技术资料

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

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

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however,its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here,we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids,revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation,expressed SOX2,and lacked CRX,consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas,particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities,demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1,Flavopiridol) and the cell-cycle inhibitor Volasertib,indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively,our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma,establishes a robust and clinically relevant human retinal organoid platform,and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype. View Publication

BackgroundDiabetic peripheral neuropathy (DPN) is one of the most prevalent and debilitating complications of diabetes,marked by chronic neuroinflammation,immune dysregulation,and progressive neuronal degeneration. Current treatments offer limited efficacy,largely focusing on symptomatic relief rather than addressing the underlying disease mechanisms. There is a critical need for disease-modifying therapies that target the molecular basis of DPN.ResultsIn this study,we developed a novel targeted nanotherapeutic system—ZH-1c-EVs@SIN—composed of neural stem cell-derived extracellular vesicles (NSC-EVs) modified with the ZH-1c aptamer and loaded with the anti-inflammatory compound sinomenine (SIN). This system was specifically designed to target microglia and inhibit the WNT5a/TRPV1 signaling pathway. Transcriptomic profiling of microglia revealed key gene networks implicated in DPN pathology and responsive to SIN treatment. Functional assays demonstrated that ZH-1c-EVs@SIN facilitated a shift in microglial phenotype from pro-inflammatory M1 to anti-inflammatory M2,significantly reduced inflammatory cytokine expression,and restored levels of neuronal regulatory proteins. Nanoparticle tracking analysis and transmission electron microscopy confirmed optimal vesicle size and morphology,while fluorescence imaging showed efficient uptake by microglia. In vivo studies in a murine model of DPN revealed marked improvements in pain-related behavior and histopathological signs of nerve damage.ConclusionZH-1c-EVs@SIN represents a promising therapeutic strategy for DPN,offering targeted immunomodulation and enhanced neural repair via regulation of the WNT5a/TRPV1 signaling axis. This nano-delivery platform introduces a novel and precise approach to intervening in diabetic neuropathy and may be applicable to other neuroinflammatory conditions.Graphical abstractMechanism of ZH-1c-EVs@SIN Mediating the WNT5a/TRPV1 Pathway to Improve Immune-Inflammatory Homeostasis in the Treatment of DPN in Mice. View Publication

Embryonic stem cells (ESC) are pluripotent,with the potential to differentiate into multiple cell types,making them a valuable tool for regenerative medicine and disease therapy. However,common culture methods face challenges,including strict operating procedures and high costs. Currently,Leukemia inhibitory factor (LIF),an indispensable bioactive protein for ESC culture,is typically applied to maintain self-renewal and pluripotency,but its instability and high cost limit its effectiveness in stable culture conditions. Hence,we have developed an innovative strategy using a soluble nanomaterial,metal-organic polyhedra (MOPs),to effectively maintain the self-renewal and pluripotency of ESC. The selected amino-modified vanadium-based MOP not only exhibits excellent biocompatibility and high stability but also possesses similar or even superior biological functions compared to commercial LIF. Due to the precise structure of MOPs,the active site responsible for maintaining ESC pluripotency has been identified and regulated at the molecular level. The new ESC culture method significantly reduces costs,simplifies preparation,and enhances the practicality of biopharmaceutical preparation and storage. This represents the first case of using MOPs to maintain self-renewal of ECS,opening an avenue for introducing advanced materials into the development of innovative ESC culture methods. Subject terms: Biomaterials - cells,Chemical biology View Publication

Craniosynostosis is a multigenic congenital condition in which one or more calvarial sutures have prematurely fused during the development of the fetus. Pathogenic variants in FGFR2 are associated with the development of syndromic craniosynostosis,such as Crouzon,Apert and Pfeifer syndromes. Investigation of FGFR2 -linked craniosynostosis is hindered by the lack of appropriate in vitro models. Patient-derived human induced pluripotent stem cell (hiPSC) in vitro disease models provide the opportunity to investigate the disease,identify molecular targets for pharmaceutical treatments,and enable the generation of autologous pluripotent stem cell catalogues. Here,we report three patient-derived hiPSC lines carrying the C342Y,S252W or E565G FGFR2 pathogenic variant. The patient hiPSC lines express characteristic pluripotency markers and display distinct phosphorylation profiles under unstimulated conditions. FGFR2 C342Y showed autophosphorylation in the absence of bFGF ligand,although downstream docking proteins PLCγ and FRS2α were not phosphorylated. FGFR2 S252W and FGFR2 E565G hiPSCs showed increased phosphorylation of docking proteins PLCγ and FRS2α,whereas FGFR2 was not phosphorylated. These patient hiPSC lines provide molecular and cellular options to investigate FGFR2 -linked craniosynostosis in the patient-specific genomic context and develop therapeutic modalities. View Publication

Transposable elements (TEs) are genomic elements present in multiple copies in mammalian genomes. TEs were thought to have little functional relevance but recent studies report roles in biological processes,including embryonic development. To investigate the expression dynamics of TEs during human early development,we generated long-read sequence data from human pluripotent stem cells (hPSCs) in vitro differentiated to endoderm,mesoderm,and ectoderm lineages to construct lineage-specific transcriptome assemblies and accurately place TE sequences. Our analysis reveals that specific TE superfamilies exhibit distinct expression patterns. Notably,we observed TE switching,where the same family of TE is expressed in multiple cell types,but originates from different transcripts. Interestingly,TE-containing transcripts exhibit distinct levels of transcript stability and subcellular localization. Moreover,TE-containing transcripts increasingly associate with chromatin in germ layer cells compared to hPSCs. This study suggests that TEs contribute to human embryonic development through dynamic chromatin interactions. Transposable elements are genetic parasites that have colonised genomes and they express as parts of coding and noncoding RNAs. Here,the authors explore how they are expressed in transcripts in normal human development,and how they alter transcript dynamics. View Publication

Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses,but have largely not addressed the innate immune cells,such as neutrophils,that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments,we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases. Hypoimmune gene editing in human pluripotent stem cells (hPSCs) provides a promising platform for cellular therapies. Here,the authors report that CRISPR mediated deletion of ICAM-1 in hPSC-derived grafts reduces immune cell adhesion,dampens T cell activation,and protects against immune rejection. View Publication

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics,yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput,low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation,allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities,demonstrating efficient delivery of various cargo,with high rates of transfection,gene knockout via non-homologous end joining,and precise knock-in through homology-directed repair. To showcase its utility in functional genomics,we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells,identifying novel regulators of exhaustion,including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13532-z. View Publication

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However,its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here,we investigated the role of CD24a,the murine CD24 gene,in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.MethodsClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations,including myeloid-derived suppressor cells (MDSCs),natural killer cells,T cells,and macrophages,within tumors,spleens,and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.ResultsKnocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity,effects that were partially reversed by re‐expression of CD24a. Moreover,in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout,underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore,in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1,CXCL5,and CXCL10,chemokines known to recruit gMDSCs,further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.ConclusionsOur findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state,thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-025-01165-3. View Publication

T-cell exhaustion contributes to immunotherapy failure in chronic lymphocytic leukemia (CLL). Here,we analyze T cells from CLL patients’ blood,bone marrow,and lymph nodes,as well as from a CLL mouse model,using single-cell RNA sequencing,mass cytometry,and tissue imaging. T cells in CLL lymph nodes show the most distinct profiles,with accumulation of regulatory T cells and CD8+ T cells in various exhaustion states,including precursor (TPEX) and terminally exhausted (TEX) cells. Integration of T-cell receptor sequencing data and use of the predicTCR classifier suggest an enrichment of CLL-reactive T cells in lymph nodes. Interactome studies reveal potential immunotherapy targets,notably galectin-9,a TIM3 ligand. Inhibiting galectin-9 in mice reduces disease progression and TIM3+ T cells. Galectin-9 expression also correlates with worse survival in CLL and other cancers,suggesting its role in immune evasion and potential as a therapeutic target. Multi-omics can be used to characterise tumour and immune cell populations. Here the authors use multi-omics to characterise CLL blood and tissue samples and use prediction models for CLL TCR specificity and implicate interactions between galectin-9 and TIM3 as involved in CLL immune escape and propose galectin-9 as a possible immunotherapy target. View Publication

AbstractObjectivesTumour necrosis factor inhibitors (TNFi) are widely used and effective as treatment for immune-mediated inflammatory diseases (IMIDs). However,TNFi therapy causes a faster waning of antibody responses following vaccination. The underlying cause by which TNFi affect humoral immunity remains to be elucidated. The formation of long-lasting,high-affinity antibodies after vaccination results from germinal centre (GC)-derived,T cell-dependent B-cell responses. Therefore,this study investigated how TNFi affect the formation and maintenance of antigen-specific B- and CD4+ T-cell responses following SARS-CoV-2 mRNA vaccination.MethodsSARS-CoV-2 spike-specific B-cell responses were characterised using spectral flow cytometry. Spike-specific CD4+ T cells were measured using an activation-induced marker assay. 15 patients with inflammatory bowel disease (IBD) treated with TNFi were compared with 9 IBD patients without systemic immunosuppression and 10 healthy controls.ResultsSpike-specific CD4+T-cell frequency and phenotype,including T follicular helper cells,were not affected by TNFi. Total spike-specific B-cell frequencies were reduced in TNFi-treated patients. Deep phenotyping revealed lower IgG+memory B-cell frequencies in TNFi-treated patients 3–6 months after vaccination. These data were confirmed in TNFi-treated rheumatoid arthritis patients. Interestingly,already at day 7 after the second vaccination,TNFi therapy reduced the induction of class-switched CD11c- CD71+activated B cells,which are believed to be GC-derived. Conversely,CD11c+B cells,associated with extrafollicular B-cell responses,were not affected by TNFi therapy.ConclusionsThese data suggest that TNFi therapy affects the differentiation of GC-derived B cells,which may explain its effect on humoral immune responses. View Publication