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

BackgroundLung cancer,particularly non-small cell lung cancer (NSCLC),has high recurrence rates and remains a leading cause of cancer-related death,despite recent advances in its treatment. Emerging therapies,such as chimeric antigen receptor (CAR)-T cell therapy,have shown promise but face significant challenges in targeting solid tumors. This study investigated the potential of combining receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeting CAR-T cells with ferroptosis inducers to promote ferroptosis of tumor cells and enhance anti-tumor efficacy.MethodsRNA-seq data and immunofluorescence analysis of relapsed NSCLC patient samples were used to explore ROR1 expression. In addition,ROR1-targeting CAR-T cells were developed to assess cytotoxic activity against ROR1+ tumor cells,and the effect of cytokine stimulation on their efficacy was evaluated. Lipidomics,immunofluorescent histochemistry,and western blotting were used to explore the observed effects. Ferroptosis indicators,including levels of reactive oxygen species,were used to detect the combined effect of CAR-T cells and ferroptosis-inducing drugs. Finally,tumor-bearing mice were used to validate the in vivo efficacy of the combination therapy strategy.ResultsTumor cells treated with ferroptosis inducers showed increased sensitivity to Interferon gamma (IFN-γ) secreted by ROR1 CAR-T cells. Furthermore,ROR1 CAR-T cells enhanced the production of phosphatidylcholine with diacyl-polyunsaturated fatty acid tails (PC-PUFA2) by working in tandem with IFN-γ. This enhancement promoted the expression of acyl-CoA synthetase long chain family member 4 (ACSL4),which in turn strengthened the overall anti-tumor response.ConclusionsCombining ROR1 CAR-T cells with ferroptosis inducers enhanced anti-tumor efficacy in NSCLC by promoting ferroptosis through increased lipid peroxidation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40364-025-00730-0. View Publication

Memory T cells are a highly heterogeneous collection of antigen-experienced cells that undergo dynamic adaptations upon antigen re-encounter and environmental signals. This heterogeneity hinders studies on memory T cell durability and age-related dysfunction. Using chronic Epstein-Barr virus (EBV) infection and barcode-enabled antigen tracing,we assess the influence of age on memory states at the level of single antigen-specific CD8+ T cells. In young adults (<40 years),EBV-specific CD8+ T cells recognizing different antigenic peptides assume divergent preferred differentiation phenotypes. In older adults (>65-years),antigen-specific cells show largely distinct phenotypic and transcriptomic aging trajectories. Common to many albeit not all antigen-specific populations are maintained TCR diversity,gained natural killer cell-like,innate signatures and lost stem-like features while no evidence is seen for cellular senescence or exhaustion. TCR avidity contributes to these phenotypic differences and aging-related changes. Collectively,our data uncover divergent antigen-guided aging shifts in memory T cell phenotypes,which are informative for antigen selection in optimizing vaccine design and adoptive T cell therapy. Homeostasis of memory T cells is modulated by each antigen encounter,thereby creating a heterogeneous population preventing precise tracking. Here,the authors use barcode-assisted tracing of Epstein-Barr virus-specific CD8+ memory T cells of young and older individuals to find antigen-guided,clonally divergent aging trajectories. View Publication

Ageing-dependent low-grade inflammation is a hallmark of central nervous system (CNS) diseases. Vascular and immune abnormalities are implicated in the progression of gliomas and occur in the early stages of Alzheimer's disease (AD); however,the mechanisms by which these alterations manifest in the brain parenchyma remain unclear. Using RNAseq,scRNAseq,bioinformatics tools and a cohort of patients with glioma and Alzheimer's disease for validation of results,we have established an analysis of blood–brain barrier (BBB) dysfunction and neuron loss. A mouse model for glioblastoma pathology was also used that reversed BBB disruption and neuron loss,with the incorporation of the IDH mutation. Finally,we established a characterization of the relevant immune populations with an IHC analysis and transcriptional profile. In this study,molecular analyses of the brain ecosystem revealed that blood–brain barrier dysfunction and neuronal synapse integrity exhibit significant threshold-dependent changes that correlate directly and inversely,respectively,with brain ageing (significant changes at 57 years) and the progression of AD and gliomas (survival of 1525 vs 4084 days for patients with High vs Low BBB dysfunction). Using human samples and mouse models,we identified immunoageing processes characterized by an imbalance between pro-inflammatory and anti-inflammatory signals. This dysregulation promotes the extravasation of monocyte-derived macrophages (85% increase of cells),particularly those with a suppressive phenotype,alongside an increase in inflammatory cytokine levels. Notably,our data show that vascular normalization in a glioma model can reverse neuronal loss and attenuate the aggressiveness of the tumours. Finally,tumour development can be prevented by reactivating the ageing immune system. We propose that the ageing brain represents a common,BBB dysfunction-associated process driving chronic inflammation. This inflammation is regulated by TREM2+/TIM3+ suppressive myeloid cells,which play a central role in disease progression. Our findings suggest that targeting these pathways could offer therapeutic strategies to mitigate CNS pathologies linked to ageing,characterized by toxic neuroinflammation and myeloid dysfunction. This study was funded by ISCIII and co-funded by the European Union. View Publication

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation,vasculopathy,and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc,playing key roles in disease pathogenesis. However,the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes,B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC),showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1 flox/flox LysMCre+/+ mice,and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I,independent of TGF$\beta$ upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation,to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc,the top most upregulated pathways were hallmark IFN-$\gamma$ and IFN-$\alpha$ response. Additionally,several genes previously linked to SSc pathogenesis and fibrosis in general were also induced,including CCL2,CCL7,MMP12,and CXCL10. ANKRD1,a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients,with higher expression of profibrotic markers and activation of interferon responsive genes,thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis. View Publication

Use of cisplatin,a chemotherapeutic agent,is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1,2,3,8,and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced,and except for caspase 1,all the other caspases tested were activated. Z-VAD,the broad-spectrum inhibitor of caspases,prevented caspase activation and apoptosis,but not c-myc and p53 induction. On the other hand,N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity. View Publication

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate,termed BODIPY trade mark -aminoacetaldehyde (BAAA),and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining,termed the SSCloALDHbr population,was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU,(2) expand in primary and secondary LTC,and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation,the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P textless 0.015 and P textless 0.003 respectively). In summary,peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation. View Publication

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting,we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC(+)) and epithelial-specific antigen (ESA(+)) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC(-)/ESA(+)). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins,claudin-1 and occludin,and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures,the MUC(+)/ESA(+) epithelial cell line was luminal epithelial restricted in its differentiation repertoire,the suprabasal-derived MUC(-)/ESA(+) epithelial cell line was able to generate itself as well as MUC(+)/ESA(+) epithelial cells and Thy-1(+)/alpha-smooth muscle actin(+) (ASMA(+)) myoepithelial cells. The MUC(-)/ESA(+) epithelial cell line further differed from the MUC(+)/ESA(+) epithelial cell line by the expression of keratin K19,a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane,the MUC(+)/ESA(+) epithelial cell line formed acinus-like spheres. In contrast,the MUC(-)/ESA(+) epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus,MUC(-)/ESA(+) epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting,but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific,but not primed-specific,proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus,identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting. View Publication

To better understand transcriptional regulation during human oogenesis and preimplantation development,we defined stage-specific transcription,which highlighted the cleavage stage as being highly distinctive. Here,we present multiple lines of evidence that a eutherian-specific multicopy retrogene,DUX4,encodes a transcription factor that activates hundreds of endogenous genes (for example,ZSCAN4,KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably,mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells,measured here by the reactivation of '2C' genes and repeat elements,the loss of POU5F1 (also known as OCT4) protein and chromocenters,and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus,we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state. View Publication

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo,we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood,spleen,and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes,which downregulate GFP expression on differentiation into macrophages in this model,CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages,allowing continued cell tracking during resolution of inflammation. In summary,this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. View Publication