技术资料

Spaceflight and microgravity environments have been shown to cause significant health impairments,including bone loss,immune dysfunction,and hematopoietic disorders. Hematopoietic stem cells (HSCs),as progenitors of the hematopoietic system,are critical for the continuous renewal and regulation of immune cells. Therefore,elucidating the regulatory mechanisms governing HSC fate and differentiation in microgravity environments is of paramount importance. In this study,hindlimb unloading (HU) was employed in mice to simulate microgravity conditions. After 28 days of HU,cells were isolated for analysis. Flow cytometry and colony-forming assays were utilized to assess changes in HSC proliferation and differentiation. Additionally,transcriptomic and untargeted metabolomic sequencing were performed to elucidate alterations in the metabolic pathways of the bone marrow microenvironment and their molecular regulatory effects on HSCs fate. Our findings revealed that 28 days of HU impaired hematopoietic function,leading to multi-organ damage and hematological disorders. The simulated microgravity environment significantly increased the HSCs population in the bone marrow,particularly within the long-term and short-term subtypes,while severely compromising the differentiation capacity of hematopoietic stem/progenitor cells. Transcriptomic analysis of HSCs,combined with metabolomic profiling of bone marrow supernatants,identified 1,631 differentially expressed genes and 58 metabolites with altered abundance. Gene set enrichment analysis indicated that HU suppressed key pathways,including hematopoietic cell lineage and MAPK signaling. Furthermore,integrated analyses revealed that metabolites affected by HU,particularly hypoxanthine enriched in the purine metabolism pathway,were closely associated with hematopoietic cell lineage and MAPK signaling pathways. Molecular docking simulations and in vitro experiments confirmed that hypoxanthine interacts directly with core molecules within these pathways,influencing their expression. These findings demonstrate that hypoxanthine in the bone marrow supernatant acts as a signaling mediator under microgravity,influencing HSCs fate by modulating hematopoietic cell lineage and MAPK signaling pathways. This study offers novel insights into the impact of microgravity on HSC fate and gene expression,underscoring the pivotal role of bone marrow microenvironmental metabolic changes in regulating key signaling pathways that determine hematopoietic destiny. The online version contains supplementary material available at 10.1186/s13287-025-04213-9. View Publication

Calcium-regulated heat-stable protein 1 (CARHSP1) has been identified as a cold shock domain (CSD) protein family member,participating in the regulation of ribosomal translation,mRNA degradation,and the rate of transcription termination. However,there is an extremely limited understanding of the function of CARHSP1 as an RNA binding protein (RBP) in prostate cancer (PCa). The expression pattern of CARHSP1 and the correlation between the CARHSP1 expression and clinical prognosis in PCa patients were analyzed by using multiple public databases. In vitro and in vivo functional assays were conducted to assess the role of CARHSP1. The mechanisms of CARHSP1 function on IL-17RA were identified by RNA pull-down and RNA stability assays. A co-culture model of Jurkat cells and PCa cells was established to investigate the potential role of CARHSP1 in tumor immunity of PCa. CARHSP1 was highly expressed in PCa,and correlated with advanced characteristics of PCa and unfavorable prognosis in PCa patients. Moreover,knockdown of CARHSP1 significantly dampened the capacity of proliferation,migration,invasion,and immune evasion of PCa cells in vitro and in vivo. Mechanistically,the RNA-binding protein CARHSP1 selectively bound to the mRNA of IL-17RA,resulting in the increased expression of both IL-17RA mRNA and protein. Downregulating expression of CARHSP1 shortened the half-life of IL-17RA mRNA and reduced its expression. Subsequently,the downstream pathways of IL-17RA,JAK-STAT3 signaling pathway and NF-κB signaling pathway,were activated by CARHSP1 and contributed to the malignant phenotype of PCa cells. In conclusion,our results demonstrated that the increased expression of CARHSP1 in PCa is correlated with advanced clinical characteristics and unfavorable prognosis,and CARHSP1 may promote the progression of PCa through enhancing the mRNA stability of IL-17RA and activating its downstream pathways. These results suggest that CARHSP1 is an important regulator of tumor microenvironment in PCa,and CARHSP1-IL-17RA axis could be potential novel therapeutic targets for PCa. The online version contains supplementary material available at 10.1186/s13578-025-01371-4. View Publication

Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms,the pathogenesis of MMA in the central nervous system is poorly understood,which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology,targeted metabolomics,and bulk transcriptomics,we expose an altered state of excitability,which is exacerbated by application of dimethyl-2-oxoglutarate,and we suggest may be connected to metabolic rewiring. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA,which through our comprehensive characterisation of this paradigmatic model,enables first steps to identifying effective therapies. Subject terms: Mechanisms of disease,Metabolic disorders,Diseases of the nervous system View Publication

Tracheal replacement is a promising approach for treating tracheal defects that are caused by conditions such as stenosis,trauma,or tumors. However,slow postoperative epithelial regeneration often leads to complications,such as infection and granulation tissue formation. Ferroptosis,which is an iron-dependent form of regulated cell death,limits the proliferation of tracheal basal cells (TBCs),which are essential for the epithelialization of tissue-engineered tracheas (TETs). This study explored the potential of ferrostatin-1 (FER-1),which is a ferroptosis inhibitor,to increase TBC proliferation and accelerate the epithelialization of 3D-printed TETs. TBCs were isolated from rabbit bronchial mucosal tissues and cultured in vitro. Ferroptosis was induced in TBCs at passage 2,as shown by increased reactive oxygen species (ROS) levels,Fe 2 ⁺ accumulation,decreased ATP contents,and mitochondrial damage. TBCs were treated with FER-1 (1 μM) for 48 h to inhibit ferroptosis. The effects on ROS levels,Fe 2 ⁺ levels,ATP contents,and mitochondrial morphology were measured. For in vivo experiments,FER-1-treated TBCs were seeded onto 3D-printed polycaprolactone (PCL) scaffolds,which were implanted into rabbits with tracheal injury. Epithelial regeneration and granulation tissue formation were evaluated 6 months after surgery. FER-1 treatment significantly reduced ferroptosis marker levels in vitro; that is,FER-1 treatment decreased ROS and Fe 2 ⁺ accumulation,ameliorated mitochondrial structures,and increased ATP levels. TBC proliferation and viability were increased after ferroptosis inhibition. In vivo,the group that received 3D-printed scaffolds seeded with TBCs exhibited accelerated TET epithelialization and reduced granulation tissue formation compared with the control groups. These results suggest that inhibiting ferroptosis with FER-1 improves TBC function,leading to more efficient tracheal repair. Ferrostatin-1 effectively inhibits ferroptosis in tracheal basal cells,promoting their viability and proliferation. This results in faster epithelialization of tissue-engineered tracheas,offering a promising strategy for improving tracheal reconstruction outcomes and reducing complications such as infection and granulation tissue formation. Future studies are needed to further investigate the molecular mechanisms underlying ferroptosis in TBCs and its potential clinical applications. The online version contains supplementary material available at 10.1186/s13287-025-04263-z. View Publication

Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS may exhibit hepatomegaly,as well as an increased risk of hepatoblastoma. To understand the impact of these 11p15 changes in the liver,we performed a multiomic study [single nucleus RNA-sequencing (snRNA-seq) + single nucleus assay for transposable-accessible chromatin-sequencing (snATAC-seq)] of both BWS-liver and nonBWS-liver tumor-adjacent tissue. Our approach uncovers hepatocyte-specific enrichment of processes related to peroxisome proliferator—activated receptor alpha (PPARA). To confirm our findings,we differentiated a BWS induced pluripotent stem cell model into hepatocytes. Our data demonstrate the dysregulation of lipid metabolism in BWS-liver,which coincides with observed upregulation of PPARA during hepatocyte differentiation. BWS hepatocytes also exhibit decreased neutral lipids and increased fatty acid β-oxidation. We also observe increased reactive oxygen species byproducts in BWS hepatocytes,coinciding with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism. Subject terms: Paediatric research,Imprinting View Publication

Ageing is the most prominent risk factor for Alzheimer’s disease (AD). However,the cellular mechanisms linking neuronal proteostasis decline to the characteristic aberrant protein deposits in the brains of patients with AD remain elusive. Here we develop transdifferentiated neurons (tNeurons) from human dermal fibroblasts as a neuronal model that retains ageing hallmarks and exhibits AD-linked vulnerabilities. Remarkably,AD tNeurons accumulate proteotoxic deposits,including phospho-tau and amyloid β,resembling those in APP mouse brains and the brains of patients with AD. Quantitative tNeuron proteomics identify ageing- and AD-linked deficits in proteostasis and organelle homeostasis,most notably in endosome–lysosomal components. Lysosomal deficits in aged tNeurons,including constitutive lysosomal damage and ESCRT-mediated lysosomal repair defects,are exacerbated in AD tNeurons and linked to inflammatory cytokine secretion and cell death. Providing support for the centrality of lysosomal deficits in AD,compounds ameliorating lysosomal function reduce amyloid β deposits and cytokine secretion. Thus,the tNeuron model system reveals impaired lysosomal homeostasis as an early event of ageing and AD. Subject terms: Organelles,Protein folding View Publication

With a high global incidence of over three million new cases in 2020 and a high mortality of over two million fatalities,ovarian cancer is one of the most common malignant tumors in gynecology. Exosomes can control the immunological condition of the tumor microenvironment (TME) by participating in intercellular interactions. Therefore,we aimed to construct an exosome-related prognostic model to predict the clinical outcomes of ovarian cancer patients. In this research,expression patterns of exosome-related genes were examined in multiple single-cell RNA-sequencing and bulk RNA-sequencing datasets. In addition,a novel exosome-related prognostic model was established by the least absolute shrinkage and selection operator (LASSO) regression method. Then,the correlations between risk score and immunological characteristics of the TME were explored. Moreover,SERPINB1,a gene in the prognostic signature,was further analyzed to reveal its value as a novel biomarker. In the current study,combined with single-cell and bulk omics datasets,we constructed an exosome-related prognostic model of four genes (LGALS3BP,SAT1,SERPINB1,and SH3BGRL3). Moreover,the risk score was associated with worse overall survival (OS) in ovarian cancer patients. Further analysis found that patients with high-risk score tended to shape a desert TME with hardly infiltration of immune cells. Then,SERPINB1,positively correlated with the favorable OS and negatively with the risk score,was chosen as the representative biomarker of the model. Moreover,SERPINB1 was positively correlated with the infiltration of immune subpopulations in both public and in-house cohort. In addition,the high-resolution analysis found that SERPINB1 + tumor cells communicated with microenvironment cells frequently,further explaining the potential reason for shaping an inflamed TME. To sum up,we established a novel exosome-related prognostic model (LGALS3BP,SAT1,SERPINB1,and SH3BGRL3) to predict the prognosis of patients with ovarian cancer and identify the immunological characteristics of the TME. In addition,SERPINB1 was identified as a promising biomarker for prognostic prediction in ovarian cancer. The online version contains supplementary material available at 10.1186/s13048-025-01589-3. View Publication

Osteosarcoma is the most prevalent primary malignant bone tumor affecting pediatric and adolescent individuals. However,despite the passage of three decades,there has been no notable enhancement in the overall survival rate of patients with osteosarcoma. In recent years,CD155 has been reported to exhibit abnormal amplification in a range of tumors,yet the precise underlying mechanism remains elusive. The objective of this study is to investigate the role of CD155 in osteosarcoma,and to identify drugs that specifically target this molecule,thereby offering a novel direction for the treatment of osteosarcoma. The prognosis of patients with osteosarcoma with high and low expression of CD155 was verified by immunohistochemistry. CCK-8 and colony formation assays were used to detect cell proliferation and drug resistance. Transwell experiments were used to detect cell migration and invasion. The sphere formation experiment was used to evaluate the stemness of tumor cells. Additionally,in vivo animal models were utilized to assess the functional role of CD155 in a biological context. RNA-seq and co-immunoprecipitation methods were used to search for downstream target molecules and signaling pathways of CD155. Finally,virtual screening was used to find drugs targeting CD155. In this study,we have established the significant amplification of CD155 in osteosarcoma. Utilizing a comprehensive array of experimental methods,including CCK-8 assay,colony formation assay,Transwell assay,and in vivo animal models,we unequivocally demonstrate that CD155 significantly potentiates the malignancy of osteosarcoma both in vitro and in vivo. Additionally,our findings reveal that CD155 promotes osteosarcoma stemness by modulating the Wnt/β-catenin signaling pathway. Advanced molecular techniques,such as RNA sequencing and co-immunoprecipitation,have been instrumental in elucidating the mechanism of CD155 in activating the Wnt/β-catenin pathway via the SRC/AKT/GSK3β signaling axis,thereby enhancing the stem-cell-like properties of osteosarcoma cells. To explore targeted therapeutic options,we conducted virtual screening and identified troxerutin as a promising CD155 inhibitor. Our findings reveal that troxerutin effectively inhibits CD155,attenuates the SRC/AKT/GSK3β signaling cascade,diminishes the nuclear localization of β-catenin,and consequently mitigates osteosarcoma stemness. These discoveries position troxerutin as a promising candidate for targeted osteosarcoma therapy. The online version contains supplementary material available at 10.1186/s11658-025-00724-8. 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

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

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

In Alzheimer’s disease,amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However,there is limited information on how endogenous levels of tau,Aβ and other biomarkers relate to patient characteristics,or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures,we report that Aβ 1-40 and tau release levels vary with donor age and brain region,respectively. Release of other biomarkers such as KLK-6,NCAM-1,and Neurogranin vary between brain region,while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta,with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast,treatment with Aβ-containing Alzheimer’s disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue. Subject terms: Alzheimer's disease,Alzheimer's disease View Publication