技术资料

Regulated cell death is a key component of the innate immune response,which provides the first line of defense against infection and homeostatic perturbations. However,cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis,necroptosis,and PANoptosis). While specific triggers are known to induce each of these cell death pathways,it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here,we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP),apoptotic (staurosporine),necroptotic (TNF-α plus z-VAD),and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts,both mouse and human innate immune cells,macrophages,expressed higher levels of cell death proteins and activated cell death effectors more robustly,including caspase-1,gasdermins,caspase-8,and RIPKs,in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases,inflammatory disorders,and cancer. View Publication

SummaryADG106,a ligand-blocking agonistic antibody targeting CD137 (4-1BB),exhibits promising results in preclinical studies,demonstrating tumor suppression in various animal models and showing a balanced profile between safety and efficacy. This phase 1 study enrolls 62 patients with advanced malignancies,revealing favorable tolerability up to the 5.0 mg/kg dose level. Dose-limiting toxicity occurs in only one patient (6.3%) at 10.0 mg/kg,resulting in grade 4 neutropenia. The most frequent treatment-related adverse events include leukopenia (22.6%),neutropenia (22.6%),elevated alanine aminotransferase (22.6%),rash (21.0%),itching (17.7%),and elevated aspartate aminotransferase (17.7%). The overall disease control rates are 47.1% for advanced solid tumors and 54.5% for non-Hodgkin’s lymphoma. Circulating biomarkers suggest target engagement by ADG106 and immune modulation of circulating T,B,and natural killer cells and cytokines interferon γ and interleukin-6,which may affect the probability of clinical efficacy. ADG106 has a manageable safety profile and preliminary anti-tumor efficacy in patients with advanced cancers (this study was registered at ClinicalTrials.gov: NCT03802955). Graphical abstract Highlights•ADG106 is a ligand-blocking agonistic antibody targeting CD137•ADG106 enhances cytotoxic T cell activity within the tumor environment•ADG106 shows manageable safety and preliminary anti-tumor efficacy in this phase 1 study Ma et al. demonstrate the safety,efficacy,and survival benefits of ADG106,a fully human agonistic monoclonal IgG4 antibody targeting a unique and crossreactive epitope of CD137,in patients with advanced solid tumors and non-Hodgkin’s lymphoma. They show that ADG106 exhibits a favorable safety profile and encourages anti-tumor activity. View Publication

The response to programmed death-1 (PD-1) blockade varies in hepatocellular carcinoma (HCC). We utilize a panel of 16 serum factors to show that a circulating level of serum amyloid A (SAA) > 20.0 mg/L has the highest accuracy in predicting anti-PD-1 resistance in HCC. Further experiments show a correlation between peritumoral SAA expression and circulating SAA levels in patients with progressive disease after PD-1 inhibition. In vitro experiments demonstrate that SAA induces neutrophils to express PD-L1 through glycolytic activation via an LDHA/STAT3 pathway and to release oncostatin M,thereby attenuating cytotoxic T cell function. In vivo,genetic or pharmacological inhibition of STAT3 or SAA eliminates neutrophil-mediated immunosuppression and enhances antitumor efficacy of anti-PD-1 treatment. This study indicates that SAA may be a critical inflammatory cytokine implicated in anti-PD-1 resistance in HCC. Targeting SAA-induced PD-L1+ neutrophils through STAT3 or SAA inhibition may present a potential approach for overcoming anti-PD1 resistance. The reasons for why hepatocellular carcinoma (HCC) is unresponsive to anti-PD-1 inhibition in some patients is not fully understood. Here the authors use human samples and mice tumour models to implicate serum amyloid A and STAT3 signalling involvement in the resistance to anti-PD1 immunotherapy in HCC. View Publication

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies,the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner,making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN),complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2,with preclinical evidence to support their efficacy in T cell malignancies. The T cell receptor β-chain is expressed in two isoforms,TRBC1 and TRBC2,with clonally expanded mature T cell lymphomas expressing one of them exclusively,while healthy T cells randomly express either TRBC1 or TRBC2. Here authors show structure-based design of a TRBC2-specific antibody,and depletion of malignant T cells carrying TRBC1 or TRBC2 with CAR-T cells against the cognate receptor chain in murine models. View Publication

The study introduces a new immunotherapy for treating melanoma and other cancers by developing a PROTAC that degrades NR4A1,an intracellular nuclear factor that plays a crucial role in immune suppression. An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME,we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here,we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC,named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level,NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8+ T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC),all of which are known to be clinically relevant immune cell populations in human melanomas. Overall,NR-V04–mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma. Graphical Abstract View Publication

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes,including SOD1. So far,clinical records,rodent studies,and in vitro models have yielded arguments for either a primary motor neuron disease,or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin,in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC,the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings,we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model,myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers,lack of sarcomeres,morphologically different nAChR clusters,and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably,trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary,a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS. View Publication

BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass,irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications,neurodegeneration,and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity,homeostasis,and repair. Here,we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome,their modulation,and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ? 5 Gy) were treated with iMSC CM,endothelial cell viability,adherence,spheroid compactness,and proangiogenic sprout formation were significantly ameliorated,and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently,iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore,iMSC CM suppressed IR-induced NF?B activation,TNF-? release,and ROS production in THP1 cells. Additionally,iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3,iMSCs,or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2,IL6,IL8/CXCL8,ANG (Angiogenin),GRO?/CXCL1,and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors,the PI3K/AKT/mTOR modulator PRAS40 and ?-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism,translation,mitochondrial respiration,DNA damage repair,and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03847-5. View Publication

Peripheral nerve injury (PNI) remains a significant clinical challenge,often leading to long-term functional impairment. Despite advances in therapies,current repair strategies offer unsatisfactory clinical outcomes. Exosomes derived from induced pluripotent stem cells (iPSC-Exos) have emerged as a promising therapeutic approach in regenerative medicine. This study assesses the efficacy and safety of iPSC-Exos in a rat model of sciatic nerve crush injury. Briefly,iPSCs were generated from peripheral blood mononuclear cells (PBMCs) of healthy donors using Sendai virus vectors and validated for pluripotency. iPSC-Exos were characterized and injected at the injury site. Functional recovery was assessed through gait analysis,grip strength,and pain response. Histological and molecular analyses were used to examine axonal regeneration,myelination,Schwann cell (SC) activation,angiogenesis,and changes in gene expression. iPSC-Exos were efficiently internalized by SC,promoting their proliferation. No adverse effects were observed between groups on body weight,organ histology,or hematological parameters. iPSC-Exos injection significantly enhanced nerve regeneration,muscle preservation,and vascularization,with RNA sequencing revealing activation of PI3K-AKT and focal adhesion pathways. These findings support iPSC-Exos as a safe and effective non-cell-based therapy for PNIs,highlighting their potential for clinical applications in regenerative medicine. View Publication

IntroductionRetinal ganglion cells (RGCs) are susceptible to degenerative conditions such as glaucoma and traumatic optic neuropathies,which lead to vision loss. MicroRNA-21-5p has demonstrated potential neuroprotective effects,but its mechanisms in optic nerve injury remain underexplored. This study evaluates the neuroprotective role of microRNA-21-5p derived from induced pluripotent stem cells (iPSCs) in an optic nerve crush (ONC) model.Materials and methodsIn vitro qPCR demonstrated that the expression of microRNA-21-5p was increased in the co-culture medium of RGCs and iPSCs. Subsequently,in the in vivo experiments,we used a microRNA-21-5p agonist to assess its protective effects on RGCs. RNA sequencing was then performed in a mouse ONC model after treatment with a microRNA-21-5p agonist to explore the mechanisms underlying its neuroprotective effects on RGCs.ResultsAs demonstrated in our previous experiments,the RGCs-iPSCs co-culture group led to a higher survival rate of RGCs,as indicated by live/dead cell staining,compared to the RGCs-only group. Quantitative PCR (qPCR) results revealed a significant increase in the expression of microRNA-21-5p in the medium of the RGCs-iPSCs co-culture group. Furthermore,the survival rate of mouse retinal RGCs treated with a microRNA-21-5p agonist was significantly greater than that of the control group. Lastly,RNA sequencing of the retina from microRNA-21-5p agonist-treated mice indicated that microRNA-21-5p plays a protective role in RGCs by downregulating the expression of several genes,including Irf1,Ccl4,Itk,Cxcr2,Dclre1c,Traf1,Traf2,Rbl1,Cxcl5,Cxcl3,Cxcl1,Cxcl9,Il2rg,Cd3e,Cd3d,Cxcl10,Ccl5,Ccl12,Tap1,and Cxcr4.ConclusionMicroRNA-21-5p derived from iPSCs can enhance the survival rate of RGCs in the ONC model. This suggests that microRNA-21-5p may represent a novel and effective strategy for repairing RGC damage. Such a strategy could potentially be realized through the modulation of apoptosis,T-cell regulatory pathways,or TNF-? signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12886-025-04244-z. View Publication

BackgroundSchizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear,despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However,mechanistic insight at the protein level is limited.MethodsHere,we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach.ResultsSCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling,protein synthesis,cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control,SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore,we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells,indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ.ConclusionsThrough targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response,cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ,particularly during early development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12888-024-06127-x. View Publication

ABSTRACTGenetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study looked at people from 12 unrelated families with variants in the gene SMPD4,a neutral sphingomyelinase that metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These individuals have severe developmental brain malformations,including microcephaly and cerebellar hypoplasia. The disease mechanism of SMPD4 was not known and so we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia,a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells lacking SMPD4 exhibit neural progenitor cell death and have shortened primary cilia,which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development. Summary: Mouse and human stem cell models of SMPD4 loss of function demonstrate that SMPD4 promotes cilia function and neural development. View Publication

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied,the role of biophysical microenvironments in early lineage commitment remains elusive. Here,we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies,facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation,suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling,DNA histone modifications,and patterned germ layer specification. Notably,altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis,offering valuable insights for material design in tissue engineering and regenerative medicine applications. Graphical abstractImage 1 View Publication