技术资料

Affinity maturation and differentiation of B cells in the germinal center (GC) are tightly controlled by epigenetically regulated transcription programs,but the underlying mechanisms are only partially understood. Here we show that Cfp1,an integral component of the histone methyltransferase complex Setd1A/B,is critically required for GC responses. Cfp1 deficiency in activated B cells greatly impairs GC formation with diminished proliferation,somatic hypermutation and affinity maturation. Mechanistically,Cfp1 deletion reduces H3K4me3 marks at a subset of cell cycle and GC-related genes and impairs their transcription. Importantly,Cfp1 promotes the expression of transcription factors MEF2B and OCA-B and the Bcl6 enhancer-promoter looping for its efficient induction. Accordingly,Cfp1-deficient GCB cells upregulate IRF4 and preferentially differentiate into plasmablasts. Furthermore,Cfp1 ablation upregulates a panel of pre-memory genes with elevated H3K4me3 and leads to markedly expanded memory B populations. In summary,our study reveals that Cfp1-safeguarded epigenetic regulation ensures proper dynamics of GCB cells for affinity maturation and prevents the pre-mature exit from GC as memory cells. Cellular differentiation decisions,such as fates of B cells following entry into the germinal centres,are governed by epigenetically and transcriptionally regulated paths for bifurcating cell fates. Here the authors show that CFP1 is a master epigenetic regulator of activated B cells and controls their hypermutation and affinity maturation via the histone methyltransferase complex Setd1A/B. View Publication

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However,the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here,we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi,DNMT3A-3L-dCas9,CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering,we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types,including primary T cells. Additionally,we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons,including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together,our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells,broadening the use of epigenome editing in fundamental research and therapeutic applications. Epigenome editing programs gene silencing without inducing DNA breaks but challenges in delivery into human cells limit its broader use. Here,the authors present the RENDER platform,which uses virus-like particles to enable CRISPR-based epigenome editing for durable gene silencing in human cells. 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

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

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis and limited therapeutic options. Leukemic stem cells (LSCs),which drive disease progression and confer resistance to therapy,pose a significant challenge to conventional treatment strategies. In this study,we identified and characterized the inhibitory mechanisms of TH37,a small molecule derived from traditional Chinese medicine,which selectively targets AML blasts and LSCs. Our analyses identified peroxiredoxin 1 (PRDX1),an enzyme that catalyzes the breakdown of hydrogen peroxide (a reactive oxygen species),as the primary molecular target of TH37. We demonstrated that TH37 directly interacts with PRDX1,inhibiting its enzymatic activity and thereby elevating intracellular reactive oxygen species levels in AML cells. PRDX1 was found to be overexpressed in AML,and its expression correlated with poor prognosis and the activation of AML- and cancer-associated pathways. Targeting PRDX1,either through lentiviral short-hairpin RNA-mediated silencing or TH37 treatment,induced apoptosis,reduced colony formation,and impaired the engraftment and growth of AML cells in immunodeficient mouse models. Furthermore,TH37 synergized with conventional chemotherapeutic agent to significantly reduce the viability and colony-forming capacity of AML cells. These findings demonstrate the critical role of PRDX1 in AML pathogenesis and highlight its potential as a key therapeutic target to improve clinical outcomes for AML patients. View Publication

Hantaviruses are zoonotically transmitted from rodents to humans through the respiratory route,with no currently approved antivirals or widely available vaccines. The recent discovery of interhuman-transmitted Andes virus (ANDV) necessitates the systematic identification of cell tropism,infective potential,and potent therapeutic agents. We utilized human primary lung endothelial cells,various pluripotent stem cell-derived heart and brain cell types,and established human lung organoid models to evaluate the tropisms of Old World Hantaan (HTNV) and New World ANDV and Sin Nombre (SNV) viruses. ANDV exhibited broad tropism for all cell types assessed. SNV readily infected pulmonary endothelial cells,while HTNV robustly amplified in endothelial cells,cardiomyocytes,and astrocytes. We also provide the first evidence of hantaviral infection in human 3D distal lung organoids,which effectively modeled these differential tropisms. ANDV infection transcriptionally promoted cell injury and inflammatory responses,and downregulated lipid metabolic pathways in lung epithelial cells. Evaluation of selected drug candidates and pharmacotranscriptomics revealed that the host-directed small molecule compound urolithin B inhibited ANDV infection and restored cellular metabolism with minimal changes in host transcription. Given the scarcity of academic BSL-4 facilities that enable in vivo hantaviral studies,this investigation presents advanced human cell-based model systems that closely recapitulate host cell tropism and responses to infection,thereby providing critical platforms to evaluate potential antiviral drug candidates. Author summaryHantaviruses are fatal human pathogens that cause hemorrhagic fevers and are classified into either Old World or New World groups. Though most hantaviruses utilize zoonotic transmission,the New World Andes virus (ANDV) is unique in its ability to spread between humans. This distinct transmission mode underscores the need to investigate its cell tropism,pathogenicity,and therapeutic targets. Thus,we performed a systems-level comparison of the Old World Hantaan virus (HTNV) and New World hantaviruses,ANDV and Sin Nombre virus (SNV),using human lung,heart,and brain cell models,alongside lipidomic and transcriptomic profiling. We observed that ANDV exhibits broad tropism,infecting all tested cell types,including lung epithelial cells. HTNV replicated in lung endothelial,heart,and brain cells,whereas SNV replication was largely confined to lung endothelial cells. Notably,ANDV infection induced stronger host transcriptional changes,promoted cell injury and inflammatory responses,and suppressed lipid metabolic pathways in lung epithelial cells. Further drug testing and pharmacotranscriptomic analysis identified effective inhibitors of ANDV infection,including urolithin B,that restored cellular metabolism with minimal transcriptional disruption. This study provides a comparative framework for understanding hantavirus cell tropism and host responses and highlights potential antiviral candidates for treating these severe viral infections. View Publication

Angelman syndrome is a rare neurodevelopmental disorder caused by the loss of function of the maternally inherited UBE3A gene within the chr15q11-q13 region. This gene is subjected to a tissue-specific form of genomic imprinting leading to the silencing of the paternal allele in neurons. Angelman syndrome can result from various (epi)genetic mechanisms,with paternal uniparental disomy of chromosome 15 (patUPD15) being one of the rarest and least studied due to the absence of suitable models. To address this gap,we generated three independent induced pluripotent stem cell (iPSC) lines from individuals with Angelman syndrome caused by patUPD15,alongside genetically matched unaffected familial controls. Peripheral blood mononuclear cells (PBMCs) were reprogrammed into iPSCs using a non-integrative Sendai virus-based approach expressing the Yamanaka factors. All iPSC lines underwent rigorous quality control,confirming stem cell identity,trilineage differentiation potential,and genetic and epigenetic integrity. This newly established iPSC toolkit provides a powerful platform to investigate the molecular underpinnings of Angelman syndrome caused by patUPD15,paving the way for future translational research and therapeutic development tailored for this understudied form of the disorder. The online version contains supplementary material available at 10.1007/s13577-025-01287-8. View Publication

5-Ethynyl-2′-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast,efficient click chemistry detection. However,commercial EdU kits suffer from high costs,proprietary formulations,limited antibody multiplexing capabilities,and difficulties with larger biological specimens. Here,we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics),an optimized,affordable,and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency,brightness,and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types,including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs,including embryonic heart,forelimbs,and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development,cell proliferation,and DNA replication dynamics with unprecedented precision and flexibility. Subject areas: Biochemistry,Cell biology,Developmental biology,Computational bioinformatics View Publication

Human induced pluripotent stem cell (hiPSC) derived neurons are powerful tools to model disease biology in the drug development space. Here we leveraged a spectrum of neurophysiological tools to characterize iPSC-derived NGN2 neurons. Specifically,we applied these technologies to detect phenotypes associated with presynaptic dysfunction and rescue in NGN2 neurons lacking a synaptic vesicle associated protein MUNC18-1,encoded by syntaxin binding protein 1 gene (STXBP1). STXBP1 homozygous knock out NGN2 neurons lacked miniature post synaptic currents and demonstrated disrupted network bursting as assayed with multielectrode array and calcium imaging. Furthermore,knock out neurons released less glutamate into culture media,consistent with a presynaptic deficit. These synaptic phenotypes were rescued by reconstitution of STXBP1 protein by AAV transduction in a dose-dependent manner. Our results identify a complementary suite of physiological methods suitable to examine the modulation of synaptic transmission in human neurons. View Publication

The principal organization of mammalian neuromuscular junctions (NMJs) shares essential features across species. However,human NMJs (hNMJs) exhibit distinct structural and physiological properties. While recent advances in stem-cell-based systems have significantly improved in vitro modeling of hNMJs,the extent to which these models recapitulate in vivo development remains unclear. Here,we performed temporal transcriptomic analysis of human three-dimensional (3D) neuromuscular co-cultures,composed of iPSC-derived motoneurons and skeletal muscle engineered from primary myoblasts. We found that the expression pattern follows a temporally coordinated gene expression program underlying NMJ maturation. The model recapitulates transcriptional features of NMJ development,including early myoblast fusion and presynaptic development,followed by a late-stage upregulation of postsynaptic markers and embryonic AChR subunits. Importantly,comparable transcriptional dynamics across two independent hiPSC lines confirm the reproducibility and robustness of this system. This study confirms on a transcriptional level that human 3D neuromuscular co-cultures are a robust and physiologically relevant model for investigating hNMJ development and function. 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

Neural crest stem cells (NCSCs) compose a highly migratory,multipotent,stem cell population arising from the neural plate border of the embryonic ectoderm. Investigating the development of NCSCs is critical in understanding both embryonic development and abnormal events that underlie neurocristopathies. Suggested seeding densities in in vitro human induced pluripotent stem cells (hiPSCs) differentiation protocols,varying between 10,000 cells/cm 2 and 200,000 cells/cm 2,demonstrate a lack of consensus on the optimal conditions to obtain NCSCs. Aiming to maximize the differentiation efficiency of hiPSCs towards the NCSCs lineage,we investigated the effect of the initial seeding density on NCSCs lineage commitment,both in fibroblast- and human peripheral blood mononuclear cell (PBMC)-derived hiPSCs. Cultures were characterized with gene and protein expression analysis assessing stemness ( OCT3/4 and NANOG ),neural crest identity ( SNAI2 and SOX10 ) and neuroectoderm identity ( PAX6 and SOX1 ). We demonstrate that reaching a confluent monolayer of cells by the end of the differentiating protocol is crucial to obtaining NCSCs from hiPSCs. To achieve this,our results indicated 17,000 cells/cm 2 is the optimal initial seeding density. Under this protocol,a confluent monolayer was reached after 8 days of differentiation and an average of 89% SOX10 positive cells were obtained. The fold change of SNAI2 and SOX10 expression was 11-fold and 17-fold higher,respectively,in cultures seeded with 17,000 cells/cm 2,compared to the highest tested density of 200,000 cells/cm 2 . In contrast,seeding 200,000 cells/cm 2 induced neuroectoderm-like cells,confirmed by an average of 45% of cells marking positive for PAX6. With this work,we demonstrate the importance of achieving cellular confluency during NCSCs differentiation. View Publication