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Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study,NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-β-receptor-knockout (PDGFR-β(-/-)) mice of postnatal day 1 (P1) and P28,and the roles of PDGFR-β were examined in these cells. In PDGFR-β-preserving control NSPCs,stem cell activities,such as numbers and diameters of secondary neurospheres,cell proliferation and survival rates,were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-β(-/-) NSPCs,most of these parameters were decreased as compared with age-matched controls. Among them,the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-β(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased,and noggin was increased in P1 PDGFR-β(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-β(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction,where phosphorylated PDGFR-β was co-localized with neuronal and astrocyte differentiation markers. In controls,the neuronal differentiation was decreased,and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls,neuronal differentiation was reduced in P1 PDGFR-β(-/-) NSPCs,whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-β signaling is important for the self-renewal and multipotency of NSPCs,particularly in neonatal NSPCs. BDNF,FGF2,and noggin may be involved in the effects of PDGFR-β signaling in these cells. Accordingly,the activation of PDGFR-β in NSPCs may be a novel therapeutic strategy of neurological diseases. View Publication

BACKGROUND Human microRNAs (miRNAs) have diverse functions in biology,and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS To examine whether there were any changes in mutation status in cells treated with 520d-5p,we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS 520d-5p induced a shift towards a wild type or non-malignant phenotype,which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further,520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS Cancer cell genomic mutations cannot be repaired in most contexts. However,these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine. View Publication

Label-free detection of biological events at single-cell resolution in the brain can non-invasively capture brain status for medical diagnosis and basic neuroscience research. NADH is an universal coenzyme that not only plays a central role in cellular metabolism but may also be used as a biomarker to capture metabolic processes in brain cells and structures. We have developed a new label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H in living cells. The imaging depth of NAD(P)H in tissues with all-optical methods is limited to ~100??m in brain tissue by the strong absorption of the near-ultraviolet fluorescence. Here,acoustic detection of the thermal signature of multi-photon (three-photon) excitation of NAD(P)H,a low quantum yield fluorophore,allows detection at an unprecedented depth while the focused excitation ensures high spatial resolution. We validated the photoacoustic detection of NAD(P)H by monitoring an increase in intracellular NAD(P)H in HEK293T cells and HepG2 cells incubated in NADH solution. We also demonstrated the detection of endogenous NAD(P)H photoacoustic signals in brain slices to 700 ?m depth and in cerebral organoids to 1100 ?m depth. Finally,we developed and demonstrated simultaneous photoacoustic and optical imaging of NAD(P)H in brain cells with a real-time image acquisition and processing pipeline. This approach could open a new door to monitor brain metabolic changes during development and disease,and changes due to neuronal activity,at single-cell level deep in the brains of both humans and animals. Label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H of neurons in brain slices and cerebral organoids. View Publication

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

UNLABELLED Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however,all patients ultimately still progress from their disease. Lack of CAR T-cell persistence,impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile,fitness,and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts,higher CD4/CD8 ratio,and expanded na{\{i}}ve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells decreased central memory phenotype and increased checkpoint inhibitory markers compared with HD-derived products which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. SIGNIFICANCE Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately patients still relapse. In this study we propose to use T cells from HD volunteers which have a stronger T-cell fitness higher cancer killing capacity and are ready to be administered when needed." View Publication

Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors,and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment,such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2,p-value = 4.9 × 10(-5)) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1,p-value = 8.6 × 10(-5)),showed transcriptional variation consistent with their known developmental roles. However,these analyses highlighted many other genes with stage-specific expression patterns,some of which may be novel drivers or markers of islet development. For example,the leptin receptor gene,LEPR,was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5,p-value = 2.0 × 10(-12)),suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes,including INS,ABCC8,and GLP1R,and enrichment of relevant GO-terms (e.g. insulin secretion"; odds ratio = 4.2 View Publication