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

Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder,which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD,existing pharmaceutical can only relieve its symptoms. Results: Here,induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene,and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro,as evidenced by mutant huntingtin protein aggregation,increased number of lysosomes/autophagosomes,nuclear indentations,and enhanced neuronal death during cell aging. Moreover,store-operated channel (SOC) currents were detected in the differentiated neurons,and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally,the quinazoline derivative,EVP4593,reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Conclusions: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks,providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug. [ABSTRACT FROM AUTHOR] View Publication

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus,we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors,has a normal karyotype,expresses pluripotency markers and can differentiate into three germ layers in vivo. View Publication

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently,neutrophils were considered homogeneous and transcriptionally inactive cells,but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However,the use of scRNA-seq to characterize neutrophils has proven technically difficult,explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline,rather than to the existing scRNA-seq chemistries,can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells,which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells,through a transitional phenotype (Nh1),into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level,we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity,with potential implications for the characterization of neutrophils in health and disease. View Publication

BackgroundMetastasis,the spread,and growth of malignant cells at secondary sites within a patient’s body,accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10–16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses,which represents a critical barrier for the development of efficient therapies for affected breast cancer patients.MethodsPrevious research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM,we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP,other cell lines of MCF-7,HCC-1806,and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid.ResultsWe found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover,MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids.ConclusionsOur co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture. View Publication

Cardiac differentiation of human induced pluripotent stem cells is readily achievable,yet derivation of mature cardiomyocytes has been a recognized limitation. Here,a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5,GATA4,TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further,Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition,transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus,Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment. View Publication

Peptide-based supramolecular nanostructures offer a versatile platform with substantial promise for clinical translation in regenerative medicine. These systems allow for the incorporation of biologically active sequences and can be engineered to modulate tissue-specific parameters such as stiffness,diffusivity,and biodegradability. We developed here a bioactive supramolecular nanostructure containing a peptide designed based on glial cell-derived neurotrophic factor. These nanostructures form scaffolds that mimic important trophic effects provided by this growth factor on iPSC-derived human dopaminergic neurons. Our in vitro data show that the nanostructures promote cell viability,confer neuroprotection against 6-hydroxydopamine toxicity,enhance neuronal morphology,facilitate electrophysiological maturation,and induce genes involved in neuronal survival. We also found that the scaffold promoted axonal extension in midbrain human organoids. These findings suggest that the supramolecular system could be useful to improve outcomes in cell-based therapies for Parkinson’s disease,where progressive dopaminergic degeneration is a hallmark. View Publication

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells,whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN,including pre-TFH cells,memory TFH cells,germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages,including multiple responses targeting internal influenza virus proteins,and found that each TFH cell state was attainable within a clonal lineage. Thus,human TFH cells form a durable and dynamic multitissue network. Schattgen et al. profiled the subsets and clonality of CD4+ TFH cells in the blood and lymph nodes of human volunteers who received two influenza vaccines 1 year apart to characterize their dynamics and clonal evolution over 2 years. View Publication

If iPS cells can be established easily and efficiently using freshly collected blood cells,it will enhance regenerative and personalized medicine. While reports of iPS derivation from blood-derived endothelial progenitor cells using RNA have been documented,none have been reported from peripheral blood-derived mononuclear cells (PBMCs). In this study,we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4,which suppresses p53,improved reprogramming efficiency. Subject terms: Reprogramming,Induced pluripotent stem cells View Publication

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN),the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4,a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein,thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells,the developmental ontology of the limb bud,or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs,lenalidomide,and pomalidomide,dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide,lenalidomide,and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen,all-trans retinoic acid (atRA). Finally,disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide,lenalidomide,and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents. View Publication

Blood oxygen saturation (SaO2) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO2 affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO2: 75{\%} to 85{\%}) than in the other 2 groups (SaO2 {\textless}75{\%} or {\textgreater}85{\%}). In iPSC-CMs 15{\%} and 10{\%} oxygen (O2) treatment increased cell cycle markers,whereas 5{\%} and rapid change of O2 decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs. View Publication

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity,yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here,we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation,which revealed distinct gene networks individually regulated by FOXP3 and PRDM1,in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2,to our knowledge not previously implicated in Treg cell function,coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling,we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies. View Publication