搜索结果: 'gmp t cell activator'

While distinct stages of natural killer (NK) cell development have been defined,the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which,through contact-dependent mechanisms,promote the generation of mature,functional human NK cells from CD34(+) precursors. We show that developing NK cells undergo unique,developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells,and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells,which we term the developmental synapse. Finally,we identify a role for CD56 in developmental synapse structure,NK cell motility and NK cell development. Thus,we define the developmental synapse leading to human NK cell functional maturation. View Publication

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse,but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division,RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. View Publication

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen,we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study,we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore,we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition,deficiency in Kin17 affects the functionality of multiple DSB repair pathways,namely homologous recombination,non-homologous end-joining,and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR. View Publication

The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI. View Publication

Mutations causing loss of the NF-$\kappa$B regulator I$\kappa$BNS,result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming,we sought to investigate processes important for this transition using the bumble mouse strain,deficient for I$\kappa$BNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels,mitochondrial accumulation,increased OXPHOS and mROS production,along with a reduced capacity for autophagy,compared to wildtype B cells. Overall,our results demonstrate that PC differentiation in the absence of I$\kappa$BNS is characterized by excessive activation during early rounds of B cell division,increased mitochondrial metabolism and decreased autophagic capacity,thus improving our understanding of the role of I$\kappa$BNS in PC differentiation. View Publication

Becker muscular dystrophy (BMD) is caused by in-frame mutations in dystrophin gene,leading to progressive muscle weakness,and cardiac and respiratory complications. Currently,there is no cure. We have recently identified the importance of poly-ubiquitination in regulating dystrophin stability through the binding of lncRNA H19 to the dystrophin C-terminal zinc-finger domain (ZNF),inhibiting TRIM63-mediated poly-ubiquitination. We also demonstrated that BMD mutations lead to conformational changes in ZNF domain,reduced lncRNA H19 binding and increased dystrophin ubiquitination. Here we used BMD iPSCs to investigate the in vitro myogenic potential of BMD myogenic cells,as well as in vitro and in vivo studies to evaluate the therapeutic efficacy of three candidate molecules targeting dystrophin ubiquitination pathway. In vitro assays indicated significant deficiencies in myogenic cell differentiation of BMD iPSCs,including reduced proliferation,cell-cycle arrest,increased apoptosis,senescence,and membrane damage,and impaired myotube formation. In vivo engraftment demonstrated significant improvement in BMD iPSC myogenic cell survival and dystrophin expression in the animals treated with two molecules: a TRIM63 inhibitor and an ?-synuclein aggregation inhibitor. These findings provide promising evidence for the potential therapeutic efficacy of these ubiquitination pathway inhibitors to improve muscle progenitor cell survival and dystrophin expression in BMD patients. Graphical abstract Regulation of dystrophin stability via poly-ubiquitination is crucial in Becker muscular dystrophy (BMD). BMD mutations impair lncRNA H19 binding,increasing dystrophin ubiquitination. Darabi and colleagues’ studies,using BMD iPSCs and in vivo models,demonstrate that inhibiting TRIM63 or ?-synuclein aggregation improves myogenic cell survival and dystrophin expression,suggesting promising therapeutic avenues for BMD. View Publication

PurposeIsolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently,the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore,we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications.MethodsWe developed a novel,efficient,user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS,a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator,remove dead cells by 4′,6-diamidino-2-phenylindole (DAPI) staining,and eliminate non-specific fluorescence using a “dump” channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells,while multiple epithelial markers (EpCAM,EGFR,and Pan-Cytokeratin) and an epithelial–mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining,immuno-staining,and whole transcriptome amplification (WTA),followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications.ResultsThe mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs,the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq.ConclusionWe developed a sensitive,specific,flexible,and affordable CTC detection/isolation technique,which is scalable to larger patient cohorts,provides a snapshot of CTC heterogeneity,isolates live CTCs ready for downstream molecular analysis,and,most importantly,is suitable for developing countries. View Publication

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway,suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1,via manipulating multiple signaling pathways,induces epithelial cell transformation,MDSC (Myeloid-derived suppressor cells) accumulation,and pRCC development. Interestingly,we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1,a recently developed TEAD inhibitor,impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC. Deletion of upstream tumor suppressors of the Hippo/YAP pathway is frequent in papillary renal cell carcinoma (pRCC). Here,the authors employ a transgenic mouse model,single-cell transcriptomics and public genomic datasets to show that targeting hyperactivated YAP1 prevents neoplastic renal epithelial cell immune evasion and impairs the development of pRCC. View Publication

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however,the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here,we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins,including PDGFR$\alpha$,Sca1,and CD81. Notably,CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with $\alpha$V/$\beta$1 and $\alpha$V/$\beta$5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly,CD81 loss causes diet-induced obesity,insulin resistance,and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis. View Publication

MUC-1 mucin is considered to be aberrantly glycosylated in breast,ovary,and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation,we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1,MTSV1-7,and HB-2) derived from cells cultured from human milk,with three breast cancer cell lines (MCF-7,BT-20,and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1,and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern,with NeuAcalpha2-3Galbeta1-3GalNAc-ol,Galbeta1-3GalNAc-ol,and GalNAc-ol predominating; in contrast,MUC-1 from MTSV1-7 had more complex structures,including a number of disialo,core 2 species,i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler,and fewer,carbohydrate chains than MUC-1 from normal breast epithelial cells,and that these differences,combined or separately,explain the differential tumor specificity of some MUC-1 antibodies and T cells. View Publication

In functional cytometric studies,cultured cells are exposed to effectors (e.g. drugs),and the heterogeneity of cell responses are studied using cytometry techniques (e.g. image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity,(ii) the presence of a subpopulation with an elevated cytoprotective capacity and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies,such as measurements of population heterogeneity of cytotoxicity,chemosensitivity and radiosensitivity. This article is protected by copyright. All rights reserved. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication