技术资料

The present study focused on the culture medium of induced pluripotent stem cells (iPSCs) prior to the use of cardiomyocytes differentiation induction medium (pre-culture medium). Seven types (Nos. 1-7) of StemFit AK03 medium (Ajinomoto) for clinical iPSCs with varying compositions were prepared as pre-culture medium. The cardiac muscle troponin T (cTnT) positivity of No. 1 (StemFit AK03 medium) was 84%,No. 3 (similar to E8 medium) was 89%,No. 2 (similar to E8 medium) was 91%,No. 5 (similar to EB Formation medium) was 95%,when using differentiation induction medium prepared with known components available for clinical cell production. The formation of cardiac tissues was assessed by evaluating the expression levels of specific markers,including cTnT,atrial natriuretic peptides (ANP),and pro-B-type natriuretic peptide (proBNP). The results demonstrated that cardiac tissue with high protein expression levels of cTnT and ANP was formed when similar to E8 medium as pre-culture medium. The online version contains supplementary material available at 10.1038/s41598-025-13259-x. View Publication

Fatty-acid-hydroxylase-associated neurodegeneration (FAHN) is a rare neurodegenerative disorder caused by loss-of-function mutations in the FA2H gene,leading to impaired enzymatic activity and resulting in myelin sheath instability,demyelination,and axonal degeneration. In this study,we established a human in vitro model using neurons and oligodendrocytes derived from induced pluripotent stem cells (hiPSCs) of a FAHN patient. This coculture system enabled the investigation of myelination processes and myelin integrity in a disease-relevant context. Analyses using immunofluorescence and Western blot revealed impaired expression and localisation of key myelin proteins in oligodendrocytes and cocultures. FA2H-deficient cells showed reduced myelination,shortened internodes,and disrupted formation of the nodes of Ranvier. Additionally,we identified autophagy defects—a hallmark of many neurodegenerative diseases—including reduced p62 expression,elevated LC3B levels,and impaired fusion of autophagosomes with lysosomes. This study presents a robust hiPSC-based model to study FAHN,offering new insights into the molecular pathology of the disease. Our findings suggest that FA2H mutations compromise both the structural integrity of myelin and the efficiency of the autophagic machinery,highlighting potential targets for future therapeutic interventions. View Publication

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication

Cancer research has long focused on mutations in normal body cells,but this approach has not produced major breakthroughs for most cancers. Our study explores a different concept that some aggressive cancers may actually arise from early reproductive cells called primordial germ cells,which normally develop into eggs and sperm. We created a new experimental model showing how a virus can transform human primordial germ cell-like cells into virus-positive Merkel cell carcinoma,a rare but deadly skin cancer. This model shows that cancers can emerge through changes in developmental states rather than relying solely on genetic mutations. By linking cancer development to early germ cells,our findings suggest a unifying explanation for both germ cell cancers and body cancers. This new perspective may guide more effective approaches to study,diagnose,and treat cancer by focusing on early human development rather than only DNA mutations and later developmental stages. View Publication

Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation,the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer's disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target,where its weak inhibition activates neuroprotective signalling,benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded mtCI inhibitors profiled in a drug discovery funnel designed to address safety,selectivity,and efficacy. The lead compound C458 is highly protective against Aβ toxicity,has favourable pharmacokinetics,and minimal off-target effects. C458 exhibited excellent brain penetrance,activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted using functional tests,metabolic assessment,in vivo 31 P-NMR spectroscopy,blood cytokine panels,ex vivo electrophysiology,and Western blotting. Chronic oral administration improved long-term potentiation,reduced oxidative stress and inflammation,and enhanced mitochondrial biogenesis,antioxidant signalling,and cellular energetics. Efficacy against Aβ and p-Tau was confirmed in human organoids. These studies provide further evidence that the restoration of mitochondrial function in response to mild energetic stress represents a promising disease-modifying strategy for AD. This research was supported by grants from NIH AG 5549-06,NS1 07265,AG 062135,UG3/UH3 NS 113776,and ADDF 291204 (all to ET); U19 AG069701 (to TK); the Alzheimer’s Association Research Fellowship grant 23AARF-1027342 (to TKON). View Publication

Tissue remodeling is a key feature of allergic rhinitis (AR),but its underlying molecular mechanisms remain unclear. Lysyl oxidase-like 2 (LOXL2),a regulator of tissue remodeling,has not been studied in AR. Proteomic analysis was performed on nasal mucosal tissues from 8 AR patients and 8 healthy controls (HCs) to identify differentially expressed proteins (DEPs). The top three upregulated DEPs and their association with tissue remodeling markers were validated by immunofluorescence,Western blot,and RT-qPCR in an independent cohort of 30 AR patients and 30 HCs. In vitro,human nasal epithelial cells (HNECs) were treated with IL-4,and the effects of candidate protein inhibitors on remodeling were assessed. An AR mouse model was used to evaluate the impact of these inhibitors on nasal inflammation and remodeling. Proteomic analysis revealed a disease-specific protein expression profile in the nasal mucosa of AR patients,with the top three upregulated proteins being LOXL2,TGF-β1,and TIRAP. Tissue validation showed that LOXL2 was significantly upregulated in the nasal mucosa of AR patients compared to HCs and was significantly correlated with EMT markers (TGF-β1,α-SMA,and E-cadherin). In vitro,IL-4 stimulation significantly upregulated LOXL2,TGF-β1,and α-SMA,while downregulating E-cadherin in a dose-dependent manner in human nasal epithelial cells. These effects were reversed by inhibition of LOXL2. Further investigations demonstrated that LOXL2 promotes tissue remodeling through activation of the TGF-β1/Smad signaling pathway. In the AR mouse model,LOXL2 inhibitors significantly reduced nasal mucosal inflammation and tissue remodeling. Our proteomic analysis suggests that LOXL2 may be involved in the pathological remodeling processes of AR,potentially through modulation of the TGF-β1/Smad signaling pathway. These findings provide preliminary evidence that LOXL2 could serve as a candidate biomarker and a possible therapeutic target in AR,warranting further investigation. View Publication

During viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi's sarcoma-associated herpesvirus interacts and inhibits the central cell death effector,Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins,like vFlip,on liver physiology in vivo,mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level,hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably,embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice,culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis. View Publication

Seasonal and pandemic influenza infection remains a major public health concern worldwide. Driving robust humoral immunity has been a challenge given preexisting,often cross-reactive,immunity and in particular,poorly immunogenic avian antigens. To overcome immune barriers,the adjuvant MF59 has been used in seasonal influenza vaccines to increase antibody titers and improve neutralizing activity,translating to a moderate increase in protection in vulnerable populations. However,its effects on stimulating antibody effector functions,including NK cell activation,monocyte phagocytosis,and complement activity,all of which have been implicated in protection against influenza,have yet to be defined. Using systems serology,we assessed changes in antibody functional profiles in individuals who received H5N1 avian influenza vaccine administered with MF59,with alum,or delivered unadjuvanted. MF59 elicited antibody responses that stimulated robust neutrophil phagocytosis and complement activity. Conversely,vaccination with MF59 recruited NK cells poorly and drove moderate monocyte phagocytic activity,both likely compromised because of the induction of antibodies that did not bind FCGR3A. Collectively,defining the humoral antibody functions induced by distinct adjuvants may provide a path to designing next-generation vaccines that can selectively leverage the humoral immune functions,beyond binding and neutralization,resulting in better protection from infection. View Publication

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments,better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study,we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology,immunohistochemistry,gene expression,DNA methylation,copy number,and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies. View Publication

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs),though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms,including platelet activation and neutrophil-extracellular trap formation; therefore,an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here,we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC),a sulfhydryl-containing compound with broad effects on glutathione replenishment,free radical scavenging,and reducing disulfide bonds,to investigate its antithrombotic effects in the context of MPN. Strikingly,NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo,we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore,NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN. View Publication

The placenta and umbilical cord are pre-eminent candidate sources of mesenchymal stem cells (MSCs). However,placenta-derived MSCs (P-MSCs) showed greater proliferation capacity than umbilical cord-derived MSCs (UC-MSCs) in our study. We investigated the drivers of this proliferation difference and elucidated the mechanisms of proliferation regulation. Proteomic profiling and Gene Ontology (GO) functional enrichment were conducted to identify candidate proteins that may influence proliferation. Using lentiviral or small interfering RNA infection,we established overexpression and knockdown models and observed changes in cell proliferation to examine whether a relationship exists between the candidate proteins and proliferation capacity. Real-time quantitative polymerase chain reaction,western blot analysis,and immunofluorescence assays were conducted to elucidate the mechanisms underlying proliferation. Six candidate proteins were selected based on the results of proteomic profiling and GO functional enrichment. Through further validation,yes-associated protein 1 (YAP1) and $\beta$-catenin were confirmed to affect MSCs proliferation rates. YAP1 and $\beta$-catenin showed increased nuclear colocalization during cell expansion. YAP1 overexpression significantly enhanced proliferation capacity and upregulated the expression of both $\beta$-catenin and the transcriptional targets of Wnt signaling,CCND1,and c-MYC,whereas silencing $\beta$-catenin attenuated this influence. We found that YAP1 directly interacts with $\beta$-catenin in the nucleus to form a transcriptional YAP/$\beta$-catenin/TCF4 complex. Our study revealed that YAP1 and $\beta$-catenin caused the different proliferation capacities of P-MSCs and UC-MSCs. Mechanism analysis showed that YAP1 stabilized the nuclear $\beta$-catenin protein,and also triggered the Wnt/$\beta$-catenin pathway,promoting proliferation. View Publication