技术资料

Strategies targeting leukemic stem and progenitor cells (LSPCs) are needed for durable remissions in acute myeloid leukemia (AML) and high-risk myelodysplastic neoplasms (MDS). While CD123 constitutes a promising target on LSPCs and leukemic blasts,previous CD123-targeting approaches showed limited efficacy and challenging safety profiles. Here,we describe the preclinical efficacy and safety of the bispecific CD123/CD16A innate cell engager “AFM28”,demonstrating superior activity against AML and MDS patient-derived LSPCs and blasts in vitro compared to an Fc-enhanced CD123-targeting antibody,especially towards CD123 low and/or CD64 + leukemic cells. AFM28 induces autologous anti-leukemic activity in fresh AML whole blood cultures,demonstrating its potential to enhance NK cell function from AML patients. Responsiveness can be further enhanced by allogeneic NK cell addition. Anti-leukemic activity of AFM28 is confirmed in xenograft mouse models. In addition,AFM28 is well tolerated and demonstrates pharmacodynamic activity in cynomolgus monkeys. Altogether,our results indicate that AFM28 has the potential to reduce relapse-inducing residual disease and promote long-term remissions for patients with AML and MDS with a favorable safety profile. Subject terms: Cancer immunotherapy,Preclinical research,Acute myeloid leukaemia,Myelodysplastic syndrome View Publication

We have developed the HybriSeq method for single-cell RNA profiling,which utilizes in situ hybridization of multiple probes for targeted transcripts,followed by split-pool barcoding and sequencing analysis of the probes. We have shown that HybriSeq can achieve high sensitivity for RNA detection with multiple probes and profile entire transcripts without an end bias. The utility of HybriSeq is demonstrated in characterizing cell-to-cell heterogeneities of a panel of 196 genes in peripheral blood mononuclear cells and the detection of missed annotations of transcripts. Subject terms: Gene expression profiling,RNA sequencing View Publication

Cytolysin A (ClyA) is a pore‐forming protein from a strongly silenced gene in non‐pathogenic Escherichia coli,including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction,ClyA‐expressing bacteria display a cytolytic phenotype. However,it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli ‐host interactions in vivo. Here,we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA + E. coli were internalised into cultured colon cancer cells. The OMV‐associated ClyA caused reduced levels of cancer‐activating proteins such as H3K27me3,CXCR4,STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non‐pathogenic E. coli can influence the stability of the EZH2 protein,reducing its activity in epigenetic regulation,causing elevated level of the tumour suppressor protein p53. View Publication

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1,a Polycomb Repressive Complex 1 member,is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts,its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts,which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy,along with repressive histone marks,are present at known BMI1 target genes,including the INK-ARF locus,consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications,including key regulators of cholesterol homeostasis,along with functional studies,suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses. Subject terms: Self-renewal,Cell growth,Stem-cell research View Publication

Anti-obesity medications (AOMs) have become one of the most prescribed drugs in human medicine. While AOMs are known to impact adult neurogenesis in the hypothalamus,their effects on the functional maturation of hypothalamic neurons remain unexplored. Given that AOMs target neurons in the Medial Basal Hypothalamus (MBH),which play a crucial role in regulating energy homeostasis,we hypothesized that AOMs might influence the functional maturation of these neurons,potentially rewiring the MBH. To investigate this,we exposed hypothalamic neurons derived from human induced pluripotent stem cells (hiPSCs) to Semaglutide and lipidized prolactin-releasing peptide (LiPR),two anti-obesity compounds. Contrary to our expectations,treatment with Semaglutide or LiPR during neuronal maturation did not affect the proportion of anorexigenic,Pro-opiomelanocortin-expressing (POMC+) neurons. Additionally,LiPR did not alter the morphology of POMC+ neurons or the expression of selected genes critical for the metabolism or development of anorexigenic neurons. Furthermore,LiPR did not impact the proportion of adult-generated POMC+ neurons in the mouse MBH. Taken together,these results suggest that AOMs do not influence the functional maturation of anorexigenic hypothalamic neurons. View Publication

Viral transduction is a critical step in the manufacturing of genetically modified T cells for immunotherapies,yet conventional transduction methods suffer from low to medium efficiency,high vector consumption,and limited scalability. To address these challenges,we introduce the Transduction Boosting Device (TransB),an innovative,automated,and closed-system platform designed to enable efficient and scalable gene delivery and overcome the limitations of conventional transduction methods. TransB improves cell-virus interactions by facilitating proximity between target cells and viral vectors. TransB demonstrated up to 1-fold decrease in processing time,3-fold reduction in viral vector consumption,and 0.7-fold increase in transduction efficiency compared to 24—well plate method for donor T cell transduction in studies evaluating its impact on transduction process. Comparison studies transducing T cells from three different donors with Lenti-GFP vectors showed that TransB achieved an average 0.5-fold improvement in transduction efficiencies while maintaining comparable post-transduction cell recovery,viability,growth,and phenotype compared to 24—well plate. Furthermore,TransB delivered consistent performance across two different input cell numbers demonstrating scalability of the process. These findings suggest that TransB could significantly shorten the transduction time,reduce the transduction cost and improve the transduction efficiency for manufacturing genetically modified T cell therapies. It shows strong potential as a robust,efficient,and scalable platform to enhance T cell therapy manufacturing and help overcome current manufacturing challenges in the field. The online version contains supplementary material available at 10.1186/s12967-025-06836-1. View Publication

Transplantation of engineered B cells has demonstrated efficacy in HIV disease models. B cell engineering may also be utilized for the treatment of cancer. Recent studies have highlighted that B cell activity is associated with favorable clinical outcomes in oncology. In mice,polyclonal B cells have been shown to elicit anti-cancer responses. As a potential novel cell therapy,we demonstrate that engineering B cells to target a tumor-associated antigen enhances polyclonal anti-tumor responses. We observe that engineered B cells expressing an anti-HPV B cell receptor internalize the antigen,enabling subsequent activation of oncoantigen-specific T cells. Secreted antibodies from engineered B cells form immune complexes,which are taken up by antigen-presenting cells to further promote T cell activation. Engineered B cells hold promise as novel,multi-modal cell therapies and open new avenues in solid tumor targeting. View Publication

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