技术资料

Brain organoids have been proposed as suitable human brain model candidates for a variety of applications. However,the lack of appropriate maturation limits the transferability of such functional tools. Here,we present a method to facilitate neuronal maturation by integrating astrocyte-secreted factors into hPSC-derived 2D and 3D neural culture systems. We demonstrate that protein- and nutrient-enriched astrocyte-conditioned medium (ACM) accelerates neuronal differentiation with enlarged neuronal layer and the overproduction of deep-layer cortical neurons. We captured the elevated changes in the functional activity of neuronal networks within ACM-treated organoids using comprehensive electrophysiological recordings. Furthermore,astrocyte-secreted cues can induce lipid droplet accumulation in neural cultures,offering protective effects in neural differentiation to withstand cellular stress. Together,these data indicate the potential of astrocyte secretions to promote neural maturation. Subject terms: Neurological models,Neuronal development View Publication

The airway epithelium represents a central barrier against pathogens and toxins while playing a crucial role in modulating the immune response within the upper respiratory tract. Understanding these mechanisms is particularly relevant for red foxes ( Vulpes vulpes ),which serve as reservoirs for various zoonotic pathogens like rabies or the fox tapeworm ( Echinococcus multilocularis ). The study aimed to develop,establish,and validate an air-liquid interface (ALI) organoid model of the fox respiratory tract using primary airway epithelial cells isolated from the tracheas and main bronchi of hunted red foxes. The resulting ALI cultures exhibited a structurally differentiated,pseudostratified epithelium,characterised by ciliated cells,mucus secretion,and tight junctions,as confirmed through histological and immunohistochemical analysis. Functional assessments using a paracellular permeability assay and measurement of transepithelial electrical resistance,demonstrated a tight epithelial barrier. The potential of model’s utility for studying innate immune responses to respiratory infections was validated by exposing the cultures to lipopolysaccharide,phorbol-12-myristate-13-acetate and ionomycin,and nematode somatic antigens. Quantitative PCR revealed notable changes in the expression of pro-inflammatory cytokines TNF and IL-33. This in vitro model represents a significant advancement in respiratory research for non-classical species that may act as important wildlife reservoirs for a range of zoonotic pathogens. View Publication

Overall survival of acute myeloid leukemia (AML) remains limited. Inhibitors of the master mitotic kinase PLK1 have emerged as promising therapeutics,demonstrating efficacy in an undefined subset of patients with AML. However,the clinical success of PLK1 inhibitors remains hindered by a lack of predictive biomarkers. The Fanconi anemia (FA) pathway,a tumor-suppressive network comprised of at least 22 genes,is frequently mutated in sporadic AML. In this study,we demonstrate that FA pathway disruption sensitizes AML cells to PLK1 inhibition. Mechanistically,we identify novel interactions between PLK1 and both FANCA and FANCD2 at mitotic centromeres. We demonstrate that PLK1 inhibition impairs recruitment of FANCD2 to mitotic centromeres,induces damage to mitotic chromosomes,and triggers mitotic collapse in FANCA-deficient cells. Our findings indicate that PLK1 inhibition targets mitotic vulnerabilities specific to FA pathway–deficient cells and implicate FA pathway mutations as potential biomarkers for the identification of patients likely to benefit from PLK1 inhibitors. This work demonstrates that FA pathway mutations,which are frequently observed in sporadic AML,induce hypersensitivity to PLK1 inhibition,providing rationale for a novel synthetic lethal therapeutic strategy for this patient population. View Publication

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by immune‐mediated demyelination of the central nervous system,resulting in extensive neurological deficit and remyelination impairment. We have previously found that interleukin‐four induced one (IL4I1) protein modulates CNS inflammation and enhances remyelination in mouse models of experimental demyelination. However,it remained unclear if IL4I1 regulates lymphocyte activity in MS. To assess the therapeutic potential of IL4I1 in MS,we investigated the impact of IL4I1 treatment on human lymphocytes from peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and MS patients. We found that IL4I1 increased the relative densities of Th2 and regulatory T‐cells,while reducing Th17 cell density in healthy control (HC) samples. Furthermore,IL4I1‐treated lymphocytes promoted CNS remyelination when grafted into demyelinated spinal cord lesions in mice. We found that baseline endogenous IL4I1 expression was reduced in people with MS. However,unlike HCs,IL4I1 treatment had no significant effect on IL17 or TOB1 expression in lymphocytes derived from MS patients. These results suggest that IL4I1 skews CD4 + T‐cells to a regulatory state in healthy human lymphocytes,which may be essential for promoting remyelination. However,IL4I1 appears unable to exert its influence on lymphocytes in MS,indicating that impaired IL4I1‐mediated activity may underlie MS pathology. 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

Pancreatic cancer is one of the most malignant cancers,and limited therapeutic options are available. The induction of ferroptosis is considered to be a novel,promising strategy that has potential in cancer treatment,and ferroptosis inducers may be new options for eradicating malignant cancers that are resistant to traditional drugs. The exact mechanism underlying the function of sorcin in the initiation and progression of pancreatic cancer remains unclear. The expression of sorcin in cancer tissues was assessed by analyzing TCGA,GEO and immunohistochemical staining data,and the function of sorcin in the induction of ferroptosis in pancreatic cancer cells was investigated. The mechanism underlying the function of sorcin was revealed through proteomics,co-IP,Ch-IP,and luciferase assays. Natural product screening was subsequently performed to screen for products that interact with sorcin to identify new ferroptosis inducers. We first showed that sorcin expression was positively correlated with the survival and tumor stages of patients with pancreatic cancer,and we revealed that sorcin inhibited ferroptosis through its noncalcium binding function. Furthermore,we discovered that sorcin interacted with PAX5 in the cytoplasm and inhibited PAX5 nuclear translocation,which in turn decreased FBXL12 protein expression and then reduced ALDH1A1 ubiquitination,thus inhibiting ferroptosis. Moreover,an in-house natural product screen revealed that celastrol inhibited the interaction of sorcin and PAX5 by directly binding to the Cys194 residue of the sorcin protein; disruption of the sorcin-PAX5 interaction promoted the nuclear translocation of PAX5,induced the expression of FBXL12,increased the ubiquitylation of ALDH1A1,and eventually induced ferroptosis in pancreatic cancer cells. In this study,we revealed the mechanism of action of sorcin,which is a druggable target for inducing ferroptosis,we identified celastrol as a novel agent that induces ferroptosis,and we showed that disrupting the sorcin-PAX5 interaction is a promising therapeutic strategy for treating pancreatic cancer. The online version contains supplementary material available at 10.1186/s13045-025-01680-8. View Publication

The clinical success of the immune checkpoint inhibitor (ICI) targeting programmed cell death protein 1 (PD-1) has revolutionized cancer treatment. However,the full potential of PD-1 blockade therapy remains unrealized,as response rates are still low across many cancer types. Interleukin-2 (IL-2)-based immunotherapies hold promise,as they can stimulate robust T cell expansion and enhance effector function - activities that could synergize potently with PD-1 blockade. Yet,IL-2 therapies also carry a significant drawback: they can trigger severe systemic toxicities and induce immune suppression by expanding regulatory T cells. To overcome the challenges of PD-1 blockade and IL-2 therapies while enhancing safety and efficacy,we have engineered a novel fusion protein,AWT020,combining a humanized anti-PD-1 nanobody and an engineered IL-2 mutein (IL-2c). The IL-2c component of AWT020 has been engineered to exhibit no binding to the IL-2 receptor alpha (IL-2Rα) subunit and attenuated affinity for the IL-2 receptor beta and gamma (IL-2Rβγ) complex,aiming to reduce systemic immune cell activation,thereby mitigating the severe toxicity often associated with IL-2 therapies. The anti-PD-1 antibody portion of AWT020 serves a dual purpose: it precisely delivers the IL-2c payload to tumor-infiltrating T cells while blocking the immune-inhibitory signals mediated by the PD-1 pathway. AWT020 showed significantly enhanced pSTAT5 signaling in PD-1 expressing cells and promoted the proliferation of activated T cells over natural killer (NK) cells. In preclinical studies using both anti-PD-1-sensitive and -resistant mouse tumor models,the mouse surrogate of AWT020 (mAWT020) demonstrated markedly enhanced anti-tumor efficacy compared to an anti-PD-1 antibody,IL-2,or the combination of an anti-PD-1 antibody and IL-2. In addition,the mAWT020 treatment was well-tolerated,with minimal signs of toxicity. Immune profiling revealed that mAWT020 preferentially expands CD8 + T cells within tumors,sparing peripheral T and NK cells. Notably,this selective tumoral T-cell stimulation enables potent tumor-specific T-cell responses,underscoring the molecule’s enhanced efficacy and safety. The AWT020 fusion protein offers a promising novel immunotherapeutic strategy by integrating PD-1 blockade and IL-2 signaling,conferring enhanced anti-tumor activity with reduced toxicity. View Publication

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

Psychological stress causes gut microbial dysbiosis and cancer progression,yet how gut microbiota determines psychological stress-induced tumor development remains unclear. Here we showed that psychological stress promotes breast tumor growth and cancer stemness,an outcome that depends on gut microbiota in germ-free and antibiotic-treated mice. Metagenomic and metabolomic analyses revealed that psychological stress markedly alters the composition and abundance of gut microbiota,especially Akkermansia muciniphila ( A. muciniphila ),and decreases short-chain fatty acid butyrate. Supplement of active A. muciniphila,butyrate or a butyrate-producing high fiber diet dramatically reversed the oncogenic property and anxiety-like behavior of psychological stress in a murine spontaneous tumor model or an orthotopic tumor model. Mechanistically,RNA sequencing analysis screened out that butyrate decreases LRP5 expression to block the activation of Wnt/β-catenin signaling pathway,dampening breast cancer stemness. Moreover,butyrate as a HDAC inhibitor elevated histone H3K9 acetylation level to transcriptionally activate ZFP36,which further accelerates LRP5 mRNA decay by binding adenine uridine-rich (AU-rich) elements of LRP5 transcript. Clinically,fecal A. muciniphila and serum butyrate were inversely correlated with tumoral LRP5/β-catenin expression,poor prognosis and negative mood in breast cancer patients. Altogether,our findings uncover a microbiota-dependent mechanism of psychological stress-triggered cancer stemness,and provide both clinical biomarkers and potential therapeutic avenues for cancer patients undergoing psychological stress. Subject terms: Cancer metabolism,Cancer stem cells View Publication

Lung transplantation is the primary treatment for end-stage lung diseases. However,ischemia-reperfusion injury (IRI) significantly impacts transplant outcomes. 4-Octyl itaconate (4-OI) has shown potential in mitigating organ IRI,although its effects in lung transplantation require further exploration. BEAS-2B cells were used to model transplantation,assessing the effects of 4-OI through viability,apoptosis,and ROS assays. qRT-PCR analyzed cytokine transcription post-cold ischemia/reperfusion (CI/R). RNA sequencing and Gene Ontology analysis elucidated 4-OI’s mechanisms of action,confirmed by Western blotting. ALI-airway and lung transplantation organoid models evaluated improvements in bronchial epithelial morphology and function due to 4-OI. ELISA measured IL-6 and IL-8 levels. Rat models of extended cold preservation and non-heart-beating transplantation assessed 4-OI’s impact on lung function,injury,and inflammation. Our findings indicate that 4-OI (100 µM) during cold preservation effectively maintained cell viability,decreased apoptosis,and reduced ROS production in BEAS-2B cells under CI/R conditions. It also downregulated pro-inflammatory cytokine transcription,including IL1B,IL6,and TNF. Inhibition of Nrf2 partially reversed these protective effects. In cold preservation solutions,4-OI upregulated Nrf2 target genes such as NQO1,HMOX1,and SLC7A11. In ALI airway models,4-OI enhanced bronchial epithelial barrier integrity and ciliary beat function after CI/R. In rat models,4-OI administration improved lung function and reduced pulmonary edema,tissue injury,apoptosis,and systemic inflammation following extended cold preservation or non-heart-beating lung transplantation. Incorporating 4-OI into cold preservation solutions appears promising for alleviating CI/R-induced bronchial epithelial injury and enhancing lung transplant outcomes via Nrf2 pathway activation. The online version contains supplementary material available at 10.1186/s12931-025-03151-7. View Publication

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that function in ribosome and spliceosome biogenesis,primarily by guiding modifying enzymes to specific sites on ribosomal RNA (rRNA) and spliceosomal RNA (snRNA). However,many orphan snoRNAs remain uncharacterized,with unidentified or unvalidated targets,and studies on additional snoRNA-associated proteins are limited. We adapted an enhanced chimeric eCLIP approach to comprehensively profile snoRNA-target RNA interactions using both core and accessory snoRNA-binding proteins as baits. Using core snoRNA-binding proteins,we confirmed most annotated snoRNA-rRNA and snoRNA-snRNA interactions in mouse and human cell lines and called novel,high-confidence interactions for orphan snoRNAs. While some of these interactions result in chemical modification,others may have modification-independent functions. We showed that snoRNA ribonucleoprotein complexes containing certain accessory proteins,like WDR43 and NOLC1,enriched for specific subsets of snoRNA-target RNA interactions with distinct roles in ribosome and spliceosome biogenesis. Notably,we discovered that SNORD89 guides 2′-O-methylation at two neighboring sites in U2 snRNA that fine-tune splice site recognition. Chimeric eCLIP of snoRNA-associating proteins enables a comprehensive framework for studying snoRNA-target interactions in an RNA-binding protein-dependent manner,revealing novel interactions and regulatory roles in RNA biogenesis. The online version contains supplementary material available at 10.1186/s13059-025-03508-7. View Publication

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) have greater potential for generating chondrocytes without hypertrophic and fibrotic phenotypes compared to bone marrow-derived mesenchymal stem/stromal cells (BMSCs). However,there is a lack of research demonstrating the use of autologous iMSCs for repairing articular chondral lesions in large animal models. In this study,we aimed to evaluate the effectiveness of autologous miniature pig (minipig) iMSC-chondrocyte (iMSC-Ch)-laden implants in comparison to autologous BMSC-chondrocyte (BMSC-Ch)-laden implants for cartilage repair in porcine femoral condyles. iMSCs and BMSCs were seeded into fibrin glue/nanofiber constructs and cultured with chondrogenic induction media for 7 days before implantation. To assess the regenerative capacity of the cells,19 skeletally mature Yucatan minipigs were randomly divided into microfracture control,acellular scaffold,iMSC,and BMSC subgroups. A cylindrical defect measuring 7 mm in diameter and 0.6 mm in depth was created on the articular cartilage surface without violating the subchondral bone. The defects were then left untreated or treated with acellular or cellular implants. Both cellular implant-treated groups exhibited enhanced joint repair compared to the microfracture and acellular control groups. Immunofluorescence analysis yielded significant findings,showing that cartilage treated with iMSC-Ch implants exhibited higher expression of COL2A1 and minimal to no expression of COL1A1 and COL10A1,in contrast to the BMSC-Ch-treated group. This indicates that the iMSC-Ch implants generated more hyaline cartilage-like tissue compared to the BMSC-Ch implants. Our findings contribute to filling the knowledge gap regarding the use of autologous iPSC derivatives for cartilage repair in a translational animal model. Moreover,these results highlight their potential as a safe and effective therapeutic strategy. The online version contains supplementary material available at 10.1186/s13287-025-04215-7. View Publication