搜索结果: 'gmp t cell activator'

Three different label-free,minimally invasive,live single cell analysis techniques were used to characterize embryonic stem cells,and the hepatocytes into which they were differentiated. Atomic Force Microscopy measures the cell's mechanical properties,Raman spectroscopy measures its chemical properties,and dielectrophoresis measures the membrane's capacitance. We were able to assign cell type of individual cells with accuracies of 96.5% (Atomic Force Microscopy),92.5 % (Raman spectroscopy),and *** % (Dielectrophoresis). These techniques,used either independently or in combination,offer label-free methods to study individual living cells. Although they can be applied to any phenotypical or environmental change,these techniques have most potential in human cell therapies where the use of biomarkers is best avoided. If all three properties are independent,then a combined accuracy of *** % can be achieved in cell characterization. We suggest how these methods could be combined into one microfluidic chip for cell sorting,and how they can be applied to cell culture. View Publication

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However,the cells generated within organoids and the extent to which they recapitulate the regional complexity,cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells,which are related to endogenous classes,including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months),allowing for the establishment of relatively mature features,including the formation of dendritic spines and spontaneously active neuronal networks. Finally,neuronal activity within organoids could be controlled using light stimulation of photosensitive cells,which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli. View Publication

Lymphoblasts from 93 children with acute lymphoblastic leukemia (ALL) were characterized by immunologic cell surface markers. These patients were treated on a single protocol,featuring adriamycin therapy during remission,and have been followed from 2 to 6.5 yr (median 4 yr). Three classes of patients were defined serologically: HTA+ Ia- CALLA-,Ia+ CALLA+ HTA-,and Ia+ CALLA- HTA-. Disease-free survival and sites of relapse were assessed within immunologic subsets. Similar to the findings of others,T-cell (HTA+ Ia-) patients fared poorly as compared to non-T-cell (Ia+ HTA-) patients (median disease-free survival was 12 and 47 mo. respectively; p = 0.0004). The majority of relapses in the HTA+ patients occurred at extramedullary sites. Late testicular relapse was rare among Ia+ patients. In addition,the common ALL antigen" (CALLA) may identify a relatively favorable subset within the Ia+ population. The prognostic value of the immunologic markers was compared with traditional clinical factors. There was much overlap between HTA+� View Publication

The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM,however,resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,000 nM) had an increase in survival compared with cells treated with lower concentrations of the drug. Prolonging the time of exposure of cells to paclitaxel from 24 to 72 h increased cytotoxicity from 5 to 200 fold in different cell lines. Exponentially growing cells were more sensitive to paclitaxel than were cells in the plateau phase of growth. Cremophor EL,the diluent in which the clinical preparation of paclitaxel is formulated,antagonised paclitaxel at concentrations of 0.135% (v/v). These data suggest that paclitaxel will be most effective clinically when there is prolonged exposure of tumour to the drug. Further,it appears that modest concentrations (i.e.,50 nM) should be as effective as higher concentrations of paclitaxel. Finally,we have noted that Cremophor EL is a biologically active diluent and,at high concentrations (0.135% v/v),can antagonise paclitaxel cytotoxicity. View Publication

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood,the more mature CD56(dim) NK cell efficiently kills malignant targets at rest,whereas the less mature CD56(bright) NK cells cannot. In this study,we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this,forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity,and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet,as in humans,did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation,polarization of the microtubule organizing center,and the convergence of cytolytic granules. In summary,our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells. View Publication

Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues,such as stem cells and cancer tumors. However,a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability,confounds studies with therapeutic outcomes for cultured cells,and represents a major cost associated with cell culture. Here we report a commercially available serum-free,albumin-free,and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages,fibroblasts,as well as specific cancer cell lines; without the use of contaminants such serum,albumin,and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture. View Publication

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent,submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages,reduces tumor cell proliferation and angiogenesis,and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33,an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11,which is required for the growth of gastric cancers in mice. Accordingly,ablation of the cognate IL-33 receptor St2 limits tumor growth,and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2,Ccl3,and Il6. Conversely,genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore,tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. View Publication

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here,we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific,CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells,which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons,we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1?Mb,including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type,implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner. Scalable CRISPRa screening of cis-regulatory elements in non-cancer cell lines has proved challenging. Here,the authors describe a scalable,CRISPR activation screening framework to identify regulatory element-gene pairs in diverse cell types including cancer cells and neurons. View Publication

SummaryCell size is a crucial physical property that significantly impacts cellular physiology and function. However,the influence of cell size on stem cell specification remains largely unknown. Here,we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly,cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore,the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements,we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically,the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation,which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together,our study has established a novel connection between cell size diminution and DE differentiation,which is mediated by AMOT nuclear translocation. Additionally,our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation. Graphical abstract Highlights•Cell size decreases during the differentiation of human pluripotent stem cells into endoderm•Hypertonic pressure is conducive to the differentiation of human definitive endoderm•Actomyosin contributes to both size diminution and endoderm promotion under hypertonic pressure•Cell size diminution represses YAP activity via promoting AMOT nuclear translocation Jiang and colleagues show that cell size exhibits a gradual decrease during human endoderm differentiation. The application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced endoderm differentiation. This enhancement is reliant on actomyosin activity and achieved by promoting the nuclear translocation of AMOT,thereby repressing YAP activity. View Publication

Alternative splicing events are a major causal mechanism for complex traits,but they have been understudied due to the limitation of short-read sequencing. Here,we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer’s disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms,providing insight into the human genome evolution. In addition,some of the isoforms are expressed in a cell-type specific manner,whose alternative 3’-UTRs usage contributes to their specificity. Further,we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing. This paper unveils the complexity of human immune cell splicing,highlighting cell-specific isoforms and establishing connections between alternative splicing and complex traits. These findings have implications for understanding diseases and the evolution of the genome. View Publication

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation,potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here,we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF,containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically,UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally,XF and SF-cultured UCMSC have comparable adipogenic,osteogenic,and endothelial differentiation potential. Interestingly,the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression,transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation,and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together,GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine. View Publication

$\$-Thalassemia ($\$-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in $\$-globin gene (HBB). It is estimated that 4.5% of the population in the world carry $\$-Thal mutants (1),posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However,homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here,we describe a robust process combining efficient generation of integration-free $\$-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly,the gene-corrected $\$-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal $\$-globin. Our studies provide an efficient and universal strategy to correct different types of $\$-globin mutations in $\$-Thal iPSCs for disease modeling and applications. View Publication