技术资料

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based,and stable mammalian systems have been challenging to establish and apply. To this end,we develop PROTein Evolution Using Selection (PROTEUS),a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS,we alter the doxycycline responsiveness of tetracycline-controlled transactivators,generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution,and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall,PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells. Subject terms: Synthetic biology,Synthetic biology,Molecular evolution,Next-generation sequencing View Publication

Tumor-associated macrophages (TAMs) are key promoters of inflammatory breast cancer (IBC),the most aggressive form of breast cancer. The receptor tyrosine kinase AXL is highly expressed in various cancer types,including IBC,but its role in TAMs remains unexplored. We examined the effects of AXL inhibitor TP-0903 on tumor growth and tumor microenvironment (TME) component M2 macrophages (CD206 + ) in IBC and triple-negative breast cancer mouse models using flow cytometry and immunohistochemical staining. Additionally,we knocked out AXL expression in human THP-1 monocytes and evaluated the effect of AXL signaling on immunosuppressive M2 macrophage polarization and IBC cell growth and migration. We then investigated the underlying mechanisms through RNA sequencing analysis. Last,we performed CIBERSORT deconvolution to analyze the association between AXL expression and tumor-infiltrating immune cell types in tumor samples from the Inflammatory Breast Cancer International Consortium. We found that inhibiting the AXL pathway significantly reduced IBC tumor growth and decreased CD206 + macrophage populations within tumors. Mechanistically,our in vitro data showed that AXL promoted M2 macrophage polarization and enhanced the secretion of immunosuppressive chemokines,including CCL20,CCL26,and epiregulin,via the transcription factor STAT6 and thereby accelerated IBC cell growth and migration. RNA sequencing analysis further indicated that AXL signaling in immunosuppressive M2 macrophages regulated the expression of molecules and cytokines,contributing to an immunosuppressive TME in IBC. Moreover,high AXL expression was correlated with larger populations of immunosuppressive immune cells but smaller populations of immunoactive immune cells in tissues from patients with IBC. AXL signaling promotes IBC growth by inducing M2 macrophage polarization and driving the secretion of immunosuppressive molecules and cytokines via STAT6 signaling,thereby contributing to an immunosuppressive TME. Collectively,these findings highlight the potential of targeting AXL signaling as a novel therapeutic approach for IBC that warrants further investigation in clinical trials. The online version contains supplementary material available at 10.1186/s13058-025-02015-8. View Publication

Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort,time,and costs – which hinders the therapy’s access to the clinic. To ease this transition,we have developed a novel and flexible manufacturing platform,Bioreactor with Expandable Culture Area (BECA),that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside. This report introduces two models in this manufacturing platform: BECA-S for manual small-scale operation at R&D phase and BECA-Auto for functionally closed and automated scaled-out operation at manufacturing phase. We employed these two models to streamline transition of the T cell culture process from manual to automated and reported insignificant differences in the culture outcome between the two. Our work represents the first detailed development and demonstration of a standalone cell manufacturing platform that facilitates a seamless transition between manual and automated processing for autologous T cell therapy manufacturing. View Publication

Mounting evidence indicates that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exosomes) combine the advantages of hucMSC pluripotency with their nanoscale dimensions,enhancing their clinical potential through prolonged circulation half-life. Despite these promising characteristics,research on their immunological toxicity remains insufficient. This study focuses on the impact of hucMSC-exosomes on the general toxicity and immunopathological indicators. When mice received tail vein injections of 6 × 10 10 hucMSC-exosomes particles,we observed no significant changes in body weight,feed intake,blood composition,organ indices,or histopathological findings throughout the 14 days observation period. Similarly,blood levels of immunoglobulins,cytokines,and lymphocyte subpopulations remained stable. The hucMSC-exosomes produced no detectable negative effects on immune organs including the thymus,spleen,and bone marrow. These findings indicate that intravenous administration of 6 × 10 10 particles of hucMSC-exosomes appears relatively safe at the murine level. This assessment of safety and immunological impact following intravenous hucMSC-exosomes infusion offers experimental support for potential clinical applications and future analyses in this field. View Publication

Microglia perform key homeostatic functions to protect the central nervous system (CNS). However,in many brain disorders their protective functions are abrogated,contributing to disease progression. Therefore,studies of microglial function are critical to developing treatments for brain disorders. Different in vitro microglia models have been established,including primary human and rodent cells,induced pluripotent stem cell (iPSC)-derived models,and immortalised cell lines. However,a direct comparative analysis of the phenotypic and functional characteristics of these models has not been undertaken. Accurate modelling of human microglia in vitro is critical for ensuring the translatability of results from the bench to the brain. Therefore,our study aimed to characterise and compare commonly utilised in vitro microglia models. We assessed four established microglia models: primary human microglia,human iPSC-derived microglia,the human microglial clone 3 (HMC3) cell line,and primary mouse microglia,with primary human brain pericytes acting as a negative control. Primary human microglia,iPSC-derived microglia,and mouse microglia stained positive for myeloid-cell markers (Iba1,CD45 and PU.1),while HMC3 cells only stained positive for mural-cell markers (PDGFRβ and NG2). Distinct secretomes were observed in all cell models in response to inflammatory treatment,with iPSC-derived microglia showing the most significant inflammatory secretions. Notably,nitric oxide was only secreted by mouse microglia. Although all cell types exhibited phagocytic capacity,primary human microglia and iPSC-derived microglia displayed significantly higher levels of phagocytosis. Overall,comparative analysis revealed notable differences between human microglia,iPSC-derived microglia,HMC3 cells and mouse microglia. Such differences should be considered when using these models to study human brain diseases. Experimental findings obtained from mouse models or cell lines should ultimately be cross validated to ensure the translatability of results to the human condition. View Publication

Cancer stem cells (CSCs) play crucial roles in tumor metastasis,therapy resistance,and immune evasion. Identifying and understanding the factors that regulate the stemness of tumor cells presents promising opportunities for developing effective therapeutic strategies. In this study on oral squamous cell carcinoma (OSCC),we confirmed the key role of KLF7 in maintaining the stemness of OSCC. Using chromatin immunoprecipitation sequencing and dual-luciferase assays,we identified ITGA2,a membrane receptor,as a key downstream gene regulated by KLF7 in the maintenance of stemness. Tumor sphere formation assays,flow cytometry analyses,and in vivo limiting dilution tumorigenicity evaluations demonstrated that knocking down ITGA2 significantly impaired stemness. Upon binding to its extracellular matrix (ECM) ligand,type I collagen,ITGA2 activates stemness-associated signaling pathways,including PI3K-AKT,MAPK,and Hippo. TC-I 15,a small-molecule inhibitor of the ITGA2-collagen interaction,significantly sensitizes oral squamous cell carcinoma (OSCC) to cisplatin in xenograft models. In summary,we reveal that the KLF7/ITGA2 axis is a crucial modulator of stemness in OSCC. Our findings suggest that ITGA2 is a promising therapeutic target,offering a novel anti-CSC strategy. Subject terms: Cancer stem cells,Cancer therapeutic resistance View Publication

The interplay between 3D genomic structure and transposable elements (TE) in regulating cell state-specific gene expression program is largely unknown. Here,we explore the utilization of TE-derived enhancers in naïve and expanded pluripotent states by integrative analysis of genome-wide Hi-C-defined enhancer interactions,H3K27ac HiChIP profiling and CRISPR-guided TE proteomics landscape. We find that short interspersed nuclear elements (SINEs) are the more involved TEs in the active chromatin and 3D genome architecture. In particular,mammalian-wide interspersed repeat (MIR),a SINE family member,is highly associated with naïve-specific genomic interactions compared to the expanded state. Primarily,in the naïve pluripotent state,MIR enhancer is co-opted by ESRRB for naïve-specific gene expression program. This ESRRB and MIR enhancer interaction is crucial for the formation of loops that build a network of enhancers and super-enhancers regulating pluripotency genes. We demonstrate that loss of a ESRRB-bound MIR enhancer impairs self-renewal. We also find that MIR is co-bound by structural protein complex,ESRRB-YY1,in the naïve pluripotent state. Altogether,our study highlights the topological regulation of ESRRB on MIR in the naïve potency state. The online version contains supplementary material available at 10.1186/s13059-025-03577-8. View Publication

Low dose methotrexate (LD‐MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti‐inflammatory effects of LD‐MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD‐MTX. This is the first study to characterize the effects of LD‐MTX on HSPC. CD34 + HSPC were isolated from healthy donors' non‐mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD‐MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability,cell cycle,surface abundance of reduced folate carrier 1 (RFC1),proliferation,reactive oxygen species (ROS) levels,DNA double‐strand breaks,p38 activation,and CD34 + subpopulations. HSPC clonogenicity was tested in colony‐forming cell assays. Our results indicate that in cycling HSPC,membrane RFC1 is upregulated and,following LD‐MTX treatment,they accumulate more intracellular MTX than resting HSPC. In cycling HSPC,LD‐MTX inhibits HSPC expansion by promoting S‐phase cell‐cycle arrest,increases intracellular HSPC ROS levels and DNA damage,and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD‐MTX are more evident in CD34 + CD38High progenitors. In non‐cycling HSPC,LD‐MTX also reduces the proliferative response while preserving their clonogenicity. In summary,HSPC uptake LD‐MTX differentially according to their cycling state. In turn,LD‐MTX results in reduced proliferation and the preservation of HSPC clonogenicity. View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling,but its function in airway epithelial responses to IL-13,a key mediator in allergic diseases,remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing,providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes,including SPDEF,MUC5AC,POSTN,ALOX15,and CCL26,confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types,suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet,club,and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types,reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner,strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7. View Publication

Development and lineage choice are driven by interconnected transcriptional,epigenetic and metabolic changes. Specific metabolites,such as α-ketoglutarate (αKG),function as signalling molecules affecting the activity of chromatin-modifying enzymes. However,how metabolism coordinates cell-state changes,especially in human pre-implantation development,remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring,characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover,increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather,it decreases acetyl-CoA availability,reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation,highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells,Embryology View Publication

Ceramides are bioactive sphingolipids that have physiological effects on inflammation,apoptosis,and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied,and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model,serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs),followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia),our data showed that following IR,there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis,such as CerS2 and CerS4 in CMs. Further,there was a considerable rise in the levels of total ceramide,long-chain (C16:0,C18:0,and C18:1),and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly,compared to CMs exposed to IR without inhibitor,our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels,reduced apoptosis,improved mitochondrial function,and enhanced survival of CMs exposed to IR. Furthermore,we used a transgenic mouse model,in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice,to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides,which causes increased apoptosis,proinflammatory cytokines,interstitial inflammatory cell infiltration,and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally,in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3. View Publication