搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored,using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony edge� View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment,low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82),little/no EpoR protein was detected and it was not functional. In contrast,EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20),and functional responses to rHuEpo were reported with MCF-7 cells. In another study,a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However,the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82,no EpoR protein was detectable in normal human and matching cancer tissues from breast,lung,colon,ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells,but no rHuEpo-induced phosphorylation of AKT,STAT3,pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein. View Publication

Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5,a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs,that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan,tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular,we found that affected aortas showed lower levels of all the PDE5A isoforms,compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms,but not that of the corresponding mRNAs. VSMC stimulation with GSNO,a nitric oxide analogue or with 8-br-cGMP,but not with 8-br-cAMP,up-regulated PDE5 and NOTCH-3 protein levels,indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally,we found that PDE5 is expressed early during human aorta development,suggesting that if loss of function mutations of PDE5 occur,they might potentially affect aortic wall development. View Publication

In hereditary hemochromatosis,mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition,HFE potentially modulates cellular iron uptake by interacting with transferrin receptor,a crucial protein during erythropoiesis. However,the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this,we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second,we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly,we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake,whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary,we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis,and impairing transferrin-bound iron uptake by erythroid cells. Moreover,our results provide novel suggestions to improve the treatment of hemochromatosis. View Publication

Autosomal recessive osteopetrosis (OP) is a rare,lethal disorder in which osteoclasts are absent or nonfunctional,resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors,allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However,high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition,colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical. View Publication

Lung cancer is the leading cause of cancer-related mortality worldwide. Recent evidence indicates that tumors contain a subpopulation of cancer stem cells (CSCs) that are responsible for tumor maintenance and spread. CSCs have recently been linked to the occurrence of epithelial-to-mesenchymal transition (EMT). Neurotrophins (NTs) are growth factors that regulate the biology of embryonic stem cells and cancer cells,but still little is known about the role NTs in the progression of lung cancer. In this work,we investigated the role of the NTs and their receptors using as a study system primary cell cultures derived from malignant pleural effusions (MPEs) of patients with adenocarcinoma of the lung. We assessed the expression of NTs and their receptors in MPE-derived adherent cultures vs. spheroids enriched in CSC markers. We observed in spheroids a selectively enhanced expression of TrkB,both at the mRNA and protein levels. Both K252a,a known inhibitor of Trk activity,and a siRNA against TrkB strongly affected spheroid morphology,induced anoikis and decreased spheroid forming efficiency. Treatment with neurotrophins reversed the inhibitory effect of K252a. Importantly,TrkB inhibition caused loss of vimentin expression as well as that of a set of transcription factors known to be linked to EMT. These ex vivo results nicely correlated with an inverse relationship between TrkB and E-cadherin expression measured by immunohistochemistry in a panel of lung adenocarcinoma samples. We conclude that TrkB is involved in full acquisition of EMT in lung cancer,and that its inhibition results in a less aggressive phenotype. View Publication

Eosinophils are associated with type 2 immune responses to allergens and helminths. They release various proinflammatory mediators and toxic proteins on activation and are therefore considered proinflammatory effector cells. Eosinophilia is promoted by the cytokines interleukin (IL)-3,IL-5,and granulocyte macrophage-colony-stimulating factor (GM-CSF) and can result from enhanced de novo production or reduced apoptosis. In this study,we show that only IL-5 induces differentiation of eosinophils from bone marrow precursors,whereas IL-5,GM-CSF,and to a lesser extent IL-3 promote survival of mature eosinophils. The receptors for these cytokines use the common β chain,which serves as the main signaling unit linked to signal transducer and activator of transcription 5,p38 mitogen-activated protein kinase,and nuclear factor (NF)-κB pathways. Inhibition of NF-κB induced apoptosis of in vitro cultured eosinophils. Selective deletion of IκBα in vivo resulted in enhanced expression of Bcl-xL and reduced apoptosis during helminth infection. Retroviral overexpression of Bcl-xL promoted survival,whereas pharmacologic inhibition of Bcl-xL in murine or human eosinophils induced rapid apoptosis. These results suggest that therapeutic strategies targeting Bcl-xL in eosinophils could improve health conditions in allergic inflammatory diseases. View Publication