技术资料

Semaphorin3A (Sema3A) is a regulatory protein found to be expressed on regulatory T and B cells and also secreted into peripheral blood. It has been identified as a potent immune regulator; however,not all its regulatory mechanisms have been evaluated. In this respect,we aim to investigate how Sema3A affects key metabolic pathways in T cells during homeostasis and rheumatoid arthritis (RA),and on the AKT/mTORC1 signaling axis. In this study,peripheral blood samples were collected from 119 healthy donors and 32 rheumatoid arthritis patients. T cells were subjected to Seahorse analysis to evaluate OXPHOS and glycolysis,live cell TMRE staining to evaluate mitochondrial activity,mass spectrometry for metabolite profiling,ATP determination to study ATP production,and Western blot analysis to investigate the signaling pathway activity. This study presents evidence showing that Sema3A inhibits the AKT/mTORC1 pathway,leading to a decreased glucose uptake and glycolysis disruption. Furthermore,we show that Sema3A reduces mitochondrial capacity and OXPHOS in activated T cells of healthy and RA donors,leading to a decreased ATP production. In contrast,Sema3A upregulates fatty acid oxidation (FA),probably as a backup pathway to ensure cell survival. Results with p values of <0.05 were considered significant. Our data may point to Sema3A’s ability to convert activated T cells’ metabolic profile back to its non-activated state. This may suggest that Sema3A might be a beneficial treatment for immune-mediated diseases by metabolically reprogramming activated T cells. View Publication

Effective communication between multipotent mesenchymal stromal cells (MSCs) and endothelial cells (ECs) plays a critical role in the regulation of angiogenesis,especially under conditions of hypoxia. In addition to paracrine stimulation,direct intercellular contacts play an important role in the angiogenic interaction between MSCs and ECs,making them an important target for modulating vascular network restoration under ischemic conditions. The aim of this study was to determine the contribution of gap junctions (GJs) to the angiogenic response of MSCs and the EA.hy926 cell line (an Endothelial Cell Model) under acute hypoxic stress. In a cell co-culture model at 0.1% O2 using a specific GJ inhibitor (carbenoxolone),molecular,cellular,and functional tests were performed: assessment of viability,proliferation,migration,secretion of angiogenic mediators,and expression of crucial genes. GJ blockade was accompanied by decreases in the proliferation and migration activity and angiogenic potential of the conditioned medium in in vitro and in ovo tests. These data highlight the importance of the GJ in coordinating the angiogenic response in conditions of acute hypoxia and can be used to develop protocols for regenerative medicine. View Publication

Oxygen tension is a key regulator of early human neurogenesis; however,quantifying intra-tissue O2 in 3D models for an extended period remains difficult. Existing approaches,such as needle-type fiber microsensors and intensity-based oxygen probes or time-domain lifetime imaging,either perturb the organoids or require high excitation doses that limit the measurement period. Here,we present a step-by-step protocol to measure intra-organoid oxygen in human cerebral organoids (hCOs) using embedded ruthenium-based CPOx microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The workflow covers dorsal/ventral cerebral organoid patterning,organoid fusion at day 12 with co-embedded CPOx beads,standardized FD-FLIM acquisition (470-nm external modulation,16 phases at 50 kHz,dual-tap camera),automated bead detection and lifetime extraction in MATLAB,and session-matched Stern–Volmer calibration with Ru(dpp)3(ClO4)2 to convert lifetimes to oxygen concentration. The protocol outputs per-bead oxygen maps and longitudinal patterns stratified by bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal),enabling direct linkage between developmental growth and oxygen dynamics. Key features • End-to-end workflow linking hCOs generation,on-gel bead embedding,and FD-FLIM oxygen readout.• Longitudinal single-organoid tracking of oxygen tension with bead-level metadata.• Reference-based lifetime calibration and reproducible camera/LED settings.• Ready-to-reuse materials,recipes,timing,and analysis logic. View Publication

BackgroundAt present,healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore,efforts to develop red blood cell (RBC) production methods have gained traction. In this work,Lin−CD45−CD133+ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.MethodsLin−CD45−CD133+ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin−CD45−CD133+ cells were confirmed using flow cytometry analysis,colony-forming unit (CFU) assays,morphological analysis,immunocytochemistry (ICC) analysis,and real-time fluorescent quantitative polymerase chain reaction (RT–PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF),interleukin-3 (IL-3),erythropoietin (EPO),and FK506 for 13 days,after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.ResultsLin−CD45−CD133+ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin−CD45−CD133+ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture,the Lin−CD45−CD133+ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71+ cells gradually increased,peaked on day 10,and then started decreasing on day 13. The percentage of CD235a+ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18,with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation,and their morphology was consistent with that of normal erythrocytes.ConclusionAn ex vivo culture system was developed that can generate human erythrocytes from Lin−CD45−CD133+ cells isolated from human UCB. View Publication

Myelodysplastic syndrome (MDS) originates from hematopoietic stem cell (HSC) clones with acquired gene mutations. However,the molecular characteristics of MDS stem cells remain poorly understood. Here,we show that the chromatin accessibility profiles of MDS stem cells more accurately reflect disease status than those of progenitor cells and reveal the process of stem cell alterations during disease progression. Characterization of differentially accessible regions (DARs) shows that MDS stem cells acquire progenitor-like chromatin accessibility during disease progression,leading to disruption of the normal stem-progenitor hierarchy. Profiling of transcription factor-binding motifs at DARs further uncovers precocious activation of myeloid transcriptional networks in MDS stem cells,with a concurrent loss of HSC-associated regulatory programs. In particular,increased chromatin accessibility at CEBP target sites represents the myeloid reprogramming status of MDS stem cells. Newly developed “progenitor scores” based on chromatin accessibility stratify disease status and correlate well with prognosis. These findings indicate that chromatin landscapes of MDS stem cells define their cell-autonomous behavior and contribute to disease progression. This study reveals that MDS stem cells progressively acquire progenitor-like chromatin features during disease progression,and that a scoring system based on chromatin accessibility in MDS stem and progenitor cells correlates strongly with disease progression. View Publication

PurposeCancer immunotherapy aims to enhance the immune system’s ability to recognize and eliminate cancer cells,providing a sustained and effective immune response. However,the tumor microenvironment (TME),characterized by an abundance of tumor-associated M2 macrophages and the presence of exhausted or naïve T cells (non-effector T cells),remains a major barrier to effective immunotherapy. Herein,inflammatory M1 macrophage-derived extracellular vesicles (M1EV) were surface-modified to display interleukin-2 (M1EV_IL2),aiming to develop a multifunctional cancer immunotherapeutic agent capable of modulating both innate and adaptive immune responses.MethodsWe engineered M1EV to label the surface with azide groups through metabolic glycoengineering and developed M1EV_IL2 that displayed IL-2 via bioorthogonal chemistry. M1EV_IL2 were purified by size-exclusion chromatography (SEC) and characterized through comprehensive analyses,including nanoparticle tracking analysis (NTA). In vitro macrophage repolarization and T cell activation were evaluated at the gene-expression level,followed by ex vivo assays assessing T-cell proliferation,cytokine secretion,and activation marker expression.ResultsM1EV_IL2 effectively retained the intrinsic physicochemical properties of EVs while displaying IL-2 stably on its surface. It upregulated M1 macrophage markers,IL-1β and CXCL10,while downregulating the M2 macrophage marker CD206,thereby inducing M2-to-M1 macrophage repolarization. In addition,M1EV_IL2 also activated CD4+ T cells and induced the activation of naïve CD8+ T cells to effector T cells,leading to enhanced cell proliferation and secretion of antitumor cytokines.ConclusionThese results indicate that M1EV_IL2 has the potential to reshape the tumor immune landscape by simultaneously activating macrophages and T cells,thereby enhancing both innate and adaptive immune responses. Unlike conventional cancer therapies,which directly target tumor cells,M1EV_IL2 is expected to enhance immune responses,potentially mitigating adverse effects while improving therapeutic efficacy. Graphical Abstract View Publication

BackgroundBispecific antibodies that redirect T cells or NK cells to tumors have demonstrated substantial therapeutic efficacy,but their broader application is often constrained by immune-related toxicities,limited effector cell availability,and suboptimal access to tumor sites. These challenges have prompted efforts to identify alternative effector cell types that are more abundant in circulation,readily accessible,and capable of cytotoxic activity in the tumor microenvironment. Neutrophils,which constitute the most prevalent circulating leukocyte population,represent a promising yet underutilized target for immune cell engager design. However,efforts to exploit neutrophil-mediated tumor killing through CD89 (FcαRI) have been limited by the inherent drawbacks of IgA-based formats,including poor stability,short serum half-life,and reduced developability.ResultsTo address these challenges,we established an engineered bispecific antibody platform that incorporates CD89 engagement into an IgG1 scaffold. This design enables neutrophil redirection while preserving the favorable pharmacokinetic and manufacturing profiles of IgG-based therapeutics. The resulting bispecific architecture allows for programmable neutrophil engagement alongside tumor antigen recognition,offering a clinically viable strategy for innate immune activation. Among the bispecific designs evaluated,ZT-8,a humanized CD89 × HER2 bispecific antibody,demonstrated potent neutrophil-mediated cytotoxicity against tumor cells even in the absence of cytokine priming,suggesting a distinct activation mechanism that operates within the tumor microenvironment. Compared to IgA-based antibodies,ZT-8 exhibited superior immune effector engagement,enhanced tumoricidal activity,and substantially prolonged in vivo half-life through FcRn-mediated recycling.ConclusionThese findings define IgG-based CD89 bispecifics as a next-generation neutrophil engager platform and exemplify how antibody engineering and synthetic immunology can be leveraged to expand the effector landscape of bispecific immunotherapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13036-025-00580-2. View Publication

Rhodehouse et al. characterize a nonhuman primate model of natural control of SIV infection in which the immune system blocks new infection events with an efficiency approaching that of antiretroviral therapy. Insights into HIV-1 pathogenesis have come from studies of viral dynamics. However,there is little information on viral dynamics in lentiviral infections in which viral replication is naturally controlled in a subset of infected individuals. We evaluated the decay of simian immunodeficiency virus (SIV) RNA and cell-associated SIV genomes in a nonhuman primate (NHP) model in which replication of an engineered SIV variant is naturally controlled by cellular immune responses in most infected animals. This variant lacks a trafficking motif in the gp41 cytoplasmic tail. A trajectory of control was evident by 21 days after infection. In animals with natural control,we observed similar biphasic decay of intact proviruses in blood and lymph nodes,at rates close to those in animals that failed to control the virus and were put on antiretroviral therapy (ART). Both natural control and ART effectively blocked viral evolution,but not persistence. Thus,in this NHP model,natural control can be nearly as effective as ART in controlling viral replication. View Publication

AbstractAcquired resistance presents a major challenge for targeted therapies,with initially responsive tumors eventually reverting underlying vulnerabilities. Our group recently reported that cancers harboring isocitrate dehydrogenase 1/2 (IDH1/2) mutations have defective recruitment of homology-directed repair (HDR) factors to sites of DNA damage and consequent sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi),a vulnerability that is being tested in clinical trials. To probe potential mechanisms by which resistance to PARPi might arise in this setting,we modeled PARPi resistance in IDH-mutant tumors via serial transplantation of patient-derived xenografts in mice treated with PARPi. An analysis of candidate DNA repair factors in these resistant tumor populations identified downregulation of two end protection factors that are negative regulators of HDR,53BP1,and REV7. Knockout of these factors by CRISPR–Cas9 in IDH1-mutant cancer cells conferred robust resistance to PARPi and restored HDR capacity. To overcome this resistance,we found that treatment with the receptor tyrosine kinase inhibitor,cediranib,previously reported to suppress expression of downstream HDR factors,resensitizes 53BP1 and REV7-knockout cells to PARPi treatment. Our findings identify key pathways driving PARPi resistance in IDH1-mutant cancers and highlight potential therapeutic strategies to overcome this resistance. Graphical Abstract Graphical Abstract View Publication

Pathogenic GRN variants that reduce progranulin (PGRN) levels cause frontotemporal dementia (FTD). To facilitate model development,we generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of two family members carrying the GRN c.1009C>T (p.Q337X) pathogenic variant—one symptomatic and one asymptomatic—as well as a non-carrier first-degree relative serving as a genetically matched control. The obtained iPSC lines were validated for pluripotency markers (Nanog,Sox2,Oct4,and TRA1-1-81),genomic integrity,and differentiation potential. The obtained iPSC lines were subsequently directed toward neuroepithelial stem (NES) cells. NES identity was confirmed by the expression of lineage-specific markers,including Nestin and Sox2 (assessed by immunocytochemistry),as well as SOX1,PLAGL1,and MKI67 (evaluated by real-time PCR). Furthermore,GRN mRNA levels were significantly reduced in iPSC and NES lines derived from mutation carriers compared to control cells. The established iPSC and NES cell lines represent a platform for modeling progranulin-deficient FTD. The symptomatic and asymptomatic carrier-derived lines obtained from the same family offer a unique opportunity to study disease progression across clinical phases. The control line,derived from a related (first-degree) non-carrier,minimizes genetic background variability. Their utility of the established cell lines extends to therapeutic drug screening and further differentiation into neuronal,non-neuronal,and organoid models. View Publication

Ovine colostrum exosomes obtained from nutritionally programmed dairy ewes (F0) may present modifications in microRNAs,thus having consequences for the intestinal barrier function and immunity parameters of lambs (F1). To test this hypothesis,colostrum exosomes from two ewe groups [F0-MET (nutritionally programmed ewes being fed methionine during early life) and F0-CTRL (ewes not supplemented with methionine during early life)] were sequenced to compare differences in the miRNAome. In addition,these exosomes were added to an in vitro co-culture in a Transwell chamber system consisting of ovine duodenum intestinal organoids and macrophages to assess the expression of genes encoding tight junction proteins in organoids and immunity parameters in macrophages. Finally,the concentrations of cytokines (e.g.,IL-12 and IL-6) were assessed by ELISA kits in the supernatants of the chamber containing macrophages. According to the miRNAome,the expression of two miRNAs (e.g.,oar_miR_376c_3p and oar_miR_432) was reduced in the colostrum exosomes obtained from dairy ewes nutritionally programmed with dietary supplementation of methionine during early life (F0-MET ewes). These changes did not seem to modify the expression of intestinal barrier and immune response marker genes when these exosomes were added to a co-culture of ovine intestinal organoids and macrophages. However,the levels of IL-12 produced by macrophages were reduced (p < 0.05),which suggests the inhibition of inflammatory pathways. Further studies using ovine colostrum exosomes obtained from nutritionally programmed ewes will help to clarify their potential to improve the health of suckling lambs. View Publication

Background: Infection with Mycobacterium avium paratuberculosis (MAP) is closely associated with Crohn's disease (CD) development,where excessive inflammation and marked intestinal damage are observed. Objectives: In this study,the role of short chain fatty acids,including propionic acid (PPA) and butyric acid (BA),was evaluated in an in vitro model,mimicking CD characteristics. Methods: MAP-infected THP-1 macrophages were treated with 1 mM and 10 mM of PPA or BA,and the conditioned media was co-cultured in Caco-2 cells. Results: Both PPA and BA caused an M2 shift with significant downregulation (p-value < 0.0001) in pro-inflammatory markers at both the RNA and protein levels. The downregulation is most likely due to the antimicrobial properties of PPA and BA. MAP growth was inhibited by several folds in MGIT (Mycobacteria Growth Indicator Tube) culture media supplemented with PPA or BA. Dysfunctional Caco-2 intestinal epithelial cells' integrity and function,due to MAP infection,were restored with PPA and BA treatment. Specifically,NOX1 expression was significantly decreased in 10 mM of PPA or BA-treated cells (p < 0.001),as validated by RT-PCR and microscopy. PPA and BA restored tight junction integrity by decreasing Claudin-2 expression in the MAP group. Conclusions: The data clearly demonstrated that short chain fatty acids contain anti-inflammatory and antimicrobial properties with downstream beneficial effects on damaged intestinal epithelial cells,suggesting potential benefits as a dietary supplement for CD patients,particularly those who are not pregnant,due to a possible increased risk of autism spectrum disorder (ASD) development in offspring associated with propionic acid exposure. View Publication