搜索结果: 'gmp t cell activator'

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines,immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere,adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity,consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes,although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly,self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures,suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia,including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like,basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype,or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall,cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells,suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells,has utility as one of a suite of functional assays that provide a read-out of progenitor activity. View Publication

GABAergic interneurons are essential for neural circuit function,and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and,ultimately,for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production,with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons,we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders. View Publication

Peripheral blood was collected from a clinically diagnosed 72-year old male patient with later onset Alzheimer's disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for studying the pathological mechanism of Alzheimer's disease. View Publication

The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells,including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus,this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia. View Publication

The translocator protein 18 kDa (TSPO) is a multifunctional outer mitochondrial membrane protein associated with various aspects of mitochondrial physiology and multiple roles in health and disease. Here,we aimed to analyse the role of TSPO in the regulation of mitochondrial and cellular functions in a human neuronal cell model. We used the CRISPR/Cas9 technology and generated TSPO knockout (KO) and control (CTRL) variants of human-induced pluripotent stem cells (hiPSCs). In a multimodal phenotyping approach,we investigated cellular and mitochondrial functions in neural progenitor cells (NPCs),astrocytes,and neurons differentiated from hiPSC CTRL and TSPO KO cell lines. Our analysis revealed reduced mitochondrial respiration and glycolysis,altered Ca2+ levels in the cytosol and mitochondrial matrix,a depolarised MMP,and increased levels of reactive oxygen species,as well as a reduced cell size. Notably,TSPO deficiency was accompanied by reduced expression of the voltage-dependent anion channel (VDAC). We also observed a reduced TSPO and VDAC expression in cells derived from patients suffering from major depressive disorder (MDD). Considering the modulatory function of TSPO and the similar functional phenotype of cells derived from patients with depression,we discuss a role of TSPO in the etiology or pathology of MDD. In summary,our findings indicate a general impairment of mitochondrial function in TSPO knockout (KO) cells. This deepens our insight into the intricate role of TSPO in a range of physiological and pathological processes. View Publication

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

The cell surface antigen,epithelial glycoprotein,defined by the monoclonal antibody HEA 125,is expressed on virtually all epithelial cell membranes but not on mesodermal or neural cell membranes. The cDNA encoding epithelial glycoprotein was isolated by HEA 125 antibody enrichment of colon tumor cDNA expressed transiently in COS cells. The sequence of the epithelial glycoprotein antigen is identical to the cell membrane protein recognized by the monoclonal antibody KS 1/4 and is homologous to the tumor-associated antigen GA733. These proteins share sequence homology to nidogen,an extracellular matrix component that appears to participate in cell-matrix adhesion. These proteins also share a homologous domain found in the B1 chain of laminin,a matrix adhesion protein,and placental protein 12,an insulin-like growth factor I binding protein secreted during pregnancy that has been implicated in regulation of fetal growth. This common domain is also repeated multiple times within the thyroglobulin precursor. These findings suggest epithelial glycoprotein is a cell surface molecule involved in cell-cell or cell-matrix interaction. View Publication

Natural killer (NK) cells play a crucial role in immune surveillance by recognizing and eliminating tumor cells. However,tumors employ various mechanisms to evade NK cell-mediated immunity. NKp30 is a potent activating receptor on NK cells,but its function can be inhibited by specific ligands secreted by cancer cells. Here,we identified dipeptidase 1 (DPEP1) as a novel ligand for NKp30 in KM12C colon cancer cells,using co-immunoprecipitation,confocal microscopy,and flow cytometry. We examined how the DPEP1–NKp30 interaction affects NK cell activity and found that NK cytotoxicity increased in KM12C cells with DPEP1 knockdown but was significantly reduced in HCT116 cells overexpressing DPEP1. We further demonstrated that DPEP1 is secreted via extracellular vesicles and that its interaction with NKp30 suppressed the expression and secretion of perforin 1,granzyme B,CD107a,and interferon-γ in NK92 cells. In a xenograft mouse model treated with NK92 cells,tumors derived from HCT116/DPEP1 cells were significantly larger than those from HCT116/mock cells. Using peripheral blood-derived human NK cells,we confirmed that DPEP1 inhibited both cytotoxicity and granzyme B secretion. These findings suggest that disrupting the DPEP1–NKp30 interaction may enhance NK cell-mediated cytotoxicity and represent a novel therapeutic strategy for cancer immunotherapy. The online version contains supplementary material available at 10.1038/s41598-025-18475-z. View Publication

Early diagnosis of psoriatic arthritis (PSA) is important for successful therapeutic intervention but currently remains challenging due,in part,to the scarcity of non-invasive biomarkers. In this study,we performed single cell profiling of transcriptome and cell surface protein expression to compare the peripheral blood immunocyte populations of individuals with PSA,individuals with cutaneous psoriasis (PSO) alone,and healthy individuals. We identified genes and proteins differentially expressed between PSA,PSO,and healthy subjects across 30 immune cell types and observed that some cell types,as well as specific phenotypic subsets of cells,differed in abundance between these cohorts. Cell type-specific gene and protein expression differences between PSA,PSO,and healthy groups,along with 200 previously published genetic risk factors for PSA,were further used to perform machine learning classification,with the best models achieving AUROC ?? 0.87 when either classifying subjects among the three groups or specifically distinguishing PSA from PSO. Our findings thus expand the repertoire of gene,protein,and cellular biomarkers relevant to PSA and demonstrate the utility of machine learning-based diagnostics for this disease. View Publication

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However,few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol. DESIGN: In vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB),which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined. RESULTS: Both hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1,TGF-beta2,TGF-beta 3,TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures,TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol,TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures,TGF-beta1 inhibited osteocalcin production. CONCLUSIONS: TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol,it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production,other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo. View Publication

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with non-integrative constructs. Numerous studies,however,including those describing disease models,are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs,but in mesenchymal and endothelial iPSC derivatives,the transgenes experienced random up-regulation of Nanog and c-Myc. Additionally,we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies,which utilize cellular products derived from iPSCs generated with retro- or lentiviruses,should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work,however,is to communicate the possibility of transgene reactivation in retro- or lenti- iPSC derivatives and the associated loss of cellular fidelity in vitro,which may impact the outcomes of disease modeling and related experimentation. View Publication