技术资料

Prenatal alcohol exposure (PAE) affects embryonic development,causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation,we used an in vitro model with subchronic moderate (20 mM) and severe (70 mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analyzed genome-wide gene expression (mRNA sequencing),DNA methylation (EPIC Illumina microarrays) and metabolome (non-targeted LC-MS) of the endodermal,mesodermal and ectodermal cells. The largest number of ethanol-induced alterations were observed in endodermal cells,whereas the most prominent changes were in ectodermal cells. Methionine metabolism and genes of the main signaling pathways involved in gastrulation and body patterning were affected by ethanol in all germ layers. Many of the altered genes,including BMP4,FGF8,SIX3 and LHX2,have previously been associated with PAE and phenotypes of FASD,like defects in heart and corpus callosum development as well as holoprosencephaly. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD. View Publication

Tumor-initiating cells (TICs) share features and regulatory pathways with normal stem cells,yet how the stem cell niche contributes to tumorigenesis remains unclear. Here,we identify CXCR4 + macrophages as a niche population enriched in normal mammary ducts,where they promote the regenerative activity of basal cells in response to luminal cell-derived CXCL12. CXCL12 triggers AKT-mediated stabilization of β-catenin,which induces Wnt ligands and pro-migratory genes,enabling intraductal macrophage infiltration and supporting regenerative activity of basal cells. Notably,these same CXCR4 + niche macrophages regulate the tumor-initiating activity of various breast cancer subtypes by enhancing TIC survival and tumor-forming capacity,while promoting early immune evasion through regulatory T cell induction. Furthermore,a CXCR4 + niche macrophage gene signature correlates with poor prognosis in human breast cancer. These findings highlight the pivotal role of the CXCL12-CXCR4 axis in orchestrating interactions between niche macrophages,mammary epithelial cells,and immune cells,thereby establishing a supportive niche for both normal tissue regeneration and mammary tumor initiation. Subject terms: Cancer stem cells,Cancer microenvironment,Tumour immunology View Publication

Human airways contain specialized rare epithelial cells including CFTR-rich ionocytes that regulate airway surface physiology and chemosensory tuft cells that produce asthma-associated inflammatory mediators. Here,using a lung cell atlas of 311,748 single cell RNA-Seq profiles,we identify 687 ionocytes (0.45%). In contrast to prior reports claiming a lack of ionocytes in the small airways,we demonstrate that ionocytes are present in small and large airways in similar proportions. Surprisingly,we find only 3 mature tuft cells (0.002%),and demonstrate that previously annotated tuft-like cells are instead highly replicative progenitor cells. These tuft-ionocyte progenitor (TIP) cells produce ionocytes as a default lineage. However,Type 2 and Type 17 cytokines divert TIP cell lineage in vitro,resulting in the production of mature tuft cells at the expense of ionocyte differentiation. Our dataset thus provides an updated understanding of airway rare cell composition,and further suggests that clinically relevant cytokines may skew the composition of disease-relevant rare cells. Subject terms: Interleukins,Systems analysis,Differentiation,Sequencing View Publication

Chromothripsis,the chaotic shattering and repair of chromosomes,is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN),we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%,driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A,a serine threonine kinase,is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo,and represents a druggable axis. Collectively,these findings define chr. 21amp as a prognostic biomarker in BP-MPN,and link chromothripsis to a therapeutic target. Subject terms: Leukaemia,DNA sequencing View Publication

Influenza virus particularly affects those with chronic lung conditions such as Chronic Obstructive Pulmonary Disease (COPD). Airway epithelial cells are the first line of defense and primary target of influenza infection and release extracellular vesicles (EVs). EVs can transfer of biological molecules such as microRNAs (miRNAs) that can modulate the immune response to viruses through control of the innate and adaptive immune systems. The aim of this work was to profile the EV miRNAs released from bronchial epithelial cells in response to influenza infection and discover if EV miRNA expression was altered in COPD. Influenza infection of air-liquid interface (ALI) differentiated BCi-NS1.1 epithelial cells were characterized by analyzing the expression of antiviral genes,cell barrier permeability and cell death. EVs were isolated by filtration and size exclusion chromatography from the apical surface wash of ALI cultured bronchial epithelial cells. The EV miRNA cargo was sequenced and reads mapped to miRBase. The BCi sequencing results were further investigated by RT-qPCR and by using healthy and COPD primary epithelial cells. Infection of ALI cultured BCi cells with IAV at 3.6 x 10 6 IU/ml for 24 h led to significant upregulation of anti-viral genes without high levels of cell death. EV release from ALI-cultured BCi cells was confirmed using electron microscopy and detection of known tetraspanin EV markers using western blot and the ExoView R100 platform. Differential expression analyses identified 5 miRNA that had a fold change of >0.6: miR-155-5p,miR-122-5p,miR-378a-3p,miR-7-5p and miR-146a-5p (FDR<0.05). Differences between EV,non-EV and cellular levels of these miRNA were detected. Primary epithelial cell release of EV and their miRNA cargo was similar to that observed for BCi. Intriguingly,miR-155 expression was decreased in EVs derived from COPD patients compared to EVs from control samples. Epithelial EV miRNA release may be a key mechanism in modulating the response to IAV in the lungs. Furthermore,changes in EV miRNA expression may play a dysfunctional role in influenza-induced exacerbations of COPD. However,further work to fully characterize the function of EV miRNA in response to IAV in both health and COPD is required. View Publication

Glioblastoma (GBM) is a rapidly growing,aggressive brain tumor with very poor prognosis without currently effective therapies. The immunosuppressive nature of the tumor microenvironment (TME) in GBM hinders the development of effective tumor-eradicating immunotherapies. This hostile TME can be modulated by administering immune-activating cytokines in combination with agents inducing tumor cell death. To achieve these objectives,we sought to harness the cancer-selective cell death-inducing properties of an enhanced “Superkine” version of melanoma differentiation associated gene-7/interleukin-24,IL-24S,and the immune-activating properties of IL-15 to modulate the TME of GBM to maximize therapeutic outcomes. A fusion “Superkine” ( FSK ) comprised of IL‐24S linked to IL-15 was generated,and antitumor effects were evaluated when transduced by a type 5 adenovirus (Ad.5) in a GBM immunocompetent mouse tumor model. To target the delivery of Ad.5 FSK systemically,we employed an innovative approach of focused ultrasound (FUS) paired with microbubbles (MBs),FUS-DMB (FUS plus double MB),to safely transport the FSK engineered Ad.5 construct into mouse brain to overcome limitations of systemic viral delivery and selectivity of the blood-brain barrier. The FSK stimulated higher tumor regression and enhanced survival in vivo than the individual “Superkine” or cytokine in GBM cancer models. Apoptosis of GBM cells was induced,as well as increased tumor infiltration of T cells,dendritic cells,macrophages and natural killer (NK) cells. The antitumor-inducing activity of FSK is a consequence of induction of cancer-specific growth suppression and induction of apoptosis (IL-24S) as well as diverse effects on immune cells (IL-15 and IL-24S). Antibody neutralization indicates that a primary immune mediator of anticancer activity of FSK is through recruitment and activation of NK cells. Global cytokine analyses indicated no changes in inflammatory cytokines during therapy,suggesting that this strategy will be safe. In summary,treatment with an FSK,consisting of a fusion of IL-24S to IL-15,promotes GBM cell killing and remodeling of the TME by recruiting and activating immune cells supporting the feasibility of developing safe and effective cancer immunotherapeutic fusion proteins and selective delivery in the brain for the therapy of GBM. 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

Several years after the COVID‐19 pandemic,the impact of SARS‐CoV‐2 on immunity and the potential protective role of Bacillus Calmette–Guérin (BCG) vaccination through trained immunity remain a subject of investigation. This study aimed to determine the long‐term impact of SARS‐CoV‐2 on immune cells and the association between BCG vaccination,latent infections and COVID‐19 severity and sepsis progression. We conducted a prospective analysis of patients who recovered from mild/severe/critical COVID‐19 ( n = 97,3–17 months after COVID‐19) and sepsis patients ( n = 64). First,we assessed the impact of COVID‐19 and its severity on immune cell frequencies and expression of functional markers. Further,we analysed plasma titres of anti‐ Toxoplasma gondii /cytomegalovirus/BCG antibodies and their association with COVID‐19 severity and sepsis outcome. To examine monocyte responses to secondary challenge,monocytes isolated from COVID‐19 convalescent patients,BCG vaccinated and unvaccinated volunteers were stimulated with SARS‐CoV‐2 and LPS. Post‐COVID‐19 patients showed immune dysregulation regardless of disease severity characterised by altered expression of activation and functional markers in myeloid (CD39,CD64,CD85d,CD11b) and lymphoid cells (CD39,CD57,TIGIT). Strikingly,post‐critical COVID‐19 patients showed elevated expression of CD57 in CD8 + T cells compared to other severity groups. A trend toward improved outcomes in BCG‐seropositive COVID‐19/sepsis patients was observed,although this may be confounded by age differences between groups. In contrast,the monocyte response to stimulation appeared unaffected by COVID‐19 severity. These findings highlight the long‐term alterations of immune cells in post‐COVID‐19 patients,emphasising the substantial impact of COVID‐19 on immune function. View Publication

The impact of exogenous stressors,such as cancer chemotherapies,on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden,producing a distinctive mutation signature,whereas other chemotherapeutic agents had minimal effects. Consequently,the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals,particularly through the progression of oligoclonal hematopoiesis,thereby suggesting that chemotherapy accelerates clonal aging. Integrated phylogenetic analysis of matched therapy-related myeloid neoplasm samples traced their clonal origin to a single-HSPC clone among multiple competing clones,supporting a model of oligoclonal to monoclonal transformation. These findings underscore the need for further systematic research on the long-term hematological consequences of cancer chemotherapy. Subject terms: Genetics research,Acute myeloid leukaemia View Publication

Simvastatin,a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor,was used in cardiovascular diseases and could decrease low-density lipoprotein cholesterol,and may have a repurposed role in cancer therapy. However,the effects of simvastatin on endometrial cancer remain controversial. We aimed to elucidate the role and mechanisms of simvastatin in regulating previously identified endometrial cancer-associated mesenchymal stem cells (EmCaMSCs)-mediated immunosuppressive effects and anti-tumor progression. Coculture of EmCaMSCs and peripheral blood mononuclear cells (PBMC) was used to assay the population of CD8 + T cells,natural killer (NK) cells,and cytotoxicity of NK cells. The mechanisms were elucidated by applying recombinant proteins and inhibitors of candidate proteins,transforming growth factor-beta 2 (TGF-β2). Finally,the humanized mouse model was generated to study the effects of simvastatin-mediated immunotherapy in treating endometrial cancer. The protein expressions of TGF-β2,CD56,CD8,and PD-L1 in xenograft tumors were analyzed by Western blot or immunohistochemistry assay. In this study,simvastatin inhibited the proliferation of endometrial cancer cells (HEC-1 A and RL95-2) and EmCaMSCs,and the half-maximal inhibitory concentration (IC50) values of EmCaMSCs were much higher. Simvastatin rescued the proliferation and the population of CD8 + T cells and natural killer (NK) cells from PBMC coculturing with EmCaMSC. Simvastatin treatment reduced the expression of TGF-β2 in EmCaMSCs at both the gene and protein levels. TGF-β2 activated the downstream SMAD2/3 signaling,and their inhibition by simvastatin could enhance the cytotoxicity of NK cells against endometrial cancer cells in vitro. Additionally,a combination of simvastatin and NK cell therapy inhibited xenograft growth,potentially by reducing TGF-β2 expression. In conclusion,simvastatin could rescue the population of CD8 + T cells and NK cells from PBMC cocultured with EmCaMSCs. Furthermore,simvastatin could enhance the cytotoxicity of NK cells in vitro and inhibit tumor growth in vivo in a humanized mouse model. These results suggested that simvastatin may be considered as a repurposed and combination drug for treating endometrial cancer. The online version contains supplementary material available at 10.1038/s41598-025-08686-9. View Publication

p18 INK4 C (CDKN2C,encoded by p18 INK4c or Cdkn2c ) is an early G1-phase cyclin-dependent kinase inhibitor protein. Previous studies demonstrated enhanced self-renewal capacity of hematopoietic stem cells (HSCs) in p18 −/− mice compared to wild-type (WT) mice. Given the critical role of bone marrow niche cells-particularly mesenchymal stromal cells (MSCs)-in hematopoiesis,this study investigated the functional alterations of p18 −/− MSCs and their impact on hematopoietic support. Bone marrow derived MSCs were isolated from p18 −/− and WT mice. Their proliferation and differentiation capacities were assessed,followed by evaluation of hematopoietic support using cobblestone area-forming cell assay and long-term culture-initiating cell assay. RNA sequencing was performed to analyze the transcriptional profile of p18 −/− MSCs,with a focus on differentially expressed genes (DEGs). Key pathways associated with hematopoietic support were identified using Ingenuity Pathway Analysis. A candidate protein was quantified by ELISA,and its functional role in hematopoietic support was validated via a modified coculture system. p18 −/− MSCs displayed an increased proliferation rate,preferential differentiation toward osteogenesis over adipogenesis,and enhanced hematopoietic support. RNA sequencing analysis identified 137 DEGs,with secreted phosphoprotein 1 ( Spp1,encoding osteopontin,Opn) being significantly upregulated in p18 −/− MSCs. Elevated Opn levels were confirmed in both bone marrow and MSC-conditioned media from p18 −/− mice. Functional validation further demonstrated that Opn enhanced the hematopoietic supportive capacity of MSCs in vitro. p18 deficiency promotes osteogenic differentiation and enhances the hematopoietic supportive function of MSCs,likely mediated by Opn upregulation. These findings suggest a potential therapeutic strategy for improving bone regeneration and HSC expansion. The online version contains supplementary material available at 10.1186/s13287-025-04402-6. View Publication

Duplication 15q (dup15q) syndrome is a leading genetic cause of autism spectrum disorder,offering a key model for studying autism-related mechanisms. Using single-cell and single-nucleus RNA sequencing of cortical organoids from dup15q patient-derived iPSCs and post-mortem brain samples,we identify increased glycolysis,disrupted layer-specific marker expression,and aberrant morphology in deep-layer neurons during fetal-stage organoid development. In adolescent-adult postmortem brains,upper-layer neurons exhibit heightened transcriptional burden related to synaptic signaling,a pattern shared with idiopathic autism. Using spatial transcriptomics,we confirm these cell-type-specific disruptions in brain tissue. By gene co-expression network analysis,we reveal disease-associated modules that are well preserved between postmortem and organoid samples,suggesting metabolic dysregulation that may lead to altered neuron projection,synaptic dysfunction,and neuron hyperexcitability in dup15q syndrome. Subject terms: Autism spectrum disorders,Autism spectrum disorders,Disease model View Publication