搜索结果: 'gmp t cell activator'

NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function,accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I-dependent manner. The presence of Ly49C impacted the NK cell repertoire in a β2m-dependent manner� View Publication

The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs,elutriation and temperature sensitive mutants. Importantly,FUCCI reporters allow cell cycle events in dynamic systems,such as differentiation,to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report,we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible. View Publication

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced,but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5),which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9),is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However,LSD1 is not essential for the maintenance of ESC identity. Instead,ESCs lacking LSD1 activity fail to differentiate fully,and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers,LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex,which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation,which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. View Publication

The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle,proliferation rate and stress. Changes in nucleolar size,number,chemical composition,and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large,well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed,but varied more in intensity,forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus,a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type,but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization,in the evaluation of drug and experimental effects on the nucleolus,and in clinical diagnostics and prognostics. View Publication

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However,current QC methods,reliant on molecular staining,pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC),a machine learning-driven,multidimensional,high-content,and high-throughput flow cytometry approach,in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975),T cells and non-T cells in white blood cells (ROC-AUC = 0.969),activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990),and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers,assess viability,identify specific cell subsets or phenotypic states,and remove impurities during cell therapy manufacturing. Thus,LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Subject terms: Biotechnology,Cell biology,Immunology,Biomedical engineering View Publication

Quantification of subcellular structures such as nuclei and cytoplasmic proteins using staining methods based on fluorescent dyes or fluorescently tagged antibodies are widely used in scientific research. Accurate high-throughput quantitation of these assays can be time consuming and challenging. Here,we present our FIJI based Semi-Automated counting Macro termed SAM,and we validate its accuracy against manual counting and other automated counting methods. By introducing this automated quantification tool,we aim to contribute to the ongoing efforts to enhance the reliability,efficiency,and standardization of immunostaining analysis in the field of diabetes research and beyond.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12144-x. View Publication

Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor,Y15,was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines,profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878,and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines,TPC1,BCPAP and K1,and 380,109,and 74 genes were significantly textgreater2-fold changed with Y15 treatment,respectively. Common up-regulated genes were involved in apoptosis,cell cycle,transcription and heat shock; down-regulated genes were involved in cell cycle,cell-to-cell interactions,and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes textgreater4-fold (ptextless0.05). Among genes changed 4 fold,11 were shared between the treatments,including those involved in metabolism,cell cycle,migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib,Sorafenib,Pazopanib,and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor,detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines,demonstrated differential response changes with Y15 and PF-04554878 treatment,and showed the synergy of Y15 with PF-04554878,Cabozantinib,Sorafenib,Pazopanib,and Sunitinib. View Publication

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues,large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells,the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs,fibroblasts,and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival,while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs. View Publication

B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1(+) B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines,application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain -3 (TIM-3) on B cells,a novel molecule that has recently been described to induce anergy in T cells. Interestingly,elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall,these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. View Publication

Polymorphonuclear neutrophils (PMNs) can contribute to the regulation of the host immune response by crosstalk with innate and adaptive leukocytes,including NK cells. Mechanisms by which this immunoregulation process occurs remain incompletely understood. Here,we focused on the effect of human neutrophil-derived serine proteases on NKp46,a crucial activating receptor expressed on NK cells. We used flow cytometry,Western blotting,and mass spectrometry (MS) analysis to reveal that cathepsin G [CG; and not elastase or proteinase 3 (PR3)] induces a time- and concentration-dependent,down-regulatory effect on NKp46 expression through a restricted proteolytic mechanism. We also used a functional assay to demonstrate that NKp46 cleavage by CG severely impairs NKp46-mediated responses of NK cells,including IFN-γ production and cell degranulation. Importantly,sputa of cystic fibrosis (CF) patients,which have high concentrations of CG,also alter NKp46 on NK cells. Hence,we have identified a new immunoregulatory mechanism of neutrophils that proteolytically disarms NK cell responses. View Publication

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However,the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study,we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically,we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0,35,70,105 and 140 days),and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue,and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05),consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together,these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes. View Publication