技术资料

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro,yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin),culture media (Neurobasal™ versus Brainphys™ Imaging media),and seeding density (1 × 105 versus 2 × 105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein,then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction,an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys™ Imaging medium was observed to support neuron viability,outgrowth,and self-organisation to a greater extent than Neurobasal™ medium with either laminin type,while the combination of Neurobasal™ medium and human laminin reduced cell survival. Further,a higher seeding density fostered somata clustering,but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments,which is positively mediated by light-protective compounds found in Brainphys™ Imaging medium.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04591-0. View Publication

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. View Publication

Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Pathogenic mutations in Bcl2-associated athanogene 3 (BAG3) cause genetic dilated cardiomyopathy (DCM),a disease characterized by ventricular dilation,systolic dysfunction,and fibrosis. Previous studies have demonstrated that BAG3 mediates sarcomeric protein turnover through chaperone-assisted selective autophagy to maintain sarcomere integrity in the human heart. Although mouse models provide valuable insights into whole-organism effects of BAG3 knockout or pathogenic variants,their utility is limited by species-specific differences in pathophysiology,which often do not translate to humans and contribute to the failure of clinical trials. As a result,the development of induced pluripotent stem cell-derived cardiomyocytes (iCM) and engineered heart tissues presents a promising alternative for studying adult-onset cardiac diseases. Here,we used genome engineering to generate an isogenic pseudo-wild-type control cell line from a patient-derived iPSC line carrying a pathogenic BAG3 variant,clinically presenting with DCM. While monolayer iCMs recapitulated some features of BAG3-mediated DCM,such as reduced autophagy,mitochondrial membrane potential,and decreased HSPB8 stability,they failed to develop the age-associated impairment in sarcomere integrity. Therefore,we developed a mature,patient-specific,human engineered heart tissue model of BAG3-mediated DCM and compared it to its isogenic healthy control. We demonstrated successful recapitulation of adult-like features of the clinically observed disorganized sarcomeres and impaired tissue contractility,thereby providing a platform to investigate BAG3-related pathophysiology and therapeutic interventions. Graphical abstract View Publication

Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them,3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However,primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues,recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study,we developed a protocol to differentiate high-purity skin cells,such as fibroblasts (hFIBROs) and keratinocytes (hKERAs),from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE),a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently,hKERAs were seeded onto the dermis,and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover,exposure to Triton X-100,a known skin irritant,led to marked epidermal damage and significantly reduced cell viability,validating the model’s functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications,including the replacement of animal testing. View Publication

Alzheimer's disease (AD) is characterized by tau pathology,leading to neurodegeneration. Astrocytes regulate central nervous system homeostasis and influence AD progression. The APOE3‐Christchurch (APOE3‐Ch) variant is linked to AD resilience,but its protective mechanisms remain unclear. Human induced pluripotent stem cell–derived astrocytes (APOE3‐Ch and wild type) were used to assess tau uptake,clearance,lipid metabolism,and transcriptomic adaptations. Fluorescently labeled 2N4R‐P301L tau oligomers were tracked,and pathway‐specific inhibitors dissected tau clearance mechanisms. Lipidomic and transcriptomic analyses were performed to identify genotype‐specific adaptations. APOE3‐Ch astrocytes exhibited enhanced tau uptake via heparan sulfate proteoglycan‐ and lipoprotein receptor‐related protein 1‐mediated pathways and superior clearance through lysosomal and proteasomal degradation. They exported less tau,limiting propagation. Transcriptomic analyses revealed upregulation of genes involved in cell projection assembly and endocytosis. Lipidomic profiling showed reduced ceramides and gamma‐linolenic acid,linked to decreased neuroinflammation and ferroptosis. APOE3‐Ch astrocytes promote tau clearance and metabolic adaptations,providing insights into genetic resilience in AD and potential therapeutic targets. APOE3‐Christchurch (APOE3‐Ch) astrocytes exhibit significantly increased tau internalization compared to wild‐type astrocytes,facilitated by upregulated heparan sulfate proteoglycan and low‐density lipoprotein receptor‐related protein 1 pathways. APOE3‐Ch astrocytes demonstrate more efficient tau degradation via both lysosomal and proteasomal pathways,while exporting significantly less tau,potentially reducing tau propagation in the central nervous system. APOE3‐Ch astrocytes show upregulation of genes involved in cell projection assembly and endocytosis,suggesting structural and functional modifications that enhance tau processing. Lipidomic profiling reveals reduced ceramide levels and gamma‐linolenic acid downregulation in APOE3‐Ch astrocytes,alterations linked to reduced neuroinflammatory and ferroptotic activity,contributing to the protective phenotype. View Publication

Brain Microphysiological Systems,including neural organoids derived from human induced pluripotent stem cells,offer a unique lens to study the intricate workings of the human brain. This paper investigates the foundational elements of learning and memory in neural organoids by quantifying immediate early gene expression in response to chemical modulation,input-specific short- and long-term synaptic plasticity,neuronal network dynamics,connectivity,and criticality to demonstrate the utility of these organoids in basic science research. Neural organoids showed synapse formation,glutamatergic and GABAergic receptor expression,immediate early gene expression basally and evoked,functional connectivity,criticality,and synaptic plasticity in response to theta-burst stimulation. In addition,pharmacological interventions on GABAergic and glutamatergic receptors and input-specific theta-burst stimulation further shed light on the capacity of neural organoids to mirror synaptic modulation,specifically short- and long-term potentiation and depression,demonstrating their potential as tools for studying neurophysiological and neurological processes and informing therapeutic strategies for diseases. Neural organoids exhibit key aspects of learning and memory,including input-specific synaptic plasticity,basal and evoked immediate early gene expression,and critical network dynamics,highlighting their value in modeling human neurophysiology. View Publication

Natural killer (NK) cells,which are key components of the innate immune response,are crucial for ensuring the efficacy of vaccines as they rapidly eliminate infected cells and enhance the adaptive immune response,ensuring robust and lasting protection. In this report,we investigated the effect of Cladophora wrightiana var. minor (CW) extract,a marine alga,in activating NK cells,as an adjuvant to inactivated A/Puerto Rico/8/34 H1N1 influenza vaccine (iPR8). In vitro,CW extract significantly enhanced the level of activation markers CD69 and CD107a on NK cells and triggered intracellular secretion of interferon gamma (IFN‐γ) and granzyme B (GrB),indicating effective NK cell stimulation and cytotoxic function. In vivo,CW extract promoted substantial NK cell recruitment and activation,resulting in higher NK cell populations and elevated post‐immunization levels of activation markers. Additionally,CW extract increased IFN‐γ and GrB production in CD8+ T cells,highlighting its broader impact on the immune response. We also found direct evidence that CW‐activated NK cells and dendritic cells (DCs) interacted with and induced the activation of immature DCs and resting NK cells,respectively. These findings suggest that CW extract is a promising adjuvant for nasal vaccines,enhancing cellular immunity by activating NK cells and supporting interactions with DCs and CD8+ T cells. Cladophora wrightiana var. minor (CW) extract,administered as an adjuvant with inactivated influenza virus (iPR8),stimulates both innate and adaptive immune responses. CW enhances NK cell activation,cytotoxic function,and reciprocal crosstalk with dendritic cells,while also promoting CD8+ T cell responses and antigen‐specific IgG production. These findings support CW as a potent nasal vaccine adjuvant capable of boosting protective immunity. https://app.biorender.com/illustrations/6893f7d09e3fc89e9d953f76?slideId=2a6a42b6‐a3d9‐400a‐b839‐fa297b3108c5. View Publication

Metastatic breast cancer remains largely incurable,and the mechanisms driving the transition from primary to metastatic breast cancer remain elusive. We analyzed the complex landscape of estrogen receptor (ER)-positive breast cancer primary and metastatic tumors using scRNA-seq data from twenty-three female patients with either primary or metastatic disease. By employing single-cell transcriptional profiling of unpaired patient samples,we sought to elucidate the genetic and molecular mechanisms underlying changes in the metastatic tumor ecosystem. We identified specific subtypes of stromal and immune cells critical to forming a pro-tumor microenvironment in metastatic lesions,including CCL2+ macrophages,exhausted cytotoxic T cells,and FOXP3+ regulatory T cells. Analysis of cell-cell communication highlights a marked decrease in tumor-immune cell interactions in metastatic tissues,likely contributing to an immunosuppressive microenvironment. In contrast,primary breast cancer samples displayed increased activation of the TNF-α signaling pathway via NF-kB,indicating a potential therapeutic target. Our study comprehensively characterizes the transcriptional landscape encompassing primary and metastatic breast cancer. View Publication

BackgroundLung cancer has become one of the most fatal cancers at present. Traditional treatments showed limited therapeutic effects on lung cancer. The phototherapy has emerged as a powerful approach for lung cancer treatment. Aggregation-induced emission luminogens (AIEgens) exhibit excellent optical performance such as strong fluorescence,enhanced reactive oxygen species (ROS) generation,and effective thermal effect after aggregation,which show great potential in phototherapy. However,the disadvantages including hydrophobicity,low specificity,and short circulation lifetime limited their efficacy on cancer therapy.MethodsWe developed a biomimetic AIEgens constructed using CD8+ T cells membrane to camouflage the AIEgen C41H37N2O3S2 (named BITT) nanoparticles (termed TB). The prepared TB improved the tumor accumulation of AIEgen by PD-1/PD-L1 recognition on the CD8+ T and LLC cell membranes,respectively.ResultsThe prepared TB showed improved binding efficiency,photothermal effects,and ROS generation ability to kill the lung cancer cells. TB also showed improved circulation lifetime and excellent tumor targeting ability,leading to effective phototherapy and immunotherapy in vivo based on BITT and the CD8+ T cell-derived membranes. Based on the AIE and immune checkpoint blockade (ICB) strategies,TB enhanced the antitumor activities of lung cancer by phototherapy and immunotherapy.ConclusionThe present work developed a type of biomimetic AIEgens,which overcame the inherent limitations of conventional AIEgens and leveraged immune recognition for targeted tumor accumulation. Furthermore,the integration of AIE-driven phototherapy with immune checkpoint blockade demonstrated potent synergistic antitumor efficacy,establishing a promising combinatorial strategy against aggressive lung malignancies. View Publication

RAB7,encoded by RAB7A in humans and Rab7 in mice,is a small GTPase that catalyzes endosome maturation. It mediates NF-κB activation through the assembly of intracellular membrane signalosomes in stimulated normal B cells and plays a B cell-intrinsic role in the antibody response in mice. Here we show RAB7A transcripts are expressed in primary diffuse large B-cell lymphomas (DLBCLs),and that RAB7 protein expression is heightened in activated human tonsil B cells as well as in DLBCL and Burkitt lymphoma cell lines. Treating these cell lines with CID1067700,a selective small-molecule RAB7 inhibitor,results in a dose-dependent decrease in cell growth,associated with impaired proliferation and survival. CID1067700 also suppressed tumor development from Daudi cells,a Burkitt lymphoma cell line,in Foxn1nu/nu nude mice. The inhibitory effect of CID1067700 on Daudi cell growth in vitro is further enhanced by methyl-β-cyclodextrin,which disrupts plasma membrane lipid rafts,and by FX1,a BCL6 inhibitor. These findings,together with the unfavorable prognosis of DLBCL patients showing high RAB7A expression,suggest that targeting RAB7 is a promising therapeutic approach for mature B cell-derived lymphomas. View Publication

BackgroundAdaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability,but its functional role in human effector and regulatory CD4⁺ T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation,particularly in helper T cells function and regulatory T cells.MethodsPrimary human CD4⁺ T cells,including thymus-derived and induced regulatory T cells (Treg),were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays,flow cytometry,cytokine quantification,and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.ResultsThymus-derived,TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells,which showed pronounced up-regulation of TRAT1 following activation. In effector T cells,deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However,this was accompanied by reduced production of interleukin-17,which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4⁺ target cells via up-regulation of LAP/GARP but reduced suppression of CD8⁺ target cells,an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.ConclusionsTRAT1 acts as a dual regulator of human CD4⁺ T cell function,limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02429-z. View Publication