参考文献

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers,it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK‐cell derived extracellular vesicles (NK‐EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential,it is necessary to standardize NK‐EV production protocols. Here,we have performed a comparative analysis of EVs from the human NK‐92 cell line cultured in five serum‐free commercial media optimized for growth of human NK cells and one serum‐free medium for growth of lymphocytes. The effect of growing the NK‐92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size‐exclusion chromatography. We found that there were no significant differences in EV yield from NK‐92 cells grown under static or dynamic conditions. However,we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK‐cell derived EVs. View Publication

Low dose methotrexate (LD‐MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti‐inflammatory effects of LD‐MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD‐MTX. This is the first study to characterize the effects of LD‐MTX on HSPC. CD34 + HSPC were isolated from healthy donors' non‐mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD‐MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability,cell cycle,surface abundance of reduced folate carrier 1 (RFC1),proliferation,reactive oxygen species (ROS) levels,DNA double‐strand breaks,p38 activation,and CD34 + subpopulations. HSPC clonogenicity was tested in colony‐forming cell assays. Our results indicate that in cycling HSPC,membrane RFC1 is upregulated and,following LD‐MTX treatment,they accumulate more intracellular MTX than resting HSPC. In cycling HSPC,LD‐MTX inhibits HSPC expansion by promoting S‐phase cell‐cycle arrest,increases intracellular HSPC ROS levels and DNA damage,and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD‐MTX are more evident in CD34 + CD38High progenitors. In non‐cycling HSPC,LD‐MTX also reduces the proliferative response while preserving their clonogenicity. In summary,HSPC uptake LD‐MTX differentially according to their cycling state. In turn,LD‐MTX results in reduced proliferation and the preservation of HSPC clonogenicity. View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However,the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs),with unique design features,facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress,using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling,flow cytometry,and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis,electron microscopy,and Western blotting. Lipidomics provided insights into EV lipid composition,while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size,but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however,distinct differences were identified in lipid chain lengths and saturation levels,affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro. Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2–3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo,showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential,paving the way for future in vivo studies. The online version contains supplementary material available at 10.1186/s13287-025-04317-2. View Publication

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling,but its function in airway epithelial responses to IL-13,a key mediator in allergic diseases,remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing,providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes,including SPDEF,MUC5AC,POSTN,ALOX15,and CCL26,confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types,suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet,club,and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types,reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner,strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7. View Publication

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome,resulting in inconsistent findings across studies. Identifying a core set of genes consistently involved in COPD pathogenesis,independent of patient variability,is essential. We integrated lung tissue sequencing data from patients with COPD across two centers. We used weighted gene co-expression network analysis and machine learning to identify 13 potential pathogenic genes common to both centers. Additionally,a gene-based model was constructed to distinguish COPD at the molecular level and validated in independent cohorts. Gene expression in specific cell types was analyzed,and Mendelian randomization was used to confirm associations between candidate genes and lung function/COPD. Preliminary in vitro functional validation was performed on prioritized core candidate genes. Tissue inhibitor of metalloproteinase 4 (TIMP4) was identified as a key pathogenic gene and validated in COPD cohorts. Further analysis using single-cell sequencing from mice and patients with COPD revealed that TIMP4 is involved in ciliated cells. In primary human airway epithelial cells cultured at the air-liquid interface,TIMP4 overexpression reduced ciliated cell numbers. We developed a 13-gene model for distinguishing COPD at the molecular level and identified TIMP4 as a potential hub pathogenic gene. This finding provides insights into shared disease mechanisms and positions TIMP4 as a promising therapeutic target for further investigation. The online version contains supplementary material available at 10.1186/s12931-025-03238-1. View Publication

Development and lineage choice are driven by interconnected transcriptional,epigenetic and metabolic changes. Specific metabolites,such as α-ketoglutarate (αKG),function as signalling molecules affecting the activity of chromatin-modifying enzymes. However,how metabolism coordinates cell-state changes,especially in human pre-implantation development,remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring,characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover,increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather,it decreases acetyl-CoA availability,reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation,highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells,Embryology View Publication

SLC26A4 is the second most common cause of hereditary hearing loss worldwide. This gene predominantly harbors pathogenic variants,including splice,nonsense,and missense. Although missense variants are relatively common,their specific effects on protein function remain unclear. Consequently,there is an urgent need to establish an in vitro system to investigate how these variants impact SLC26A4 protein function. Genetic testing was conducted to determine the specific types of underlying genetic variants in patients. Following this,we employed plasmid transfection to evaluate the effects of the variants on both protein expression levels and the protein's subcellular localization. Thereafter,we transformed peripheral blood mononuclear cells (PBMCs) from the proband into induced pluripotent stem cells (iPSCs) through Sendai virus‐mediated transduction. Genetic testing revealed that the proband carried compound heterozygous variants: SLC26A4 c.919‐2A > G and c.317C > A. The c.317C > A variant markedly decreased the expression levels of SLC26A4 mRNA and its encoded protein. Additionally,it led to the protein's accumulation in the cytoplasm as aggregates. We successfully reprogrammed peripheral blood mononuclear cells from the proband into induced pluripotent stem cells (iPSCs) and verified that these iPSCs retained their pluripotency,differentiation potential,and genetic integrity. These results provide important insights into the mechanisms by which SLC26A4 gene variants lead to hearing loss. View Publication

Human brain development requires generating diverse cell types,a process explored by single-cell transcriptomics. Through parallel meta-analyses of the human cortex in development (seven datasets) and adulthood (16 datasets),we generated over 500 gene co-expression networks that can describe mechanisms of cortical development,centering on peak stages of neurogenesis. These meta-modules show dynamic cell subtype specificities throughout cortical development,with several developmental meta-modules displaying spatiotemporal expression patterns that allude to potential roles in cell fate specification. We validated the expression of these modules in primary human cortical tissues. These include meta-module 20,a module elevated in FEZF2 + deep layer neurons that includes TSHZ3,a transcription factor associated with neurodevelopmental disorders. Human cortical chimeroid experiments validated that both FEZF2 and TSHZ3 are required to drive module 20 activity and deep layer neuron specification but through distinct modalities. These studies demonstrate how meta-atlases can engender further mechanistic analyses of cortical fate specification. Subject terms: Developmental neurogenesis,Gene regulatory networks View Publication

Ceramides are bioactive sphingolipids that have physiological effects on inflammation,apoptosis,and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied,and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model,serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs),followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia),our data showed that following IR,there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis,such as CerS2 and CerS4 in CMs. Further,there was a considerable rise in the levels of total ceramide,long-chain (C16:0,C18:0,and C18:1),and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly,compared to CMs exposed to IR without inhibitor,our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels,reduced apoptosis,improved mitochondrial function,and enhanced survival of CMs exposed to IR. Furthermore,we used a transgenic mouse model,in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice,to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides,which causes increased apoptosis,proinflammatory cytokines,interstitial inflammatory cell infiltration,and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally,in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3. View Publication

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

SOT201 and its murine surrogate mSOT201 are novel cis-acting immunocytokines consisting of a humanized/murinized/,Fc-silenced anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb) fused to an attenuated human interleukin (IL)-15 and the IL-15Rα sushi+ domain. Murine mPD1-IL2v is a conjugate of a murinized,Fc silenced anti-PD-1 mAb bearing human IL-2 with abolished IL-2Rα binding. These immunocytokines spatiotemporally reinvigorate PD-1 + CD8 + tumor-infiltrating lymphocytes (TILs) via cis-activation and concomitantly activate the innate immunity via IL-2/15Rβγ signaling. Human peripheral blood mononuclear cell and cell lines were used to evaluate cis/trans activity of SOT201. Anti-PD-1 mAb responsive (MC38,CT26) and resistant (B16F10,CT26 STK11 KO) mouse tumor models were used to determine the anticancer efficacy,and the underlying immune cell activity was analyzed via single-cell RNA sequencing and flow cytometry. The expansion of tumor antigen-specific CD8 + T cells by mSOT201 or mPD1-IL2v and memory CD8 + T-cell generation in vivo was determined by flow cytometry. SOT201 delivers attenuated IL-15 to PD-1 + T cells via cis-presentation,reinvigorates exhausted human T cells and induces higher interferon-γ production than pembrolizumab in vitro. mSOT201 administered as a single dose exhibits strong antitumor efficacy with several complete responses in all tested mouse tumor models. While mPD1-IL2v activates CD8 + T cells with a 50-fold higher potency than mSOT201 in vitro,mSOT201 more effectively reactivates effector exhausted CD8 + T cells (Tex),which demonstrate higher cytotoxicity,lower exhaustion and lower immune checkpoint transcriptional signatures in comparison to mPD1-IL2v in MC38 tumors in vivo. This can be correlated with a higher rate of complete responses in the MC38 tumor model following mSOT201 treatment when compared with mPD1-IL2v. mSOT201 increased the relative number of tumor antigen-specific CD8 + T cells,and unlike mPD1-IL2v stimulated greater expansion of adoptively transferred ovalbumin-primed CD8 + T cells simultaneously limiting the peripheral CD8 + T-cell sink,leading to the development of memory CD8 + T cells in vivo. SOT201 represents a promising therapeutic candidate that preferentially targets PD-1 + TILs,delivering balanced cytokine activity for reviving CD8 + Tex cells in tumors. SOT201 is currently being evaluated in the Phase I clinical study VICTORIA-01 ( NCT06163391 ) in patients with advanced metastatic cancer. View Publication