参考文献

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

Accumulating evidence suggests that mitogenic signaling during cell cycle arrest can lead to severe cytotoxic outcomes,such as senescence,though the underlying mechanisms remain poorly understood. Here,we explored the link between cell cycle dynamics and the formation of PML-nuclear bodies (PML-NBs),intranuclear structures known to mediate cellular stress responses. Our findings demonstrate that PML-NBs increase their number during interphase arrest. Moreover,the activation of mitogenic ERK signaling by all-trans retinoic acid (ATRA) during CDK4/6 inhibitor-induced cell cycle arrest synergistically enhances the formation of larger PML-NBs by associating with SUMO. This enlargement,triggered by the simultaneous engagement of opposing cell cycle signals,leads to potent cytotoxicity accompanied by either terminal differentiation or apoptosis,depending on the cell type,across multiple acute myeloid leukemia (AML) cell lines. Importantly,in an AML mouse model,this combination treatment significantly improved therapeutic efficacy with minimal effects on normal hematopoiesis. Our results introduce conflicting cell cycle signal-induced cytotoxicity as a promising therapeutic strategy for AML. Subject terms: PML bodies,Apoptosis View Publication

Induced pluripotent stem cells (iPSCs) are widely used to model human genetic diseases. The most common strategy involves collecting cells from relevant individuals and then reprogramming them into iPSCs. This strategy is very powerful,but finding enough individuals with a specific genetic disease can be challenging,especially since most are rare. In addition,making numerous iPSC lines is time-consuming and expensive. As a result,most studies have included relatively small numbers of iPSC lines,sometimes from the same individual. Considering the experimental variability obtained using different iPSC lines,there has been great interest in delineating the most efficient number of lines needed to achieve a robust and reproducible result. Several recommendations have been published,although most conclusions have been based on methods where experimental variance from individual cases is difficult to separate from technical issues related to the preparation of iPSCs. The current study used gene expression profiles determined by RNA sequencing (RNAseq) to empirically evaluate the impact of the number of unique individuals and the number of replicate iPSC lines from each individual for modeling Lesch-Nyhan disease (LND). This disease is caused by mutations in the HPRT1 gene,which encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase. Results for detecting disease-relevant changes in gene expression depended on the analytical method employed,and whether or not statistical procedures were used to address multiple iPSC lines from the same individual. In keeping with prior studies,the best results were obtained with iPSC lines from 3-4 unique individuals per group. In contrast to prior studies,results were improved with 2 lines per individual,without statistical corrections for duplicate lines from the same individual. In the current study where all lines were produced in parallel using the same methods,most variance in gene expression came from technical factors unrelated to the individual from whom the iPSC lines were prepared. View Publication

Type 1 diabetes (T1D) results from the autoimmune-mediated loss of pancreatic β-cells. Current insulin therapies offer symptomatic relief but fall short of providing a definitive cure. Islet cell transplantation,while promising,faces limitations related to donor scarcity,procedural complexities,and the necessity for long-term immunosuppression. Consequently,there is an urgent need for innovative strategies aimed at β-cell regeneration. Patient-derived induced pluripotent stem cells (iPSCs),obtained from peripheral blood mononuclear cells (PBMCs) of T1D patients,hold great potential as a source of cells for therapeutic purposes. Therefore,the differentiation of T1D-iPSCs into functional pancreatic β-cells is a critical step toward effective β-cell replacement therapy. To assess the potential of patient-derived T1D-β-like cells (differentiated from T1D-iPSCs reprogrammed from T1D-PBMCs) for restoring β-cell function in T1D. T1D-iPSCs were reprogrammed from T1D-PBMCs using an episomal vector-based approach. Pluripotency was confirmed by flow cytometry (FCM),quantitative real-time polymerase chain reaction,genomic stability analysis,and teratoma formation assays. Differentiation into pancreatic β-cells was optimized using triiodothyronine (T3),vitamin C (Vc),and an adenovirus (M3C) encoding pancreatic duodenal homeobox-1,neurogenin 3 ( Ngn3 ),and MAF bZIP transcription factor A ( MafA ). Following characterization of β-cell features by immunofluorescence,quantitative real-time polymerase chain reaction,and flow cytometry,therapeutic efficacy was assessed through blood glucose monitoring after transplantation under the renal capsule of streptozotocin-induced diabetic mice. T1D-iPSCs were successfully generated from T1D-PBMCs. These cells exhibited the hallmark characteristics of pluripotent stem cells,including appropriate morphology,differentiation potential,genomic integrity,and expression of pluripotency-associated genes. Differentiation yielded insulin-positive (insulin + ) pancreatic β-like cells that,at the mRNA level,expressed key β-cell markers such as pancreatic duodenal homeobox-1,Ngn3,MafA,NeuroD,glucagon-like peptide-1 receptor,Nkx6.1,glucose transporter 2,and Kir6.2 . Notably,the T3 + Vc group displayed the lowest Ngn3 expression (1.31 ± 0.38 vs 1.96 ± 0.25 vs 2.51 ± 0.24,P < 0.01),while the M3C + T3 + Vc group exhibited the highest MafA expression (0.49 ± 0.11 vs 0.32 ± 0.06 vs 0.29 ± 0.08,P < 0.05). Both in vitro and in vivo assessments confirmed the insulin secretion ability of the generated β-like cells; however,they did not demonstrate appropriate modulation of insulin release in response to variations in extracellular glucose concentrations. T1D-iPSCs derived from T1D-PBMCs can be differentiated into insulin + β-like cells,albeit with functional immaturity. These cells represent a potential source of seed cells for β-cell replacement therapy in T1D. View Publication

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve,demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses,therapeutic treatments,and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic,with new subvariants continuously emerging,many harboring spike protein mutations linked to immune evasion. In this study,we characterized a panel of live SARS-CoV-2 strains,including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants,particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC),we established both monoinfections and coinfections involving SARS-CoV-2,Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably,Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020,Washington strain). Furthermore,the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead,it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6,IFN-β,and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together,our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants,as well as their interactions with other respiratory viruses,carrying important implications for therapeutic development and public health preparedness. View Publication

Immunotherapy has emerged as a key modality in cancer treatment,yet its effectiveness varies significantly among patients,often due to the metabolic stress imposed by the tumor microenvironment. Hypoxia,a major factor in the tumor microenvironment,results from the high metabolic rate of tumor cells and inadequate vascularization,impairing immune cells’ function and potentially influencing gene expression profiles. Despite the widespread use of quantitative real-time PCR in immunological studies,to the best of our knowledge,data on reference gene stability in human peripheral blood mononuclear cells under hypoxic conditions is limited. In our study,we assessed the expression stability of commonly used reference genes ( S18,HPRT,IPO8,RPL13A,SDHA,PPIA,and UBE2D2 ) in both non-stimulated and CD3/CD28-activated peripheral blood mononuclear cells cultured under normoxic,hypoxic (1% O 2 ),and chemically induced hypoxic conditions for 24 h. Analysis using four different algorithms—delta Ct,geNorm,NormFinder,and BestKeeper—identified RPL13A,S18,and SDHA as the most suitable reference genes for human peripheral blood mononuclear cells under hypoxic conditions. In contrast,IPO8 and PPIA were found to be the least suitable housekeeping genes. The study provides essential insights into the stability of reference genes in peripheral blood mononuclear cells under hypoxic conditions,a critical but understudied aspect of immunological research. Given the significant impact of hypoxia on T cell metabolism and function in the tumor microenvironment,selecting reliable reference genes is crucial for accurate gene expression analysis. Our findings will be valuable for future studies investigating hypoxia-driven metabolic reprogramming in immune cells,ultimately contributing to a better understanding of T cell responses in cancer immunotherapy. View Publication

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed,and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites,here,we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells,PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs,and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible,concentration-dependent,and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid,propionic acid,itaconic acid,and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids,including arachidonic acid. Although lactic acid inhibited PAC,it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude,weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids. View Publication

Arteriovenous malformations (AVMs) are congenital vascular anomalies defined by abnormal direct connections between arteries and veins due to their complex structure or endovascular approaches. Pharmacological strategies targeting the underlying molecular mechanisms are thus gaining increasing attention in an effort to determine the mechanism involved in AVM regulation. In this study,we examined 30 human tissue samples,comprising 10 vascular samples,10 human fibroblasts derived from AVM tissue,and 10 vascular samples derived from healthy individuals. The pharmacological agents thalidomide,U0126,and rapamycin were applied to the isolated endothelial cells (ECs). The pharmacological treatments reduced the proliferation of AVM ECs and downregulated miR-135b-5p,a biomarker associated with AVMs. The expression levels of angiogenesis-related genes,including VEGF,ANG2,FSTL1,and MARCKS,decreased; in comparison,CSPG4,a gene related to capillary networks,was upregulated. Following analysis of these findings,skin samples from 10 AVM patients were reprogrammed into induced pluripotent stem cells (iPSCs) to generate AVM blood vessel organoids. Treatment of these AVM blood vessel organoids with thalidomide,U0126,and rapamycin resulted in a reduction in the expression of the EC markers CD31 and α-SMA. The establishment of AVM blood vessel organoids offers a physiologically relevant in vitro model for disease characterization and drug screening. The authors of future studies should aim to refine this model using advanced techniques,such as microfluidic systems,to more efficiently replicate AVMs’ pathology and support the development of personalized therapies. View Publication

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro,we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7,MDA-MB-231,and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study,we showed that stromal cells promoted tumor cell aggregation into spheroids,regardless of the initial proliferation rates,with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids,whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions. View Publication

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain,HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies,independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro,HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation,cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies,HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens,in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation,cytokine secretion,and cytotoxic activity in vitro compared to either compound alone. In conclusion,HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner,without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade. View Publication