技术资料

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However,cellular and humoral immunogenicity of genome editing tools,which originate from bacteria,complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases,SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis,we identify putative immunogenic epitopes on each nuclease. Using computational modeling,we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components,including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses,yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9,but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection. Subject terms: Protein design,Immunogenetics,CRISPR-Cas9 genome editing View Publication

Optical aberrations hinder fluorescence microscopy of thick samples,reducing image signal,contrast,and resolution. Here we introduce a deep learning-based strategy for aberration compensation,improving image quality without slowing image acquisition,applying additional dose,or introducing more optics. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks,making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations and experiments to show that applying the trained ‘de-aberration’ networks outperforms alternative methods,providing restoration on par with adaptive optics techniques; and subsequently apply the networks to diverse datasets captured with confocal,light-sheet,multi-photon,and super-resolution microscopy. In all cases,the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation,including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos. Subject terms: Microscopy,Fluorescence imaging View Publication

Th2 inflammation and epithelial-mesenchymal transition (EMT) play crucial roles in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to investigate the hypothesis that MMP-12,produced by M2 macrophages,induces EMT in nasal epithelial cells,thereby contributing to airway inflammation and remodeling in CRSwNP. The expression levels of MMP-12 were measured by RT-PCR in CRS nasal mucosa and THP-1 cells. mRNA and protein levels of E-cadherin,vimentin,α-SMA,and fibronectin were determined using RT-PCR,western blotting,and immunofluorescence staining in primary nasal epithelial cells and air-liquid interface culture. The expression of MMP-12 was significantly increased in CRSwNP and M2-like THP-1 cells. In co-culture with primary nasal epithelial cells and M2-like THP-1 cells,E-cadherin expression was inhibited,and fibronectin,vimentin,and α-SMA expression were increased. MMP-12 decreased E-cadherin but induced fibronectin,vimentin,and α-SMA mRNA and protein expression in primary nasal epithelial cells and air-liquid interface culture. MMP408,an MMP-12 inhibitor,inhibited EMT-related factors. These findings suggest that MMP-12 expression in M2 macrophages induces EMT in nasal epithelial cells and may contribute to the pathogenesis of CRSwNP. View Publication

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However,the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells,and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover,MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC. View Publication

The lack of a robust system to reproducibly propagate HRV-C,a family of viruses refractory to cultivation in standard cell lines,has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C,and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression,whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly,upon HRV-C infection,we observe an innate immune response that is stronger in airway organoids than in nasal organoids,which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally,an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively,we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C,followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly,our study has opened an avenue for propagating and studying other uncultivable human and animal viruses. Subject terms: Virus-host interactions,Viral pathogenesis,Respiratory tract diseases View Publication

Metaplastic breast cancer (MpBC) is a highly chemoresistant subtype of breast cancer with no standardized therapy options. A clinical study in anthracycline-refractory MpBC patients suggested that nitric oxide synthase (NOS) inhibitor NG-monomethyl-l-arginine (L-NMMA) may augment anti-tumor efficacy of taxane. We report that NOS blockade potentiated response of human MpBC cell lines and tumors to phosphoinositide 3-kinase (PI3K) inhibitor alpelisib and taxane. Mechanistically,NOS blockade leads to a decrease in the S-nitrosylation of c-Jun NH 2 -terminal kinase (JNK)/c-Jun complex to repress its transcriptional output,leading to enhanced tumor differentiation and associated chemosensitivity. As a result,combined NOS and PI3K inhibition with taxane targets MpBC stem cells and improves survival in patient-derived xenograft models relative to single-/dual-agent therapy. Similarly,biopsies from MpBC tumors that responded to L-NMMA+taxane therapy showed a post-treatment reversal of epithelial-to-mesenchymal transition and decreased stemness. Our findings suggest that combined inhibition of iNOS and PI3K is a unique strategy to decrease chemoresistance and improve clinical outcomes in MpBC. Subject terms: Breast cancer,Cell signalling,Cancer therapy View Publication

Mild therapeutic hypothermia showed potential neuroprotective properties during and after cerebral hypoxia or ischemia in experimental animal studies. However,in clinical trials,where hypothermia is mainly applied after reperfusion,results were divergent and neurophysiological effects unclear. In our current study,we employed human-derived neuronal networks to investigate how treatment with hypothermia during hypoxia influences neuronal functionality and whether it improves post-hypoxic recovery. We differentiated neuronal networks from human induced pluripotent stem cells on micro-electrode arrays (MEAs). We studied the effect of hypothermia (34°C)–as well hyperthermia (39°C) ‐ on neuronal functionality during and after hypoxia using MEAs. We also studied the effects on the number of synaptic puncta and cell viability by immunocytochemistry. In comparison to neuronal networks under normothermia,we found that hypothermia during hypoxia improved functional neuronal network recovery,expressed as enhanced neuronal network activity. This was associated with prevention of synaptic loss during and after the hypoxic phase. Furthermore,hypothermia improved cell viability after the hypoxic phase. Instead,hyperthermia during hypoxia had detrimental effects,with an irreversible loss of neuronal network function,loss of synaptic puncta and decreased cell viability. Our results show potential neuroprotective properties of hypothermia occurring during hypoxia,indicating that administering hypothermia to bridge the time to reperfusion may be beneficial in clinical settings. View Publication

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of pathological proteins and synaptic dysfunction. This study aims to investigate the molecular and functional differences between human induced pluripotent stem cells (hiPSCs) derived from patients with sporadic AD (sAD) and age-matched controls (healthy subjects,HS),focusing on their neuronal differentiation and synaptic properties in order to better understand the cellular and molecular mechanisms underlying AD pathology. Skin fibroblasts from sAD patients ( n = 5) and HS subjects ( n = 5) were reprogrammed into hiPSCs using non-integrating Sendai virus vectors. Through karyotyping,we assessed pluripotency markers (OCT4,SOX2,TRA-1–60) and genomic integrity. Neuronal differentiation was evaluated by immunostaining for MAP2 and NEUN. Electrophysiological properties were measured using whole-cell patch-clamp,while protein expression of Aβ,phosphorylated tau,Synapsin-1,Synaptophysin,PSD95,and GluA1 was quantified by western blot. We then focused on PAK1-LIMK1-Cofilin signaling,which plays a key role in regulating synaptic structure and function,both of which are disrupted in neurodegenerative diseases such as AD. sAD and HS hiPSCs displayed similar stemness features and genomic stability. However,they differed in neuronal differentiation and function. sAD-derived neurons (sAD-hNs) displayed increased levels of AD-related proteins,including Aβ and phosphorylated tau. Electrophysiological analyses revealed that while both sAD- and HS-hNs generated action potentials,sAD-hNs exhibited decreased spontaneous synaptic activity. Significant reductions in the expression of synaptic proteins such as Synapsin-1,Synaptophysin,PSD95,and GluA1 were found in sAD-hNs,which are also characterized by reduced neurite length,indicating impaired differentiation. Notably,sAD-hNs demonstrated a marked reduction in LIMK1 phosphorylation,which could be the underlying cause for the changes in cytoskeletal dynamics that we found,leading to the morphological and functional modifications observed in sAD-hNs. To further investigate the involvement of the LIMK1 pathway in the morphological and functional changes observed in sAD neurons,we conducted perturbation experiments using the specific LIMK1 inhibitor,BMS-5. Neurons obtained from healthy subjects treated with the inhibitor showed similar morphological changes to those observed in sAD neurons,confirming that LIMK1 activity is crucial for maintaining normal neuronal structure. Furthermore,administration of the inhibitor to sAD neurons did not exacerbate the morphological alterations,suggesting that LIMK1 activity is already compromised in these cells. Our findings demonstrate that although sAD- and HS-hiPSCs are similar in their stemness and genomic stability,sAD-hNs exhibit distinct functional and structural anomalies mirroring AD pathology. These anomalies include synaptic dysfunction,altered cytoskeletal organization,and accumulation of AD-related proteins. Our study underscores the usefulness of hiPSCs in modeling AD and provides insights into the disease's molecular underpinnings,thus highlighting potential therapeutic targets. The online version contains supplementary material available at 10.1186/s13195-024-01632-3. View Publication

Cancer-associated fibroblasts (CAFs) play pivotal roles in solid tumor initiation,growth,and immune evasion. However,the optimal biomimetic modeling conditions remain elusive. In this study,we investigated the effects of 2D and 3D culturing conditions on human primary CAFs integrated into a modular tumor microenvironment (TME). Using single-nucleus RNA sequencing (snRNAseq) and Proteomics’ Proximity Extension Assays,we characterized CAF transcriptomic profiles and cytokine levels. Remarkably,when cultured in 2D,CAFs exhibited a myofibroblast (myCAF) subtype,whereas in 3D tumor spheroid cultures,CAFs displayed a more inflammatory (iCAF) pathological state. By integrating single-cell gene expression data with functional interrogations of critical TME-related processes [natural killer (NK)-mediated tumor killing,monocyte migration,and macrophage differentiation],we were able to reconcile form with function. In 3D TME spheroid models,CAFs enhance cancer cell growth and immunologically shield cells from NK cell-mediated cytotoxicity,in striking contrast with their 2D TME counterparts. Notably,3D CAF-secreted proteins manifest a more immunosuppressive profile by enhancing monocyte transendothelial migration and differentiation into M2-like tumor-associated macrophages (TAMs). Our findings reveal a more immunosuppressive and clinically relevant desmoplastic TME model that can be employed in industrial drug discovery campaigns to expand the cellular target range of chemotherapeutics. View Publication

The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCs OCT4 ) and pre-inducing with FLT3LG/SCF,and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs,making them promising for artificial haematopoiesis. However,further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs. hHFMSCs OCT4 were pre-treated with FLT3LG and SCF cytokines,followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining,RT-qPCR,and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8,colony formation assays,Ki67 index,and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa,Coomassie brilliant blue,and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR,Western blotting,and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton,proliferation,and the AJ pathway. Student’s t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05. Pre-treatment of hHFMSCs OCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small,globoid morphology,suspended above adherent cells,forming colonies,and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCs OCT4 . Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling,with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin,E-cadherin,and β-catenin genes,as well as the nuclear positioning of β-catenin,leading to variations in the cytoskeleton and cell cycle. Finally,a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner. ZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway,suggesting floating hHFMSCs OCT4 as the promising seed cells for artificial hematopoiesis. The online version contains supplementary material available at 10.1186/s13287-024-04080-w. View Publication

Inflammation in cystic fibrosis (CF) airways is difficult to treat with well-established regimens often including azithromycin (AZ) as an immunomodulatory drug. As AZ has been reported to require CF transmembrane conductance regulator (CFTR) to be able to reduce interleukin (IL)-8 and given the emergence of highly effective CFTR “triple” modulator therapy (elexacaftor/tezacaftor/ivacaftor; ETI),the aim of this study was to investigate the effect of AZ and ETI,singly and in combination,on ion channel activity and to assess the potential anti-inflammatory effects. Electrophysiological assessment of ETI and AZ was performed on three-dimensional cultures of primary CF human bronchial epithelial (HBE) cells using a Multi Trans-Epithelial Current Clamp. IL-8 from NuLi-1 (non-CF) and CuFi-1 (CF) cells treated with AZ was measured by ELISA. Inflammatory mediators from primary CF HBE cells exposed to tumour necrosis factor-α in the presence of AZ,ETI and their combination,were screened using the Proteome Profiler™ Human Cytokine Array Kit,with selected targets validated by ELISA. AZ did not alter CFTR chloride efflux,nor did it have any synergistic/antagonistic effect in combination with ETI. AZ reduced IL-8 in NuLi-1 but not CuFi-1 cells. The Proteome Profiler™ screen identified several disease-relevant cytokines that were modulated by treatment. Subsequent analysis by ELISA showed IL-8,IL-6,CXCL1 and granulocyte–macrophage colony-stimulating factor to be significantly reduced by treatment with ETI,but not by AZ. Incorporating ETI into the standard of CF care provides an opportunity to re-evaluate therapeutic regimens to reduce treatment burden and safely discontinue chronic treatments such as AZ,without loss of clinical benefit. Identification of redundant treatments in the era of CFTR modulation may improve medication adherence and overcome potential adverse effects associated with the chronic use AZ and other drugs. View Publication

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications,including gene therapy. We discovered a hyperactive variant of the SB100X transposase,called SB200X. This mutant,resulting from a specific amino acid replacement (Q124C),showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases,we show that the hyperactivity of Q124C manifests at high concentrations of the transposase,suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely,Q124C improves the transposition efficiency of the previously described K248R variant,while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications. View Publication