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

The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb),which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound,3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516),which selectively inhibits cdk2 activity (Lane et al.,Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29,SW480,and RKO human colon cancer cell extracts. Treatment with 5 microM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29textgreaterRKOtextgreaterSW480). Treatment effects were time-dependent,demonstrating greater inhibition at 48 hr versus 24hr in HT-29 cells. Furthermore,E2F species were sequestered in complexes with p107,p130,DP-1,and cyclins A and E. After a 24-hr treatment with 5 microM SU9516,cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1,inhibition of cdk2 levels and activity,and pan-sequestration of E2F. View Publication

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells,activated,phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent,but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1,STAT3,and STAT5 occurred with diverse viral envelope proteins,independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells. View Publication

Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus,efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA),bone morphogenetic protein-4 (BMP4),and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA,AA,and BMP4; the third was commercial-defined keratinocyte serum-free medium,which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR,immunofluorescence,and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology,while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12,all P textless 0.05) After hESC induction by AA+BMP4+RA,CK14 mRNA expression was upregulated after day 21,peaking by 35 d of differentiation. Combined RA,BMP4,and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering. View Publication

Human natural killer (NK) cells express an abundant level of 2B4 and CD2 on their surface. Their counter-receptors,CD48 and CD58,are also expressed on the NK cell surface,raising a question about the functional consequences of potential 2B4/CD48 and CD2/CD58 interactions. Using blocking antibodies specific to each receptor,we demonstrated that both 2B4/CD48 and CD2/CD58 interactions were essential for the development of NK effector functions: cytotoxicity and cytokine secretion. However,only 2B4/CD48,but not CD2/CD58,interactions were shown to be critical for the optimal NK cell proliferation in response to interleukin (IL)-2. IL-2-activated NK cells cultured in the absence of 2B4/CD48 or CD2/CD58 interactions were severely impaired for their ability to induce intracellular calcium mobilization and subsequent ERK activation upon tumor target exposure,suggesting that the early signaling pathway of NK receptors leading to impaired cytolysis and interferon (IFN)-γ secretion was inhibited. Nevertheless,these defects did not fully account for the reduced proliferation of NK cells in the absence of 2B4/CD48 interactions,because anti-CD2 or anti-CD58 monoclonal antibody (mAb)-treated NK cells,showing defective signaling and effector functions,displayed normal proliferation upon IL-2 stimulation. These results propose the signaling divergence between pathways leading to cell proliferation and cytotoxicity/cytokine release,which can be differentially regulated by 2B4 and CD2 during IL-2-driven NK cell activation. Collectively,these results reveal the importance of homotypic NK-to-NK cell cross-talk through 2B4/CD48 and CD2/CD58 pairs and further present their differential and overlapping roles in human NK cells. View Publication

Ionizing radiation (IR) is a known mutagen that is widely employed for medical diagnostic and therapeutic purposes. To study the extent of genetic variations in DNA caused by IR,we used IR-sensitive human embryonic stem cells (hESCs). Four hESC cell lines,H1,H7,H9,and H14,were subjected to IR at 0.2 or 1 Gy dose and then maintained in culture for four days before being harvested for DNA isolation. Irradiation with 1 Gy dose resulted in significant cell death,ranging from 60% to 90% reduction in cell population. Since IR is often implicated as a risk for inducing cancer,a primer pool targeting genomic hotspot" regions that are frequently mutated in human cancer genes was used to generate libraries from irradiated and control samples. Using a semiconductor-based next-generation sequencing approach� View Publication

Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection,antibodies likely function in the presence of large quantities of virus. In this study,we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula,inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted,peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However,enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells,which express Fc receptors for IgG (FcgammaRs),abrogated the enhanced antibody inhibition,whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines,most likely through FcgammaR triggering of NK cells,contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo,FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection. View Publication

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems,we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas,high ALDH activity identifies the tumorigenic cell fraction,capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas,expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts. View Publication

PURPOSE Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard,stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study,we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer,170 keV/μm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug,allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF,PlGF,SDF-1,and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling,a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control,20.71 ± 1.55; X-ray,36.44 ± 3.44; carbon ion,16.33 ± 1.03; number per microscopic field). Concordantly,irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray,36.44 ± 3.44; X-ray and ISCK03,4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells,a phenomenon that could not be observed with carbon ion irradiation. Thus,in this model system evaluating angiogenesis,carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models. View Publication

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study,we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that,due to the absence of p21/Waf1/Cip1,cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage,the majority of hESCs succumb to apoptosis; however,a subpopulation continues to proliferate,carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that,due to deficient DNA damage response,the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs,paving the way for further major genetic changes. View Publication

Activin A belongs to the superfamily of transforming growth factor beta (TGF$\beta$) and is a critical regulatory cytokine in breast cancer and inflammation. However,the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here,a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast,activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally,activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively,our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers. View Publication

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently,advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method,referred to as Patch-seq,enables a thorough,multimodal profiling of neurons and permits us to expose the links between functional properties,morphology,and gene expression. Here,we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects,but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders. View Publication

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs),high-proliferative potential-CFCs (HPP-CFCs),and long-term culture-initiating cells (LTC-ICs). In mice,both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs,whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold,respectively,as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone,suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys,rhPlGF-1 alone had no mobilization effect,whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs,HPP-CFCs,and LTC-ICs per milliliter of blood by 5-,7-,and 15-fold,respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion,our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting. View Publication