技术资料

DNA repair mechanisms in human primary cells,including error-free repair,and,recurrent nuclease cleavage events,remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR),an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs,small indels,large deletions,and other aberrations in absolute terms in clinically relevant edited cells,including HSPCs,iPSCs,and T-cells. By quantifying up to 90% of loci with unresolved DSBs,CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore,we accurately quantify DNA repair precision,revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics,paving the way for mechanistic studies to advance gene therapy,designer editors,and small molecule discovery. Quantifying genomic aberrations resulting from designer nucleases activity is essential for gene therapy clinical translation. Here,the authors present a modular digital PCR technique that profiles DNA repair precision and cut-repair cycles at the edited loci,exposing current evaluation biases. View Publication

Since the COVID-19 pandemic,there has been a documented rise in the incidence of neurological manifestations among individuals complicated with encephalitis or myelitis. The spectrum of neurological symptoms associated with HCoVs infections is expanding. However,the infection characteristics and pathogenesis of seasonal HCoVs to the central nervous system remain obscure. No pharmacological agents have demonstrated the capacity to specifically and efficaciously mitigate the neurological symptoms induced by HCoVs infections to date. Methods: We developed human cerebral organoids (HCOs) derived from human induced pluripotent stem cells and established a blood–brain barrier (BBB) HCOs co-culture model. We subjected these models to seasonal human coronavirus (HCoV) infections to investigate the viral characteristics within the central nervous system (CNS). Utilizing RNA sequencing,we conducted a preliminary exploration of the mechanisms underlying virus-induced inflammatory responses in the CNS. Furthermore,we assessed the efficacy of antiviral and anti-inflammatory drugs using the HCO model. Results: Our results showed that among seasonal coronaviruses,HCoV-OC43 replicates efficiently within the organoids,primarily targeting neurons and astrocytes,and disrupts the barrier function of the BBB. RNA sequencing analysis revealed that HCoV-OC43 infection triggers an inflammatory response through the TNF and NF-κB signaling pathways,leading to cell death,impaired neuronal function,and disrupted interneuron signaling. Interestingly,Bardoxolone methyl (CDDO-Me) demonstrated antiviral effects comparable to remdesivir,reducing both inflammation and cell death. Conclusions: Conclusively,HCOs infected with HCoV-OC43 offer valuable insights into the pathogenesis of HCoVs in central nervous system (CNS),and might serve as a tool for developing novel therapeutic strategies for HCoVs infections,including COVID-19,especially on exploring treatment candidates.Graphical abstract View Publication

BackgroundCancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy,but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells,making them easier targets of anti-tumor immunity. Methods: Prexasertib was used to block CHK1 in OC cell lines and xenografts,and its cytotoxicity was assessed in vitro and in vivo. In vitro tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR,Western blot,flow cytometry,and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth,tumor cell stemness,and tumor infiltrating lymphocytes via tumor volume monitoring,immunohistochemistry,and flow cytometry. Results: Prexasertib effectively inhibited CHK1 phosphorylation,exhibited significant anti-tumor effects in vitro and in vivo,accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system,and Prexasertib treatment suppressed sphere formation and reduced the ALDH+ cell fraction. Unexpectedly,Prexasertib upregulated PD-L1 expression in tumor cells. In vivo,combining Prexasertib with Atezolizumab led to more remarkable remission of tumors,when compared with Prexasertib or Atezolizumab alone. Meanwhile,the tumor-infiltrating CD8+ T cells significantly increased in the combination group,while exhausted T cells decreased; the treatments did not affect CD4+ cell infiltration. Conclusions: Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity. View Publication

Hepatocellular carcinoma (HCC) is the most prevalent primary malignancy of the liver. Characterized by rapid progression and poor overall survival rates,HCC requires effective and streamlined treatment regimens. It predominantly occurs in East Asia and sub-Saharan Africa,where it has historically been managed with herbal formulas. We previously observed that the herbal formula HO-1089 exerts potent anti-HCC effects both in vitro and in vivo. In this study,we investigated the anticancer efficacy and mechanisms of HO-1197,a reconstituted herbal formulation derived from HO-1089. HO-1197 selectively inhibited the viability of HCC cell lines without hepatotoxicity and demonstrated superior anticancer activity compared with both HO-1089 and sorafenib. Mechanistically,HO-1197 induced apoptosis and G2/M arrest through reactive oxygen species-mediated DNA damage,independent of p53 status. Transcriptomic analysis revealed downregulation of mitosis-related genes,particularly those regulated by FOXM1,a key driver of HCC proliferation and metastasis. HO-1197 suppressed FOXM1 expression and nuclear translocation,reducing its downstream targets and diminishing angiogenic and metastatic potential. Furthermore,HO-1197 modulated the tumor immune microenvironment by promoting pro-inflammatory macrophage polarization and enhancing natural killer cell-mediated cytotoxicity. HO-1197 exhibited potent antitumor efficacy,and combination therapy with HO-1197 and sorafenib exhibited synergistic effects in both two-dimensional and immune-activated multicellular spheroid models. These findings suggest that HO-1197 is a promising multifunctional therapeutic candidate with antitumor,antiangiogenic,antimetastatic,and immunomodulatory properties. Its combination with sorafenib may offer effective treatment for HCC. HO-1197,which demonstrated strong efficacy,is a novel herbal medicine developed by H&O Biosis and is referred to as an Integrated Natural Medicine. View Publication

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However,access to such cells is limited,and studies systematically mapping the spectrum of their inflammatory states are scarce. Here,we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust,accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling,revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli,including pro-inflammatory cytokines (TNFα,interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1),TLR3 (Poly(I:C)),TLR4 (lipopolysaccharide,LPS),and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g.,oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally,the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1,LPS,or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism,highlighting the role of soluble mediators in the signal propagation. Altogether,this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation. View Publication

The differentiation of pluripotent stem cells into neurons is an essential area of biomedical research,with significant implications for understanding neural development and treating neurological diseases. This study compares neural cultures derived from a common induced pluripotent stem cell line (KYOU-DXR0109B) generated by two widely adopted methods: DUAL SMAD inhibition and exogenous NGN2 overexpression. The DUAL SMAD inhibition method,which differentiates through the neural stem cell stage,produces heterogeneous cultures containing a mix of neurons,neural precursors,and glial cells. Conversely,NGN2 overexpression generates more homogeneous cultures composed predominantly of mature neurons. Transcriptomic analysis revealed significant differences in neural gene markers expression profiles,with cultures from the DUAL SMAD inhibition method enriched in neural stem cell and glial markers,while NGN2 overexpression cultures showed elevated markers for cholinergic and peripheral sensory neurons. This study underscores the importance of choosing appropriate differentiation protocols based on the desired cell types,as each method yields neural cultures with distinct cellular compositions. Understanding these differences can help optimize protocols for specific research and therapeutic applications. View Publication

Retinal neurogenesis is mediated by the coordinated activities of a complex gene regulatory network (GRN) of transcription factors (TFs) in multipotent retinal progenitor cells (RPCs). How this GRN mechanistically guides neural competence remains poorly understood. In this study,we present integrated transcriptional,genetic and genomic analyses to uncover the regulatory mechanisms of SOX2,a key factor in establishing neural identity in RPCs. We show that SOX2 is preferentially enriched in the RPC-specific enhancer landscape associated with essential regulators of retinogenesis. Disruption of SOX2 expression impairs retinogenesis,marked by a selective loss of enhancer activity near genes essential for RPC proliferation and lineage specification. We identified the RPC transcription factor VSX2 as a binding partner for SOX2 and,together,SOX2 and VSX2 co-target a core,retina-specific chromatin repertoire characterized by enhanced TF binding and robust chromatin accessibility. This cooperative binding establishes a shared SOX2-VSX2 transcriptional code that promotes the expression of crucial regulators of neurogenesis while repressing the acquisition of alternative lineage cell fate. Our data illuminate fundamental biological insights on how transcription factors act in concert to drive chromatin-based genetic programs underlying retinal neural identity. View Publication

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs),only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients,the antitumor immune response is defective,conferring resistance and promoting tumor progression. Therefore,the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs),at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate,progression-free survival (PFS),overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy,higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs,identified patients with significantly worse survival. Furthermore,CD137⁺Tregs resulted also positively correlated with worse PS,while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab,with a significant increase in non-responsive patients during therapy. Interestingly,both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137−Tregs and LOX-1⁻PMN-MDSCs,respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis,as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy,improving patient selection and suggesting novel personalized therapies. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. View Publication

The T-cell receptor (TCR) initiates T-lymphocyte activation,but the mechanism of TCR activation remains uncertain. Here,we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent,the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast,the HLA-bound complex adopts the open and extended conformation,and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together,these results establish allosteric conformational change during TCR activation,reveal avenues for immunotherapeutic engineering,and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here,the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs,revealing large conformational changes during activation. View Publication

Staphylococcal enterotoxins (SE) crosslink the MHC‐II on antigen‐presenting cells (APC) with the T‐cell receptor,inducing a polyclonal T‐cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T‐cell activation,the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte‐derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA‐induced enrichment of gene pathways associated with inflammation,infection,and dermatitis,effects that were amplified in the presence of T cells. Phenotypic and functional characterization showed that SEA‐primed monocytes differentiated into MDM with an altered polarization,deviating from classical M1/M2 pathways. SEA‐primed MDM exhibited downregulation of key markers,including HLA‐DR,CD80,CD86,and PD‐L1. Functional assays demonstrated that SEA‐primed MDM pushed hyperinflammatory T‐cell responses,with significantly enhanced proliferation and IFN‐γ secretion. In contrast,following SEA‐priming,moDC retained robust antigen‐presenting capabilities and displayed enhanced expression of molecules involved in T‐cell interactions. These findings provide mechanistic insights into SEA‐mediated immune modulation,illustrating how SEA reprograms MDM functions and amplifies proinflammatory T‐cell responses. This advances our understanding of superantigen‐driven immune interactions,offering a foundation for developing therapeutic strategies to mitigate superantigen‐mediated immune conditions. Staphylococcal enterotoxin A (SEA) alters monocyte differentiation and function,while preserving T cell stimulatory capacity. SEA‐primed macrophages downregulate antigen‐presenting markers yet drive heightened T‐cell proliferation and IFN‐γ secretion. These findings reveal mechanisms of SEA‐mediated immune modulation and superantigen‐driven inflammation. View Publication