD34+细胞的转染效率为97.9%;Real-Time PCR检测结果显示:转染CCR5-shRNA慢病毒的CD34+细胞回输后,小鼠外周血CD34+细胞CCR5 mRNA表达显著下降.结论 成功制备高滴度的CCR5-shRNA重组慢病毒,该病毒在体内能有效下调小鼠外周血CD34+细胞CCR5 mRNA的表达,为治疗器官移植后排斥反应的研究奠定了基础.%Objective To construct the recombinant lentivirus carrying chemokine receptor 5 (CCR5)-short hairpin RNA ( shRNA), and investigate its effects on the expression of CCR5 mRNA of CD34+ hematopoietic stem cells ( CD34+ cells) in peripheral blood of mice. Methods RNA interference ( RNAi) target sequence was designed by Ambion RNAi target sequence and references. The target sequence was amplified after transduction into plasmid pBSHHl, and was transducted into FG12 lentiviral vector containing enhanced green fluorescent protein ( EGFP). The recombinant lentivirus of CCR5-shRNA was packaged, and the virus titer was determined. The recombinant lentivirus was transducted into CD34+ hematopoietic stem cells in peripheral blood of mice, and the transduction efficiency was measured. Then, CD34

  6. In vitro modulation of radiation-induced FAS-related apoptosis in CD34{sup +} progenitor cells by combination cytokines; Reduction de l'apoptose radio-induite impliquant le recepteur FAS au niveau des cellules hematopoietiques CD34{sup +} par une combinaison de cytokines

    Energy Technology Data Exchange (ETDEWEB)

    Drouet, M.; Mathieu, J.; Grenier, N.; Soutif, A.; Herodin, F

    1998-07-01

    Combination cytokines such as SCF, Flt-3 ligand, IL-3 and thrombopoietin can modulate Fas mRNA expression by in vitro irradiated CD34{sup +} cells which results in a moderate decrease of apoptotic ratio and an improved rate of clonogenicity of the irradiated progenitors. (authors)

  7. Positive selection on gene expression in the human brain

    DEFF Research Database (Denmark)

    Khaitovich, Philipp; Tang, Kun; Franz, Henriette

    2006-01-01

    Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues [1] . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage [1] , [2] and [3] . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...

  8. Position and locality constrained soft coding for human action recognition

    Science.gov (United States)

    Wang, Bin; Liu, Yu; Xiao, Wenhua; Xu, Wei; Zhang, Maojun

    2013-10-01

    Although the traditional bag-of-words model has shown promising results for human action recognition, in the feature coding phase, the ambiguous features from different body parts are still difficult to distinguish. Furthermore, it also suffers from serious representation error. We propose an innovative coding strategy called position and locality constrained soft coding (PLSC) to overcome these limitations. PLSC uses the feature position in a human oriented region of interest (ROI) to distinguish the ambiguous features. We first construct a subdictionary for each feature by selecting the bases from their spatial neighbor in human ROI. Then, a modified soft coding with locality constraint is adopted to alleviate the quantization error and preserve the manifold structure of features. This novel coding algorithm increases both the representation accuracy and discriminative power with low computational cost. The human action recognition experimental results on KTH, Weizmann, and UCF sports datasets show that PLSC can achieve a better performance than previous competing feature coding methods.

  9. Human Capital and Optimal Positive Taxation of Capital Income

    OpenAIRE

    Jacobs, B.; Bovenberg, A.L.

    2005-01-01

    This paper analyzes optimal linear taxes on capital and labor incomes in a life-cyclemodel of human capital investment, financial savings, and labor supply with heteroge-nous individuals. A dual income tax with a positive marginal tax rate on not onlylabor income but also capital income is optimal. The positive tax on capital incomeserves to alleviate the distortions of the labor tax on human capital accumulation.The optimal marginal tax rate on capital income is lower than that on labor inco...

  10. Performance of humans in concurrent avoidance/positive-reinforcement schedules

    OpenAIRE

    Ruddle, H. V.; Bradshaw, C M; Szabadi, E; Foster, T M

    1982-01-01

    Performance maintained under concurrent schedules consisting of a variable-interval avoidance component and a variable-interval positive-reinforcement component was studied in three human subjects using points exchangeable for money as the reinforcer. The rate of responding in the avoidance component increased, and the rate of responding in the positive-reinforcement component declined, as a function of the frequency of point-losses avoided in the avoidance component. The performance of all t...

  11. Human habitat positioning system for NASA's space flight environmental simulator

    Science.gov (United States)

    Caldwell, W. F.; Tucker, J.; Keas, P.

    1998-01-01

    Artificial gravity by centrifugation offers an effective countermeasure to the physiologic deconditioning of chronic exposure to microgravity; however, the system requirements of rotational velocity, radius of rotation, and resultant centrifugal acceleration require thorough investigation to ascertain the ideal human-use centrifuge configuration. NASA's Space Flight Environmental Simulator (SFES), a 16-meter (52-foot) diameter, animal-use centrifuge, was recently modified to accommodate human occupancy. This paper describes the SFES Human Habitat Positioning System, the mechanism that facilitates radius of rotation variability and alignment of the centrifuge occupants with the artificial gravity vector.

  12. Phenotypic changes of human cells in human-rat liver during partial hepatectomy-induced regeneration

    Institute of Scientific and Technical Information of China (English)

    Yan Sun; Dong Xiao; Hong-An Li; Jin-Fang Jiang; Qing Li; Ruo-Shuang Zhang; Xi-Gu Chen

    2009-01-01

    AIM: To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera (HRC) generated by in utero transplantation of human cells during partial hepatectomy (PHx)-induced liver regeneration. METHODS: Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow cytometry, in situ hybridization and immunohistochemistry. RESULTS: ISH analysis demonstrated human Alupositive cells in hepatic parenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein (AFP)+, CD34+ and CD45+ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen (PCNA) showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver.

  13. False-positive Human Papillomavirus DNA tests in cervical screening

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Pribac, Igor; Lynge, Elsebeth

    2011-01-01

    Based on data from randomised controlled trials (RCT) on primary cervical screening, it has been reported that the problem of more frequent false-positive tests in Human Papillomavirus (HPV) DNA screening compared to cytology could be overcome. However, these reports predominantly operated...

  14. School Health Education about Human Sexuality. Position Statement. Revised

    Science.gov (United States)

    Bradley, Beverly J.; Mancuso, Patty; Cagginello, Joan B.; Board, Connie; Clark, Sandra; Harvel, Robin; Kelts, Susan

    2012-01-01

    It is the position of the National Association of School Nurses (NASN) that age-appropriate health education about human sexuality should be included as part of a comprehensive school health education program and be accessible to all students in schools. NASN recognizes the role of parents and families as the primary source of education about…

  15. Human capital and optimal positive taxation of capital income

    NARCIS (Netherlands)

    B. Jacobs (Bas); A.L. Bovenberg (Lans)

    2010-01-01

    textabstractThis paper analyzes optimal linear and non-linear taxes on capital and labor incomes in a life-cycle model of human capital investment, financial savings, and labor supply with heterogenous individuals. A dual income tax with a positive marginal tax rate on not only labor income but also

  16. Human Capital and Optimal Positive Taxation of Capital Income

    NARCIS (Netherlands)

    B. Jacobs (Bas); A.L. Bovenberg (Lans)

    2005-01-01

    textabstractThis paper analyzes optimal linear taxes on capital and labor incomes in a life-cycle model of human capital investment, financial savings, and labor supply with heteroge- nous individuals. A dual income tax with a positive marginal tax rate on not only labor income but also capital inco

  17. Human Capital and Optimal Positive Taxation of Capital Income

    NARCIS (Netherlands)

    B. Jacobs (Bas); A.L. Bovenberg (Lans)

    2005-01-01

    textabstractThis paper analyzes optimal linear taxes on capital and labor incomes in a life-cycle model of human capital investment, financial savings, and labor supply with heteroge- nous individuals. A dual income tax with a positive marginal tax rate on not only labor income but also capital inco

  18. Differences in distribution and regulation of astrocytic aquaporin-4 in human and rat hydrocephalic brain

    DEFF Research Database (Denmark)

    Skjolding, Anders Daehli; Holst, Anders Vedel; Broholm, Helle

    2013-01-01

    in human hydrocephalic cortex relative to controls was quantified by western blotting (n=28). A second biopsy (n=13) was processed for immunohistochemistry (GFAP, CD68, CD34 and aquaporin-4) and double immunofluorescence (aquaporin-4+GFAP and aquaporin-4+CD34). Brain tissue from human controls and kaolin...

  19. Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

    DEFF Research Database (Denmark)

    Wu, Xue; Yin, Tieying; Tian, Jie

    2015-01-01

    It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34...... experiment using a rabbit model in which the coated stents with different substrates were implanted showed that anti-CD34 and anti-CD133 antibody-coated stents markedly reduced the intima area and restenosis than bare mental stents (BMS) and gelatin-coated stents. Compared with the anti-CD34 antibody...

  20. Principle of relative positioning of structures in the human body

    Institute of Scientific and Technical Information of China (English)

    Buliang Meng; Ailan Pang; Ming Li

    2013-01-01

    The arrangement of various biological structures should generally ensure the safety of crucial structures and increase their working efficiency; however, other principles governing the relative positions of structures in humans have not been reported. The present study therefore investigated other principles using nerves and their companion vessels in the human body as an example. Nerves and blood vessels usually travel together and in the most direct way towards their targets. Human embryology, histology, and gross anatomy suggest that there are many possible positions for these structures during development. However, for mechanical reasons, tougher or stronger structures should take priority. Nerves are tougher than most other structures, followed by arteries, veins, and lymphatic vessels. Nerves should therefore follow the most direct route, and be followed by the arteries, veins, and lymphatic vessels. This general principle should be applicable to all living things.

  1. Principle of relative positioning of structures in the human body.

    Science.gov (United States)

    Meng, Buliang; Pang, Ailan; Li, Ming

    2013-03-25

    The arrangement of various biological structures should generally ensure the safety of crucial structures and increase their working efficiency; however, other principles governing the relative positions of structures in humans have not been reported. The present study therefore investigated other principles using nerves and their companion vessels in the human body as an example. Nerves and blood vessels usually travel together and in the most direct way towards their targets. Human embryology, histology, and gross anatomy suggest that there are many possible positions for these structures during development. However, for mechanical reasons, tougher or stronger structures should take priority. Nerves are tougher than most other structures, followed by arteries, veins, and lymphatic vessels. Nerves should therefore follow the most direct route, and be followed by the arteries, veins, and lymphatic vessels. This general principle should be applicable to all living things.

  2. Effects of student ontological position on cognition of human origins

    Science.gov (United States)

    Ervin, Jeremy Alan

    In this study, the narratives from a hermeneutical dialectic cycle of three high school students were analyzed to understand the influences of ontological position on the learning of human origins. The interpretation of the narratives provides the reader an opportunity to consider the learning process from the perspective of worldview and conceptual change theories. Questions guiding this research include: Within a context of a worldview, what is the range of ontological positions among a high school AP biology class? To what extent does ontological position influence the learning of scientific concepts about human origins? If a student's ontological position is contradictory to scientific explanation of human origins, how will learning strategies and motivations change? All consenting students in an AP biology class were interviewed in order to select three students who represented three different ontological positions of a worldview: No Supernatural, Supernatural Without Impact, or Supernatural Impact. The issue of worldview is addressed at length in this work. Consenting students had completed the graduation requirements in biology, but were taking an additional biology course in preparation for college. Enrollment in an AP biology course was assumed to indicate that the selected students have an understanding of the concept of human origins at a comprehensive level, but not necessarily at an apprehension level, both being needed for conceptual change. Examination of the narratives reveals that students may alternate between two ontological positions in order to account for inconsistencies within a situation. This relativity enables the range of ontological positions to vary depending on concepts being considered. Not all Supernatural Impact positions conflict with biological understanding of human origins due to the ability of some to create a dichotomy between religion and school. Any comprehended concepts within this dichotomy lead to plagiaristic knowledge

  3. IL-7Rα and L-selectin, but not CD103 or CD34, are required for murine peanut-induced anaphylaxis

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    Maltby Steven

    2012-08-01

    Full Text Available Abstract Background Allergy to peanuts results in severe anaphylactic responses in affected individuals, and has dramatic effects on society and public policy. Despite the health impacts of peanut-induced anaphylaxis (PIA, relatively little is known about immune mechanisms underlying the disease. Using a mouse model of PIA, we evaluated mice with deletions in four distinct immune molecules (IL7Rα, L-selectin, CD34, CD103, for perturbed responses. Methods PIA was induced by intragastric sensitization with peanut antigen and cholera toxin adjuvant, followed by intraperitoneal challenge with crude peanut extract (CPE. Disease outcome was assessed by monitoring body temperature, clinical symptoms, and serum histamine levels. Resistant mice were evaluated for total and antigen specific serum IgE, as well as susceptibility to passive systemic anaphylaxis. Results PIA responses were dramatically reduced in IL7Rα−/− and L-selectin−/− mice, despite normal peanut-specific IgE production and susceptibility to passive systemic anaphylaxis. In contrast, CD34−/− and CD103−/− mice exhibited robust PIA responses, indistinguishable from wild type controls. Conclusions Loss of L-selectin or IL7Rα function is sufficient to impair PIA, while CD34 or CD103 ablation has no effect on disease severity. More broadly, our findings suggest that future food allergy interventions should focus on disrupting sensitization to food allergens and limiting antigen-specific late-phase responses. Conversely, therapies targeting immune cell migration following antigen challenge are unlikely to have significant benefits, particularly considering the rapid kinetics of PIA.

  4. Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages

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    Rossi John

    2004-12-01

    Full Text Available Abstract Background RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys, and small interfering RNAs (siRNAs. Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages. Results High efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection. Conclusions Viral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

  5. The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

    Science.gov (United States)

    Roeven, Mieke W H; Thordardottir, Soley; Kohela, Arwa; Maas, Frans; Preijers, Frank; Jansen, Joop H; Blijlevens, Nicole M; Cany, Jeannette; Schaap, Nicolaas; Dolstra, Harry

    2015-12-15

    Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

  6. [The comparative characteristics of antibacterial properties of the peptides of the active site of GM-CSF, and substances delivered from supernatants of hematopoietic progenitor CD34+45- cells].

    Science.gov (United States)

    Zurochka, A V; Zurochka, V A; Kostolomova, E G; Dobrynina, M A; Sykhoveĭ, Iu G; Gritsenko, V A

    2012-01-01

    The antibacterial activity of synthetic peptides of the active site of GM-CSF and supernatants of CD34+45- hematopoietic progenitor cells has been investigated GM-CSF peptides and cell supernatants were found to possess pronounced antibacterial activity, at that a combination of these substances has a more pronounced activity in comparison with the single substances. Possible mechanisms of the identified effects of synthetic peptides and substances from the supernatants of CD34+5- cells are discussed.

  7. Analysis of CD45- [CD34+/KDR+] endothelial progenitor cells as juvenile protective factors in a rat model of ischemic-hemorrhagic stroke.

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    Julius L Decano

    Full Text Available BACKGROUND: Identification of juvenile protective factors (JPFs which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. A priori, if EPC aging plays a 'master-switch JPF-role' in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset. METHODOLOGY/PRINCIPAL FINDINGS: FACS analysis revealed that CD45- [CD34+/KDR+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[CD34+/KDR+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003. Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in CD45-[CD34+/KDR]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001, suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age

  8. Controls of nucleosome positioning in the human genome.

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    Daniel J Gaffney

    Full Text Available Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase-seq from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase-seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7% of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned--in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.

  9. Human rights: common meaning and differences in positioning

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    Willem Doise

    Full Text Available Human rights are defined as normative social representations embedded in institutional juridical definitions. Research findings show that human rights can be studied as normative social representations implying a degree of common understanding across cultures together with organized differences within and between cultures. Important factors in modulating individual positioning in the realm of human rights are experiences of social conflict and injustice, beliefs about the efficiency of various social actors to have rights enforced and attitudes of liberalism or collectivism. On the other hand, an ethnocentric use of human rights is well documented and has been experimentally studied. Generally, concerns about these rights expressed by citizens of Western countries become much stronger when non-Western countries are involved, whereas violations of these rights in their own country are often not severely condemned.

  10. Human and non-human primate genomes share hotspots of positive selection.

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    David Enard

    2010-02-01

    Full Text Available Among primates, genome-wide analysis of recent positive selection is currently limited to the human species because it requires extensive sampling of genotypic data from many individuals. The extent to which genes positively selected in human also present adaptive changes in other primates therefore remains unknown. This question is important because a gene that has been positively selected independently in the human and in other primate lineages may be less likely to be involved in human specific phenotypic changes such as dietary habits or cognitive abilities. To answer this question, we analysed heterozygous Single Nucleotide Polymorphisms (SNPs in the genomes of single human, chimpanzee, orangutan, and macaque individuals using a new method aiming to identify selective sweeps genome-wide. We found an unexpectedly high number of orthologous genes exhibiting signatures of a selective sweep simultaneously in several primate species, suggesting the presence of hotspots of positive selection. A similar significant excess is evident when comparing genes positively selected during recent human evolution with genes subjected to positive selection in their coding sequence in other primate lineages and identified using a different test. These findings are further supported by comparing several published human genome scans for positive selection with our findings in non-human primate genomes. We thus provide extensive evidence that the co-occurrence of positive selection in humans and in other primates at the same genetic loci can be measured with only four species, an indication that it may be a widespread phenomenon. The identification of positive selection in humans alongside other primates is a powerful tool to outline those genes that were selected uniquely during recent human evolution.

  11. Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34(+) bone marrow cells, following gamma irradiation in cynomolgus macaques

    Energy Technology Data Exchange (ETDEWEB)

    Derdouch, S.; Gay, W.; Prost, S.; Le Dantec, M.; Delache, B.; Auregan, G.; Andrieu, T.; Le Grand, R. [CEA, DSV, Serv Immunovirol, Inst Maladies Emergentes et Therapies Innovantes, Fontenay Aux Roses (France); Derdouch, S.; Gay, W.; Prost, S.; Le Dantec, M.; Delache, B.; Auregan, G.; Andrieu, T.; Le Grand, R. [Univ Paris 11, UMR E01, Orsay (France); Negre, D.; Cosset, F. [Univ Lyon, UCB Lyon 1, IFR 128, F-69007 Lyon (France); Negre, D.; Cosset, F. [INSERM, U758, F-69007 Lyon (France); Negre, D.; Cosset, F.L. [Ecole NormaleSuper Lyon, F-69007 Lyon (France); Leplat, J.J. [CEA, DSV, IRCM, SREIT, Lab Radiobiol, F-78352 Jouy En Josas (France); Leplat, J.J. [CEA, DSV, IRCM, SREIT, Etude Genome, F-78352 Jouy En Josas (France); Leplat, J.J. [INRA, DGA, Radiobiol Lab, F-78352 Jouy En Josas (France); Leplat, J.J. [INRA, DGA, Etude Genome, F-78352 Jouy En Josas (France)

    2008-07-01

    Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myelo-ablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34{sup +} bone marrow cells following gamma irradiation in cynomolgus macaques. The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% {+-} 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudo-typed and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% {+-} 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producing eGFP were detected as early as three days after transplantation,and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. Conclusion: The transplantation of CD34{sup +} bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T-and B-lymphocyte, thrombocyte and red blood cell compartments. (authors)

  12. Comparison of gene expression of mitogenic kinin path in adherent and non-adherent CD 34-stem cells using oligonucleotide microarrays.

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    Krzysztof Machaj

    2008-02-01

    Full Text Available One of the more interesting cells present in the umbilical cord blood - as far as their potential clinical use is concerned - are stem cells not presenting the CD34 antigen. These are the pluripotential cells with their biological properties similar to mesenchymal stem cells, with the ability to differentiate into such tissue types as bone, cartilage, nervous (to some extent, glia and muscle. The authors compared the activity of genes coding the proteins in mitogenic signal paths activated by kinin receptors using oligonucleotide microarrays in adherent and non-adherent CD 34- cells derived from umbilical cord blood. In the linear regression model with a 95% prognosis area for differentiating genes outside this area, the following genes were selected: c-jun (present in 3 isoforms and c-fos. The fos and jun genes create the AP-1 transcriptive factor which regulates the expression of genes taking part in numerous cellular processes, including the cell cycle and mitosis. The obtained results shed some light on the molecular processes behind the MSC proliferation and are a starting point for further studies on the mesenchymal stem cell biology.

  13. Effects of Simian Betaretrovirus Serotype 1 (SRV1) Infection on the Differentiation of Hematopoietic Progenitor Cells (CD34+) Derived from Bone Marrow of Rhesus Macaques (Macaca mulatta)

    Science.gov (United States)

    Montiel, Nestor A; Todd, Patricia A; Yee, JoAnn; Lerche, Nicholas W

    2012-01-01

    Peripheral blood cytopenias, particularly persistent anemia and neutropenia, are commonly associated with simian betaretrovirus infection of Asian monkeys of the genus Macaca. The pathogenetic mechanisms underlying these hematologic abnormalities are not well understood. The current study investigated the in vitro tropism of simian betaretrovirus (SRV) for both hematopoietic progenitor (CD34+) and stromal cells obtained from rhesus macaque bone marrow and assessed the effects of infection on hematopoietic progenitor cell differentiation in vitro. After in vitro exposure, SRV proviral DNA could be demonstrated by real-time PCR in cells and the reverse transcriptase assay in supernatants from SRV-exposed progenitor-associated stroma, but not in differentiated colonies derived from SRV-exposed progenitors. Furthermore, in vitro exposure involving cell–cell contact of uninfected CD34+ progenitor cells with SRV-infected stromal cells resulted in a statistically significant reduction in granulocyte–macrophage colony formation in absence of detectable SRV-infection of progenitor cells. Reduction in colony formation occurred in a ‘dose-dependent’ fashion with increasing contact time. No effects on erythroid lineages and RBC differentiation were noted. Our results suggest that hematologic abnormalities observed during SRV disease (natural or experimental) of rhesus macaques may not result from direct effects of viral infection of progenitor cell populations, but rather be (at least in part) a consequence of SRV infection of supportive bone marrow stroma with secondary effects on differentiation of associated progenitor cells. PMID:22330653

  14. Efficient transduction and engraftment of G-CSF-mobilized peripheral blood CD34+ cells in nonhuman primates using GALV-pseudotyped gammaretroviral vectors.

    Science.gov (United States)

    Beard, Brian C; Mezquita, Pau; Morris, Julia C; Kiem, Hans-Peter

    2006-08-01

    The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype. Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient transduction and engraftment of G-PBSC using Phoenix-GALV-pseudotyped vectors. We transplanted three baboons with G/only mobilized CD34(+) cells transduced with GALV-pseudotyped retroviral vectors. We observed high-level, persistent engraftment of gene-modified G-PBSC in all animals with gene marking levels in granulocytes up to 60%. We analyzed amphotropic (PIT2) and GALV (PIT1) receptor expression in G/only cells and found preferential expression of PIT1 after G/only, which may explain the inferior results with amphotropic pseudotypes. These findings demonstrate that high stem cell gene transfer levels can be achieved using G-CSF-mobilized PBSC with Phoenix-GALV-pseudotyped vectors.

  15. Endothelial progenitor cells (CD34+KDR+) and monocytes may provide the development of good coronary collaterals despite the vascular risk factors and extensive atherosclerosis.

    Science.gov (United States)

    Kocaman, Sinan Altan; Yalçın, Mehmet Rıdvan; Yağcı, Münci; Sahinarslan, Asife; Türkoğlu, Sedat; Arslan, Uğur; Kurşunluoğlu, Nevruz; Ozdemir, Murat; Timurkaynak, Timur; Cemri, Mustafa; Abacı, Adnan; Boyacı, Bülent; Cengel, Atiye

    2011-06-01

    Endothelial progenitor cells (EPC) have a regenerative role in the vascular system. In this study, we aimed to evaluate simultaneously the effects of EPC and inflammatory cells on the presence and the extent of coronary artery disease (CAD) and the grade of coronary collateral growth in patients with clinical suspicion of CAD. This study has a cross-sectional and observational design. We enrolled 112 eligible patients who underwent coronary angiography consecutively (mean age: 59±9 years). The association of circulating inflammatory cells and EPC (defined by CD34+KDR+ in the lymphocyte and monocyte gate) with the presence, severity and extent of CAD and the degree of collateral growth were investigated. Logistic regression analysis was used to define the predictors of collateral flow. Of 112 patients 30 had normal coronary arteries (NCA, 27%, 55±9 years) and 82 had CAD (73%, 61±8 years). Among the patients with CAD, the percent degree of luminal stenosis was <50% in 12 patients; 50-90% in 35 patients; and ≥90% in the other 35 patients. Circulating inflammatory cells were higher (leukocytes, 7150±1599 vs 8163±1588 mm(-3), p=0.001; neutrophils, 4239±1280 vs 4827±1273 mm(-3), p=0.021; monocytes, 512±111 vs 636±192 mm(-3), p=0.001) and EPCs were lower (0.27±0.15% vs 0.17±0.14%, p<0.001; 21±15 vs 13±12 mm(-3), p=0.004) in CAD group than NCA group. When we investigated the collateral growth in patients having ≥90% stenosis in at least one major coronary artery, we found that the patients with good collateral growth had significantly higher EPC (0.22±0.17% vs 0.10±0.05%, p=0.009; 18±15 vs 7±3 mm(-3), p=0.003) in comparison to patients with poor collateral growth. Presence of EPC was associated with reduced risk for coronary artery disease (OR: 0.934, 95%CI: 0.883-0.998, p=0.018) and was an independent predictor for good collateral growth (OR: 1.295, 95%CI: 1.039-1.615, p=0.022). A sum of CD34+KDR-, CD34+KDR+ and CD34-KDR+ cells (192±98 mm(-3)), and a

  16. Sensing Super-Position: Human Sensing Beyond the Visual Spectrum

    Science.gov (United States)

    Maluf, David A.; Schipper, John F.

    2007-01-01

    The coming decade of fast, cheap and miniaturized electronics and sensory devices opens new pathways for the development of sophisticated equipment to overcome limitations of the human senses. This paper addresses the technical feasibility of augmenting human vision through Sensing Super-position by mixing natural Human sensing. The current implementation of the device translates visual and other passive or active sensory instruments into sounds, which become relevant when the visual resolution is insufficient for very difficult and particular sensing tasks. A successful Sensing Super-position meets many human and pilot vehicle system requirements. The system can be further developed into cheap, portable, and low power taking into account the limited capabilities of the human user as well as the typical characteristics of his dynamic environment. The system operates in real time, giving the desired information for the particular augmented sensing tasks. The Sensing Super-position device increases the image resolution perception and is obtained via an auditory representation as well as the visual representation. Auditory mapping is performed to distribute an image in time. The three-dimensional spatial brightness and multi-spectral maps of a sensed image are processed using real-time image processing techniques (e.g. histogram normalization) and transformed into a two-dimensional map of an audio signal as a function of frequency and time. This paper details the approach of developing Sensing Super-position systems as a way to augment the human vision system by exploiting the capabilities of Lie human hearing system as an additional neural input. The human hearing system is capable of learning to process and interpret extremely complicated and rapidly changing auditory patterns. The known capabilities of the human hearing system to learn and understand complicated auditory patterns provided the basic motivation for developing an image-to-sound mapping system. The

  17. PEG10 Activation by Co-Stimulation of CXCR5 and CCR7 Essentially Contributes to Resistance to Apoptosis in CD19+CD34+ B Cells from Patients with B Cell Lineage Acute and Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    ChunsongHu; JeiXiong; LinjeiZhang; BaojunHuang; QiupingZhang; QunLi; MingzhenYang; YaouWu; QunWu; QianShen; QingpingGao; KejianZhang; ZhiminSun; JunyanLin; YouxinJin

    2004-01-01

    We investigated CD19+CD34+ and CD19+CD34 B cells from cord blood (CB) and typical patients with B cell lineage acute and chronic lymphocytic leukemia (B-ALL and B-CLL) in terms of expression and functions of CXCR5/CXCL13 and CCR7/CCL19. CXCR5 and CCR7 were selectively frequent expressed on B-ALL, B-CLL and CB CD19+CD34+ B cells, but not on CD19+CD34- B cells. Instead of induction of impressive chemotactic responsiveness, CXCL13 and CCL19 together induced significant resistance to TNF-α-mediated apoptosis in B-ALL and B-CLL but not CB CD19+CD34+ B cells. B-ALL and B-CLL CD19+CD34+ B cells expressed elevatedlevel of Paternally Expressed Gene 10 (PEG10), and CXCL13 and CCL19 together significantly up-regulated PEG10 expression in the cells. We found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulated PEG10 expression and function, subsequent stabilized caspase-3 and caspase-8 in B-ALL and B-CLL CD19+CD34+ B cells, and rescued the cells from TNF-α-mediated apoptosis. We suggested that normal lymphocytes, especially naive B and T cells, utilized CXCR5/CXCL13 and CCR7/CCL19 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. Meanwhile certain malignant cells took advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration, resistance to apoptosis, and inappropriate proliferation. Cellular & Molecular Immunology.

  18. PEG10 Activation by Co-Stimulation of CXCR5 and CCR7 Essentially Contributes to Resistance to Apoptosis in CD19+CD34+ B Cells from Patients with B Cell Lineage Acute and Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    Chunsong Hu; Qian Shen; Qingping Gao; Kejian Zhang; Zhimin Sun; Junyan Liu; Youxin Jin; Jinquan Tan; Jei Xiong; Linjei zhang; Baojun Huang; Qiuping Zhang; Qun Li; Mingzhen Yang; Yaou Wu; Qun Wu

    2004-01-01

    We investigated CD19+CD34+ and CD19+CD34- B cells from cord blood (CB) and typical patients with B cell lineage acute and chronic lymphocytic leukemia (B-ALL and B-CLL) in terms of expression and functions of CXCR5/CXCL13 and CCR7/CCL19. CXCR5 and CCR7 were selectively frequent expressed on B-ALL, B-CLL and CB CD19+CD34+ B cells, but not on CD19+CD34- B cells. Instead of induction of impressive chemotactic responsiveness, CXCL13 and CCL19 together induced significant resistance to TNF-α-mediated apoptosis in B-ALL and B-CLL but not CB CD19+CD34+ B cells. B-ALL and B-CLL CD19+CD34+ B cells expressed elevated level of Paternally Expressed Gene 10 (PEG10), and CXCL13 and CCL19 together significantly up-regulated PEG10 expression in the cells. We found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulated PEG10 expression and function, subsequent stabilized caspase-3 and caspase-8 in B-ALL and B-CLL CD19+CD34+ B cells, and rescued the cells from TNF-α-mediated apoptosis. We suggested that normal lymphocytes, especially na(I)ve B and T cells, utilized CXCR5/CXCL13 and CCR7/CCL19 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis.Meanwhile certain malignant cells took advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration,resistance to apoptosis, and inappropriate proliferation.

  19. Transplantation of Highly Purified Autologous CD34+ Cells from the Peripheral Blood for Treatment of Progressive Multiple Sclerosis%自体外周血纯化CD34+细胞移植治疗进展型多发性硬化

    Institute of Scientific and Technical Information of China (English)

    冀冰心; 徐娟; 苏力; 刘聪艳; 董会卿

    2007-01-01

    目的 评价自体外周血纯化CD34+细胞移植治疗进展型多发性硬化(PMS)的安全性和疗效.方法 2002-09―2006-03期间15例PMS患者在首都医科大学宣武医院接受了自体外周血纯化CD34+细胞移植.单独使用粒细胞集落刺激因子(G-CSF)动员造血干细胞,全部回输采集物进行CD34+细胞纯化.预处理采用BEAM(卡氮芥、依托泊甙、阿糖胞苷、马法兰)方案.中位随访期为21(3~45)个月,移植前后应用扩充神经功能残疾量表(EDSS)、年平均发病次数进行疗效评价. 结果分选后中位CD34+细胞纯度为93.2 (78.6~97.7)%,中位回收率为67.0(22.4~79.8)%,相当于减少了4个对数级的T细胞.无移植相关死亡,造血重建时间与其自体外周造血干细胞移植(APBSCT)相当,未出现严重的毒性反应及并发症.患者移植后12个月EDSS评分(3.95±2.55)较移植前(5.64±0.71)降低(P<0.05),年平均发病次数移植后(0.45±0.82)较移植前(1.31±0.71)减少(P<0.05).移植后45个月疾病无活动者生存率为(47.01±17.87)%,EDSS评分无进展者(包括稳定和改善)生存率为(57.69±20.24)%. 结论自体外周血纯化CD34+细胞移植治疗PMS安全有效.

  20. African signatures of recent positive selection in human FOXI1

    Directory of Open Access Journals (Sweden)

    Moreno-Estrada Andrés

    2010-09-01

    Full Text Available Abstract Background The human FOXI1 gene codes for a transcription factor involved in the physiology of the inner ear, testis, and kidney. Using three interspecies comparisons, it has been suggested that this may be a gene under human-specific selection. We sought to confirm this finding by using an extended set of orthologous sequences. Additionally, we explored for signals of natural selection within humans by sequencing the gene in 20 Europeans, 20 East Asians and 20 Yorubas and by analysing SNP variation in a 2 Mb region centered on FOXI1 in 39 worldwide human populations from the HGDP-CEPH diversity panel. Results The genome sequences recently available from other primate and non-primate species showed that FOXI1 divergence patterns are compatible with neutral evolution. Sequence-based neutrality tests were not significant in Europeans, East Asians or Yorubas. However, the Long Range Haplotype (LRH test, as well as the iHS and XP-Rsb statistics revealed significantly extended tracks of homozygosity around FOXI1 in Africa, suggesting a recent episode of positive selection acting on this gene. A functionally relevant SNP, as well as several SNPs either on the putatively selected core haplotypes or with significant iHS or XP-Rsb values, displayed allele frequencies strongly correlated with the absolute geographical latitude of the populations sampled. Conclusions We present evidence for recent positive selection in the FOXI1 gene region in Africa. Climate might be related to this recent adaptive event in humans. Of the multiple functions of FOXI1, its role in kidney-mediated water-electrolyte homeostasis is the most obvious candidate for explaining a climate-related adaptation.

  1. A compatitive research on the application of bone marrow CD34+ stem cells vs. bone marrow mononuclear cells in small vascular endothelialization tissue engineering%骨髓CD34+干细胞与单核细胞应用于组织工程小血管内皮化的比较

    Institute of Scientific and Technical Information of China (English)

    耿纪群; 谢宗涛; 蔡铭; 李刚; 韩悦; 游庆军

    2012-01-01

    Objective To compare the application of bone marrow CD34+ stem cells vs.bone marrow mononuclear cells (MNCs) in vascular tissue prosthesis engineering.Methods We gathered dog bone marrow,segregated endothelia progenitor cells (EPCs) through immunomagnetic beads,and induced them to differentiate to endothelia cells (ECs) by endothelial cell growth facter(VEGF).Then we identified ECs through inverted phase contrast microscope,immunocytochemistry and tube-like structure formed test.Then we take some unfractioned MNCs,after routine cultivation implant them in the vascular prosthesis,and compare them under the microscope to the previous group.Results CD34+ cells being segregated were (78.46 ±6.37) % in content evaluated by flowcytometry,and were spread on the undersurface of culture flask 2 weeks after being cultured,and lined up as slabstones,being masccline for CD31 and Ⅷ factor in immunocytochemical staining.Under scanning eletron microscope,ECs tiled on the surface of vascular prosthesis,with their pseudopodia stretching out to micropore of vascular internalsurface; in the MNCs group no reticular structure can be readily found.For the CD34 cell group,the vascular prosthesis endothelialization area is (68.4 ±2.3)%,which is obviously more than that for the MNCs cell group (41.3 ±3.4) %,P < 0.01.Conclusion Highly purified bone marrow CD34+ cells can be obtained through immunomagnetic beads,cultured in vitro and differentiated to ECs directionally by VEGF.Better endothelialization effect can be gained from cell plantation on internal surface of vascular prosthesis than MNCs.%目的 探讨骨髓CD34+干细胞与骨髓单核细胞(MNCs)应用于组织工程小血管内皮化的效果,并作比较.方法 采集犬骨髓,经免疫磁珠分离出CD34+细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,行细胞免疫荧光、免疫组织化学和体外成血管实验鉴定;将培养后的细胞和未经分离的MNCs分别种植于人工

  2. Effects of Serum from Aplastic Anemia patients on the Expression of Cyclin D3 Isoform in Umbilical Cord Blood CD34+ Cells

    Institute of Scientific and Technical Information of China (English)

    孟凡凯; 谭细友; 刘文励; 孙汉英; 周剑锋; 李春蕊; 刘丹; 何莉; 孙岚

    2004-01-01

    Summary: The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDIMACS Semi-solid methylcellulose culture technique was used to measure the formation of CFUGM;The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.

  3. Bone Marrow CD34(+) Progenitor Cells from HIV-Infected Patients Show an Impaired T Cell Differentiation Potential Related to Proinflammatory Cytokines.

    Science.gov (United States)

    Bordoni, Veronica; Bibas, Michele; Viola, Domenico; Sacchi, Alessandra; Cimini, Eleonora; Tumino, Nicola; Casetti, Rita; Amendola, Alessandra; Ammassari, Adriana; Agrati, Chiara; Martini, Federico

    2017-06-01

    The impact of HIV infection on the frequency and differentiation capability of CD34(+) bone marrow hematopoietic progenitor cells (BM-HPCs) is still debated, having a possible primary role in antiretroviral-induced immunoreconstitution. We investigated the influence of HIV replication or proinflammatory cytokines on lymphopoietic capability of BM-HPCs from seven viremic (VR) and five nonviremic (NVR) HIV-infected patients. We found that BM-HPCs from VR patients were unable to differentiate in vitro toward T cells, and produced proinflammatory cytokines in the absence of viral replication. In contrast, the lymphoid differentiation potential of BM-HPCs was partially restored in successfully antiretroviral therapy-treated patients. We also showed that TLR8 triggering induced BM-HPCs from healthy donors to release proinflammatory cytokines affecting T cell differentiation. These data suggest that in HIV-infected patients, the lymphopoiesis capability of BM-HPCs may be modulated by a virus-driven autocrine mechanism involving proinflammatory cytokines.

  4. Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture.

    Science.gov (United States)

    Glen, Katie E; Workman, Victoria L; Ahmed, Forhad; Ratcliffe, Elizabeth; Stacey, Adrian J; Thomas, Robert J

    2013-09-01

    Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Peran Ekstrak Etanol Topikal Daun Mengkudu (Morinda citrifolia L. pada Penyembuhan Luka Ditinjau dari Imunoekspresi CD34 dan Kolagen pada Tikus Galur Wistar

    Directory of Open Access Journals (Sweden)

    Indah Puti Rahmayani S

    2013-12-01

    Full Text Available Problems in wound healing occurred if proper care is not given and the wound develops into a chronic wound. Noni (Morinda citrifolia L. is one of the most common plants in tropical areas, including Indonesia, which fruit, leaves dan root are used in traditional treatment, for example wound healing. This experimental research with post test-only control group design identified the effect of topical application of noni leaves ethanol extract on wound healing by examining the histopathological appearance of fibroblas count, inflammatory cell infiltration, cluster of differentiation 34 (CD34 immunoexpression, and collagen deposition. The research was performed between November 2010 until September 2011 at the Pharmacology and Pathology Anatomy laboratories of Dr. Hasan Sadikin General Hospital Bandung. Excisions were made on each back of the rat of the 36 rats that were divided into control and test groups where the test group received topical application of noni leaves ethanol extract. The wound was examined on day 3, 7, and 14 using a light microscope. The result showed a tendency of better wound healing in the test group for all parameters based on the data on day 3, 7, and 14. Mann-Whitney Test with 95% confidence interval (p<0.05 showed that the p value for fibroblast count, inflammation cell infiltration, CD34 immunoexpression and collagen subsequently were p=0.319, p=0.290, p=0.251, and p=0.245, respectively. In conclusion, topical application of noni leaves ethanol extract has a benefit on wound healing although the results are not statistically significant.

  6. A map of recent positive selection in the human genome.

    Directory of Open Access Journals (Sweden)

    Benjamin F Voight

    2006-03-01

    Full Text Available The identification of signals of very recent positive selection provides information about the adaptation of modern humans to local conditions. We report here on a genome-wide scan for signals of very recent positive selection in favor of variants that have not yet reached fixation. We describe a new analytical method for scanning single nucleotide polymorphism (SNP data for signals of recent selection, and apply this to data from the International HapMap Project. In all three continental groups we find widespread signals of recent positive selection. Most signals are region-specific, though a significant excess are shared across groups. Contrary to some earlier low resolution studies that suggested a paucity of recent selection in sub-Saharan Africans, we find that by some measures our strongest signals of selection are from the Yoruba population. Finally, since these signals indicate the existence of genetic variants that have substantially different fitnesses, they must indicate loci that are the source of significant phenotypic variation. Though the relevant phenotypes are generally not known, such loci should be of particular interest in mapping studies of complex traits. For this purpose we have developed a set of SNPs that can be used to tag the strongest approximately 250 signals of recent selection in each population.

  7. Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Lecourt, Severine, E-mail: severine.lecourt@sls.aphp.fr [UPMC/AIM UMR S 974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); INSERM U974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); CNRS UMR 7215, Groupe Hospitalier Pitie-Salpetriere, Paris (France); Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Marolleau, Jean-Pierre, E-mail: Marolleau.Jean-Pierre@chu-amiens.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); CHU Amiens Hopital Sud, Service d' Hematologie Clinique, UPJV, Amiens (France); Fromigue, Olivia, E-mail: olivia.fromigue@larib.inserm.fr [INSERM U606, Universite Paris 07, Hopital Lariboisiere, Paris (France); Vauchez, Karine, E-mail: k.vauchez@institut-myologie.org [UPMC/AIM UMR S 974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); INSERM U974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); CNRS UMR 7215, Groupe Hospitalier Pitie-Salpetriere, Paris (France); Genzyme S.A.S., Saint-Germain en Laye (France); Andriamanalijaona, Rina, E-mail: rinandria@yahoo.fr [Laboratoire de Biochimie des Tissus Conjonctifs, Faculte de Medecine, Caen (France); Ternaux, Brigitte, E-mail: brigitte.ternaux@orange.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Lacassagne, Marie-Noelle, E-mail: mnlacassagne@free.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Robert, Isabelle, E-mail: isa-robert@hotmail.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Boumediene, Karim, E-mail: karim.boumediene@unicaen.fr [Laboratoire de Biochimie des Tissus Conjonctifs, Faculte de Medecine, Caen (France); Chereau, Frederic, E-mail: fchereau@pervasistx.com [Myosix S.A., Saint-Germain en Laye (France); Marie, Pierre, E-mail: pierre.marie@larib.inserm.fr [INSERM U606, Universite Paris 07, Hopital Lariboisiere, Paris (France); and others

    2010-09-10

    Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56{sup +} cells grew rapidly, a population of CD15{sup +} cells emerged, partly from CD56{sup +} cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56{sup +} and CD15{sup +} cells shared osteogenic and chondrogenic abilities, while CD56{sup +} cells presented a myogenic capacity and CD15{sup +} cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

  8. Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

    DEFF Research Database (Denmark)

    Wu, Xue; Yin, Tieying; Tian, Jie;

    2015-01-01

    It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34......-coated stents, the time of cells adhesion was longer and earlier present in the anti-CD133 antibody-coated stents and anti-CD133 antibody-coated stents have superiority in re-endothelialization and inhibition of ISR. In conclusion, this study demonstrated that anti-CD133 antibody as a stent coating...... for capturing EPCs is better than anti-CD34 antibody in promoting endothelialization and reducing ISR....

  9. Measuring domestic violence in human immunodeficiency virus-positive women.

    Science.gov (United States)

    Patrikar, Seema; Verma, Ak; Bhatti, Vk; Shatabdi, S

    2012-04-01

    Violence affects the lives of millions of women worldwide, in all socioeconomic classes. Violence and the fear of violence are emerging as important risk factor contributing to the vulnerability to human immunodeficiency virus (HIV) infection for women. The objective of the present cross sectional study is to compare the experiences of domestic violence between HIV-positive and HIV-negative married women seeking treatment in a tertiary care hospital. The study is conducted in a tertiary care hospital in Pune on a randomly selected 150 married women (75 HIV-positive and 75 HIV-negative). Informed consent was obtained from all the women and also a trained counsellor was present during the process of data collection. The data was collected by interview method by taking precautions as laid down in the World Health Organization's ethical and safety recommendations for research on domestic violence and using modified conflict tactics scale (CTS). The definition of violence followed is as per the Declaration on the Elimination of Violence against Women, adopted by the United Nations General Assembly in 1993. The percentage of women reporting domestic violence is 44.7% (95% confidence interval [CI] = 36.84-52.68). The proportion of physical, emotional and sexual violence reported is 38% (95% CI = 30.49-45.96), 24% (95% CI = 17.67-31.31), and 14.7% (95% CI = 9.66-21.02), respectively. The odds of reporting violence of all forms is significantly higher among HIV-positive women than among HIV-negative women (Pviolence. The findings suggest high proportion of HIV-positive women report violence then HIV-negative women which must be addressed through multilevel prevention approaches.

  10. Expression of P21 and its correlation with CD34+cells in mobilized peripheral blood stem/progenitor cells%动员的外周血干/祖细胞中P21的表达与CD34+细胞的相关性

    Institute of Scientific and Technical Information of China (English)

    孙玲; 马杰; 杨红旗; 孙慧; 张茵; 刘林湘; 邢莹

    2004-01-01

    目的:检测动员的外周血干/祖细胞中P21的表达及与CD34+细胞的相关性,探讨其在动员过程中的变化及意义.方法:选取异基因外周血干细胞移植正常供者9例和自体外周血干细胞移植者(急性白血病患者)6例,运用流式细胞术检测动员的外周血干/祖细胞中CD34+细胞及其亚群,用免疫细胞化学的方法检测外周血动员前后P21的表达.结果:动员的外周血干/祖细胞中P21的表达明显高于动员前,二者间差异有统计学意义(P<0.05);动员的外周血干/祖细胞中CD34+细胞比例与P21的表达率呈正相关(r=0.553).结论:外周血干/祖细胞动员过程中P21表达增高,可能与其促进祖细胞增殖、调控分化过程及抗凋亡活性有关.

  11. Developing positive leadership in health and human services

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Shannon

    2013-03-01

    Full Text Available Orientation: Measuring the target outcomes of leadership development programmes provides evidence for the effectiveness of these interventions and the validity of their theoretical underpinnings.Research purpose: The aim of this study was to determine whether staff from the Tasmanian Department of Health and Human Services (Australia experienced increased levels of self-efficacy, social support within the workplace and positive affect, following participation in a leadership development programme.Research design, approach and method: Quantitative and qualitative methods were used, allowing for triangulation of results. The General Self-Efficacy Scale and the Berlin Social-Support Scale (perceived available support, instrumental were applied in an online survey administered before and nine months following the programme. Participant satisfaction surveys captured immediate responses and semi-structured interviews captured longer-term reflections.Main findings: Descriptive statistics indicated a moderate overall increase in self-efficacy, with strong increases in resilience, dealing with opposition, resourcefulness and problem solving. There was some evidence of greater overall social support and a strong increase in the development of social support networks. There was no support for an increase in participants’ positive orientation towards their jobs in the quantitative data. The impact of adverse environmental factors on participants’ perceptions also became evident through the interviews.Practical implications: Leadership development programmes that strengthen positive psychological resources provide participants with confidence and resilience in times of change. Organisations benefit from increased levels of employee self-efficacy as engagement and problem-solving abilities are enhanced.Contribution/value-add: These results contribute to the body of knowledge associated with effective leadership development.

  12. 电磁辐射对CD34+造血干细胞JAK2和STAT5蛋白活化的影响及意义%Effect of electromagnetic exposure on activation of JAK2 and STAT5 in CD34 + cord blood stem cells

    Institute of Scientific and Technical Information of China (English)

    张勇; 王劲; 钟大平; 裴莉

    2010-01-01

    目的 观察电磁辐射对CD34+造血干细胞JAK2和STAT5蛋白活化的影响,探讨JAK2和STAT5蛋白在电磁辐射致造血干细胞损伤中的作用.方法 从脐血中分离培养CD34+造血干细胞,分别用平均功率为1、5、25 mW/cm~2的电磁波辐射20 min,干预组(平均辐射强度为25 mW/cm~2)使用AG490(终浓度10~(-6)mol/L)预处理CD34+造血干细胞30 min.检测正常组、辐射组和干预组12、24和48 h时细胞活力(MTT法)、细胞凋亡率(流式细胞术)、Caspase-3蛋白水平(WB法)、磷酸化JAK2和STAT5蛋白水平(WB法).结果 与正常组比较,辐射组细胞12和24 h活力降低、细胞凋亡率升高,具有剂量效应,磷酸化JAK2和STAT5蛋白表达呈升高后降低的趋势.干预组12和24 h细胞活力显著高于辐射25 mW/cm~2组,细胞凋亡率显著低于辐射25 mW/cm~2组,磷酸化JAK2和STAT5蛋白水平较辐射25 mW/cm~2组低.结论 JAK2和STAT5蛋白参与了1-25 mW/cm~2功率范围内的电磁辐射对CD34+造血干细胞的损伤过程,阻断JAK2和STAT5蛋白活化可缓解CD34+造血干细胞凋亡的发生.%Objective To observe the effect of electromagnetic radiation on the activation of JAK2 and STAT5 in the CD34 + cord blood stem cell.Methods CD34 + cord blood stem cells were isolated from the umbilical cord blood.Three exposed groups were administered with 1,5,25 mW/cm~2 electromagnetic exposure for 20 minutes,respectively.In the intervention group with the radiation power of 25 mW/cm~2,AG490 of the final concentration of 10~(-6) mol/L was pretreated for 30 minutes.The cell viabilitv (MTT),the rate of apoptosis (FCM),Caspase-3 protein level (WB) and the activation of JAK2 and STAT5 (WB) were assayed.Results Compared with the normal group,the cell viability of the electromagnetic group decreased at 12 h and 24 h,while apoptosis rate increased with dose.The activation of JA K2 and STAT5 increased at 12 h and 24 h and then decreased.The cell viability of the intervention group was

  13. Metabolic syndrome in human immunodeficiency virus positive patients

    Directory of Open Access Journals (Sweden)

    Sarita Bajaj

    2013-01-01

    Full Text Available Aims and Objectives : To assess the prevalence of metabolic syndrome (MetS in human immunodeficiency virus (HIV positive patients. Prevalence of MetS was compared in patients who were not on highly active antiretroviral therapy (HAART to patients who were on HAART. Materials and Methods: Seventy HIV positive cases were studied. Pregnant and lactating women, patients on drugs other than HAART known to cause metabolic abnormalities and those having diabetes or hypertension were excluded. Cases were evaluated for MetS by using National Cholesterol Education Program Adult Treatment Panel-III. Results: 47 cases were on HAART and 23 cases were not on HAART. Fasting Blood Glucose ≥100 mg/dl was present in 28.6% cases, out of whom 27.7% were on HAART and 30.4% were not on HAART (P = 0.8089. 12.9% cases had BP ≥130/≥85 mm Hg, out of whom 14.9% were on HAART and 8.7% were not on HAART (P = 0.4666. 42.9% cases had TG ≥150 mg/dl, out of whom 44.7% were on HAART and 39.1% were not on HAART (P = 0.6894. HDL cholesterol was low (males <40 mg/dl, females <50 mg/dl in 50% cases, out of whom 55.3% were on HAART and 39.1% were not on HAART (P = 0.2035. Conclusions: Prevalence of MetS was 20%. Majority of patients had only one component of MetS (32.9%. Low HDL was present in 50%, followed by raised triglycerides in 42.9%. Waist circumference was not increased in any of the patients. There was no statistically significant difference between those on HAART and those not on HAART in distribution of risk factors and individual components of MetS.

  14. Is there any role of mast cell density and microvessel density in cervical squamous cell carcinoma? A histologic study with special reference to CD-34 immunomarker staining

    Directory of Open Access Journals (Sweden)

    Santosh Kumar Mondal

    2014-01-01

    Full Text Available Background: Mast cells are involved in induction of angiogenesis in the early-stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression. Aims and Objectives: This study was carried out to evaluate the association between mast cell density (MCD and microvessel density (MVD in carcinoma in situ (CIS, microinvasive carcinoma (CA and invasive squamous cell CA of cervix. Materials and Methods: Six cases of CIS, four cases of microinvasive CA and 38 cases of invasive CA were studied over a period of 2 years from August, 2011 to June, 2013. Ten control samples were included in the study. Routine histologic examination was done. Toluidine blue stain was used for MCD determination. Immunohistochemical analysis with CD-34 was done for assessing MVD. Student′s t-test was used to calculate the statistical significance of MCD and MVD. Results: Both MCD and MVD increased from normal samples through CIS to invasive cervical CA. In the four cases of microinvasive CA, the MCD and MVD were more than that of the control samples, but less than that of the six cases of CIS. Conclusion: There is a correlation between mast cell accumulation and angiogenesis in CIS, microinvasive CA and invasive cervical squamous cell CA. MCD and MVD in invasive CA exceed those in CIS and microinvasive CA. It gives us an opportunity to postulate that therapeutic strategies against mast cell mediators and angiogenesis may be of benefit in patients of early-stage cervical CA.

  15. A phase 3, randomized, double-blinded, active-controlled, unblinded standard of care study assessing the efficacy and safety of intramyocardial autologous CD34+ cell administration in patients with refractory angina: design of the RENEW study.

    Science.gov (United States)

    Povsic, Thomas J; Junge, Candice; Nada, Adel; Schatz, Richard A; Harrington, Robert A; Davidson, Charles J; Fortuin, F David; Kereiakes, Dean J; Mendelsohn, Farrell O; Sherman, Warren; Schaer, Gary L; White, Christopher J; Stewart, Duncan; Story, Kenneth; Losordo, Douglas W; Henry, Timothy D

    2013-06-01

    Preclinical trials indicate that CD34+ cells represent an effective angiogenic stem cell component. Early-phase clinical trials suggest that intramyocardial administration of autologous CD34+ cells may improve functional capacity and symptoms of angina. RENEW is a pivotal phase 3 trial designed to determine the efficacy of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ stem cells for the treatment for patients with refractory angina and chronic myocardial ischemia. Patients (n = 444) receiving maximally tolerated antianginal therapies and lacking conventional revascularization options with Canadian Cardiovascular Society class III or IV angina and ischemia on stress testing will be randomized 2:1:1 to cell therapy (G-CSF-mediated stem cell mobilization, apheresis, and intramyocardial injection of 1 × 10(5) autologous CD34(+) cells/kg), active control (G-CSF-mediated stem cell mobilization, apheresis, and intramyocardial placebo injection), or open-label standard of care. The primary efficacy end point is change in exercise treadmill time in the treated vs active control patients, with 90% power to detect a 60-second difference in exercise time between cell-treated (n = 200) and active control (n = 100) patients. Key secondary end points include total number of anginal episodes per week and the incidence of independently adjudicated major adverse cardiac events and serious adverse events. RENEW will be the first adequately powered study aimed at definitively determining the efficacy of a cell therapy (intramyocardially delivered autologous CD34+ cells) for improvement of functional capacity in patients with refractory angina.

  16. CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

    Science.gov (United States)

    Boiret, N; Kanold, J; Fouassier, M; Bons, J M; Halle, P; Rapatel, C; Berger, J; Pireyre, P; Blanzat, V; Travade, P; Bonhomme, J; Demeocq, F; Berger, M G

    2000-08-01

    Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

  17. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing, E-mail: wangstella5@163.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Yang, Qifeng, E-mail: qifengy@gmail.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Haffty, Bruce G., E-mail: hafftybg@umdnj.edu [Department of Radiation Oncology, UMDNJ-Robert Wood Johnson School of Medicine, Cancer Institute of New Jersey, NB (United States); Li, Xiaoyan, E-mail: xiaoyanli1219@gmail.com [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Moran, Meena S., E-mail: meena.moran@yale.edu [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States)

    2013-02-08

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  18. Position stand on androgen and human growth hormone use.

    Science.gov (United States)

    Hoffman, Jay R; Kraemer, William J; Bhasin, Shalender; Storer, Thomas; Ratamess, Nicholas A; Haff, G Gregory; Willoughby, Darryn S; Rogol, Alan D

    2009-08-01

    Hoffman, JR, Kraemer, WJ, Bhasin, S, Storer, T, Ratamess, NA, Haff, GG, Willoughby, DS, and Rogol, AD. Position stand on Androgen and human growth hormone use. J Strength Cond Res 23(5): S1-S59, 2009-Perceived yet often misunderstood demands of a sport, overt benefits of anabolic drugs, and the inability to be offered any effective alternatives has fueled anabolic drug abuse despite any consequences. Motivational interactions with many situational demands including the desire for improved body image, sport performance, physical function, and body size influence and fuel such negative decisions. Positive countermeasures to deter the abuse of anabolic drugs are complex and yet unclear. Furthermore, anabolic drugs work and the optimized training and nutritional programs needed to cut into the magnitude of improvement mediated by drug abuse require more work, dedication, and preparation on the part of both athletes and coaches alike. Few shortcuts are available to the athlete who desires to train naturally. Historically, the NSCA has placed an emphasis on education to help athletes, coaches, and strength and conditioning professionals become more knowledgeable, highly skilled, and technically trained in their approach to exercise program design and implementation. Optimizing nutritional strategies are a vital interface to help cope with exercise and sport demands (). In addition, research-based supplements will also have to be acknowledged as a strategic set of tools (e.g., protein supplements before and after resistance exercise workout) that can be used in conjunction with optimized nutrition to allow more effective adaptation and recovery from exercise. Resistance exercise is the most effective anabolic form of exercise, and over the past 20 years, the research base for resistance exercise has just started to develop to a significant volume of work to help in the decision-making process in program design (). The interface with nutritional strategies has been less

  19. Identification of a novel population of human cord blood cells with hematopoietic and chondrocytic potential

    Institute of Scientific and Technical Information of China (English)

    Karen E JAY; Anne ROULEAU; T Michael UNDERHILL; Mickie BHATIA

    2004-01-01

    With the exception of mature erythrocytes, cells within the human hematopoietic system are characterized by the cell surface expression of the pan-leukocyte receptor CD45. Here, we identify a novel subset among mononuclear cord blood cells depleted of lineage commitment markers (Lin-) that are devoid of CD45 expression. Surprisingly, functional examination of Lin-CD45- cells also lacking cell surface CD34 revealed they were capable of multipotential hematopoietic progenitor capacity. Co-culture with mouse embryonic limb bud cells demonstrated that Lin-CD45-CD34- cells were capable of contributing to cartilage nodules and differentiating into human chondrocytes. BMP-4, a mesodermal factor known to promote chondrogenesis, significantly augmented Lin-CD45-CD34- differentiation into chondrocytes.Moreover, unlike CD34+ human hematopoietic stem cells, Lin-CD45-CD34- cells were unable to proliferate or survive in liquid cultures, whereas single Lin-CD45-CD34- cells were able to chimerize the inner cell mass (ICM) of murine blastocysts and proliferate in this embryonic environment. Our study identifies a novel population of Lin-CD45-CD34-cells capable of commitment into both hematopoietic and chondrocytic lineages, suggesting that human cord blood may provide a more ubiquitous source of tissue with broader developmental potential than previously appreciated.

  20. Head Position Preference in the Human Newborn: A New Look.

    Science.gov (United States)

    Ronnqvist, Louise; Hopkins, Brian

    1998-01-01

    Studied head position preference in 20 newborns differing by Cesarean or vaginal delivery and sex. Found that neither factor accounted for differences. The head turned right more often and was maintained longer in this position during quiet wakefulness, regardless of scoring method. When using global scoring, duration of midline position was…