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

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

MicroRNAs and chromatin remodeling complexes represent powerful epigenetic mechanisms that regulate the pluripotent state. miR-302 is a strong inducer of pluripotency,which is characterized by a distinct chromatin architecture. This suggests that miR-302 regulates global chromatin structure; however,a direct relationship between miR-302 and chromatin remodelers has not been established. Here,we provide data to show that miR-302 regulates Brg1 chromatin remodeling complex composition in human embryonic stem cells (hESCs) through direct repression of the BAF53a and BAF170 subunits. With the subsequent overexpression of BAF170 in hESCs,we show that miR-302's inhibition of BAF170 protein levels can affect the expression of genes involved in cell proliferation. Furthermore,miR-302-mediated repression of BAF170 regulates pluripotency by positively influencing mesendodermal differentiation. Overexpression of BAF170 in hESCs led to biased differentiation toward the ectoderm lineage during EB formation and severely hindered directed definitive endoderm differentiation. Taken together,these data uncover a direct regulatory relationship between miR-302 and the Brg1 chromatin remodeling complex that controls gene expression and cell fate decisions in hESCs and suggests that similar mechanisms are at play during early human development. 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

Insights into the expression of pacemaker-speci�?c markers in human induced pluripotent stemcell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentia-tion and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date,no study hasdirectly assessed gene expression in each pacemaker-,atria-,and ventricular-like cardiomyocytesubtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytescan only be identi�?ed by action potential pro�?les. Traditional acquisition of action potentialsusing patch-clamp recordings renders the cells unviable for subsequent analysis. We circum-vented these issues by acquiring the action potential pro�?le of a single cell optically followedby assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the �?rst time revealed expression of proposed pacemaker-speci�?cmarkers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet(Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expressionwas found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- andventricular-like subtypes but its downregulation over time in all subtypes diminished the differ-ences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statisti-cally different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentiallyexpressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes,thesemarkers alone are insuf�?cient in identifying hiPSC-derived pacemaker-like cardiomyocytes. View Publication

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs),representing a potential unlimited cell source for disease modeling,cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined,their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed,the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions,hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized,and therefore not representative of native conditions. Here,we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV),resembling the native human V anisotropic ratio (AR=LCV/TCV=1.8-2.0). Importantly,the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17-23% of aligned preparations,thereby providing a physiological baseline for assessing arrhythmogenicity. As such,promotion of pro-arrhythmic effect (e.g.,spatial dispersion by ?? adrenergic stimulation) could be better predicted. Mechanistically,such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion,not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors),but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias. ?? 2013. View Publication

While readily achieved in cell lines,the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here,we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein. View Publication

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus,restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis,in addition to standard clinical care. Herein,we evaluated the effects of the SPM lipids,lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ),on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production,and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst,a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls,indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin,which plays a central role in clot activation and atherosclerosis. Finally,LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy,as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function,LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual. View Publication

SummaryCytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore,while mutations in TARDBP (encoding TDP-43) have been associated with ALS,the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9,we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C,two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization,phosphorylation,solubility,or aggregation compared with isogenic control MNs. However,our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures,as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively,our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease. Graphical abstract Highlights•Mutant MNs maintain viability but are more vulnerable to cellular stress•Mutant MNs do not show TDP-43 pathology•TDP-43 variants lead to a progressive decline in spontaneous neuronal activity•Functional impairments are accompanied by abnormal synaptic marker expression Molecular neuroscience; Cellular neuroscience View Publication

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

The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics,force generation,and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness,we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly,our approach has the potential for broad application in the study of cardiac disease,drug discovery,and cardiotoxicity screening. View Publication

With the advent of induced pluripotent stem cells and directed differentiation techniques,it is now feasible to derive individual-specific cardiac cells for human heart tissue engineering. Here we report the generation of functional engineered human cardiac patches using human induced pluripotent stem cells-derived cardiac cells and decellularized natural heart ECM as scaffolds. The engineered human cardiac patches can be tailored to any desired size and shape and exhibited normal contractile and electrical physiology in vitro. Further,when patching on the infarct area,these patches improved heart function of rats with acute myocardial infarction in vivo. These engineered human cardiac patches can be of great value for normal and disease-specific heart tissue engineering,drug screening,and meet the demands for individual-specific heart tissues for personalized regenerative therapy of myocardial damages in the future. View Publication