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

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q),screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders,and identified 19 patients with del20q (2.9%),of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F,we performed colony assays in methylcellulose,picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies,and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients,we found that some colonies with del20q carried only wild-type JAK2,whereas other del20q colonies were JAK2-V617F positive,indicating that del20q occurred before the acquisition of JAK2-V617F. However,in colonies from 3 of 9 patients,we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L,microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events,such as del20q or 9pLOH,occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations. View Publication

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM,BCR cross-linking led to a quick burst of Syk,ERK1/2,and p38 signaling. In contrast,IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders,such as autoimmunity and cancer. View Publication

Costimulation by the chemokine,stromal cell-derived factor-1 (SDF-1)/CXCL12,has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however,the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082,-819,and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) (memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker� View Publication

Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors,nerve growth factor and brain-derived neurotrophic factor,share substantial amino acid sequence identity. We have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene,neurotrophin-3,that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain,suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development. View Publication

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4,KChIP2 and Kv4.3 were quantified from BCs. Results: BCs exhibited spontaneous beating (60.5??2.6bpm) and maximum-diastolic-potential (MDP) of 67.8??0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7??1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at -80mV) showed slow kinetics (??1=200??110ms (12%) and ??2=2380??240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. ?? 2013 Elsevier Ltd. View Publication

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system,but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells,we assessed fucosylated (2′FL and 3FL),sialylated (3′SL and 6′SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity,to stimulate the secretion of chemokines from intestinal epithelial cells,and to modulate cytokine release from LPS-activated dendritic cells (DCs),M1 macrophages (MØs),and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10,CCL20 and CXCL8 from intestinal epithelial cells,promoted the secretion of several cytokines (including IL-10,IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs,and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus,3′SL and 6′SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively,our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life. View Publication

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However,only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So,we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts. Human dermal fibroblasts in vitro were treated with various concentrations of KGN,with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction,Western blot,and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson's trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN. Results showed that without obvious influence on fibroblasts' apoptosis and viability,KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner,but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1),MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice. Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing,tissue engineering of fibroblasts,or aesthetic and reconstructive procedures. View Publication

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement,these requirements are met by an increased glycolysis,due,at least in part,to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia,we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably,Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore,TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics,irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen,Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition,augmented proliferation,and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover,Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus,Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. View Publication

Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations,convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors,measuring yield and purity outputs of undifferentiated,neuroectoderm,mesendoderm,and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically,small interfering RNA knockdown of RAPTOR,a component of mTOR complex 1,phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold,with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives. View Publication

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T-cell responses has been associated with the immunological control of HIV-1 replication; however,the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4(+) T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells,each of the individuals in the present study exhibited progressive disease,with one individual showing rapid progression. In this rapid progressor,three IL-2-producing HIV-1 Gag-specific CD4(+) T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER,REPRGSDIAGT,and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to textgreater1 year postinfection,and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence,FRDYVDQFYKT,was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4(+) T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost,and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus,our data support that IL-2-producing HIV-1-specific CD4(+) T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4(+) T cells rather than to the fixation of escape mutations at high frequencies. View Publication

Stem cell-based therapies have been reported in protecting cerebral infarction-induced neuronal dysfunction and death. However,most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here,we used human embryonic stem cell-derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen-glucose deprivation (OGD) for 1 h. For treatment group,different exosomes were derived from neuron,embryonic stem cell,neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD,while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD-induced neuronal death,Mammalian target of rapamycin (mTOR),pro-inflammatory and apoptotic signaling pathway changes,as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD-induced neuronal death and dysfunction in human embryonic stem cell-derived neurons,potentially through their modulation on mTOR,pro-inflammatory and apoptotic signaling pathways. View Publication