技术资料

Telomeres are terminal protective chromosome structures. Genetic variants in genes coding for proteins required for telomere maintenance cause rare,life-threatening Telomere Biology Disorders (TBDs) such as dyskeratosis congenita,aplastic anemia or pulmonary fibrosis. The more frequently used mice strains have telomeres much longer than the human ones which question their use as in vivo models for TBDs. One mice model with shorter telomeres based on the CAST/EiJ mouse strain carrying a mutation in the Terc gene,coding for the telomerase RNA component,has been studied in comparison with C57BL/6J mice,carrying the same mutation and long telomeres. The possible alterations produced in lungs and the haematopoietic system,frequently affected in TBD patients,were determined at different ages of the mice. Homozygous mutant mice presented a very shortened life span,more notorious in the short-telomeres CAST/EiJ strain. The lungs of mutant mice presented a transitory increase in fibrosis and a significant decrease in the relative amount of the alveolar epithelial type 2 cells from six months of age. This decrease was larger in mutant homozygous animals but was also observed in heterozygous animals. On the contrary the expression of the senescence-related protein P21 increased from six months of age in mutant mice of both strains. The analysis of the haematopoietic system indicated a decrease in the number of megakaryocyte-erythroid progenitors in homozygous mutants and an increase in the clonogenic potential of bone marrow and LSK cells. Bone marrow cells from homozygous mutant animals presented decreasing in vitro expansion capacity. The alterations observed are compatible with precocious ageing of lung alveolar cells and the bone marrow cells that correlate with the alterations observed in TBD patients. The alterations seem to be more related to the genotype of the animals that to the basal telomere length of the strains although they are more pronounced in the short-telomere CAST/EiJ-derived strain than in C57BL/6J animals. Therefore,both animal models,at ages over 6–8 months,could represent valuable and convenient models for the study of TBDs and for the assay of new therapeutic products. View Publication

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study,we have created a comprehensive resource of high-quality genomic,epigenomic,and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC),motor neuron,monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage,and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally,cell type is a stronger source of epigenetic variation than genetic variation. Further,we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics,Genomics,Transcriptomics View Publication

Respiratory viruses pose an ongoing threat to human health with excessive cytokine secretion contributing to severe illness and mortality. However,the relationship between cytokine secretion and viral infection remains poorly understood. Here we elucidate the role of CXCL8 as an early response gene to EV-D68 infection. Silencing CXCL8 or its receptors,CXCR1/2,impedes EV-D68 replication in vitro. Upon recognition of CXCL8 by CXCR1/2,the MAPK pathway is activated,facilitating the translocation of nuclear hnRNP-K to the cytoplasm. This translocation increases the recognition of viral RNA by hnRNP-K in the cytoplasm,promoting the function of the 5′ untranslated region in the viral genome. Moreover,our investigations also reveal the importance of the CXCL8 signaling pathway in the replication of both influenza virus and rhinovirus. In summary,our findings hint that these viruses exploit the CXCL8/MAPK/hnRNP-K axis to enhance viral replication in respiratory cells in vitro. Subject terms: Virus-host interactions,Chemokines,Antimicrobial responses,Viral host response View Publication

In contrast to mammals,adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly,we found that regenerating cardiomyocytes experience DNA replication stress,which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes,human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication. Subject terms: Cardiac regeneration,DNA damage and repair,Ageing View Publication

Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity,scalability,throughput,and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However,these models have faced challenges associated with their poorly scalable creation,sourcing suitable cells,and the extraction of reliable,quantifiable metrics. We present a turnkey iPSC-based model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 ± 22.7 µN in response to blue light,with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC50 - 0.11 ± 0.015 µg). Variability of the EC50 values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ,providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories. View Publication

Coronary artery disease (CAD) is linked to atherosclerosis plaque formation. In pro-inflammatory conditions,human Natural Killer (NK) cell frequencies in blood or plaque decrease; however,NK cells are underexplored in CAD pathogenesis,inflammatory mechanisms,and CAD comorbidities,such as human cytomegalovirus (HCMV) infection and diabetes. Analysis of PBMC CITE-seq data from sixty-one CAD patients revealed higher blood NK cell SPON2 expression in CAD patients with higher stenosis severity. Conversely,NK cell SPON2 expression was lower in pro-inflammatory atherosclerosis plaque tissue with an enriched adaptive NK cell gene signature. In CAD patients with higher stenosis severity,peripheral blood NK cell SPON2 expression was lower in patients with high HCMV-induced adaptive NK cell frequencies and corresponded to lower PBMC TGFβ transcript expression with dependency on diabetes status. These results suggest that high NK cell SPON2 expression is linked to atherosclerosis pro-homeostatic status and may have diagnostic and prognostic implications in cardiovascular disease. View Publication

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells and a differentiation block,highlighting the urgent need for novel differentiation-inducing therapies. This study evaluated Adina rubella Hance (ARH) stem as a potent differentiation inducer by systematically screening 200 plant extracts. ARH stem promoted phenotypic differentiation in AML cells. In addition to its differentiation-inducing effects,ARH stem exhibited strong antileukemic activities,such as inhibiting cell proliferation,inducing cell death,and enhancing mitochondrial reactive oxygen species (mtROS) levels,the latter of which is critical for its differentiation-promoting activity. Comparative analysis with the extracts from other parts of the plant confirmed the superior efficacy of the stem extract because of its unique chemical composition. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry analysis identified Picroside III as a major active compound within the stem extract,capable of recapitulating ARH stem-induced differentiation and demonstrating significant antileukemic properties. These findings underscore the therapeutic potential of ARH stem and its active component,Picroside III,as promising agents for differentiation-based treatment strategies in AML. View Publication

Genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) are becoming increasingly important in the treatment of hematologic malignancies and are also intensively being investigated for other diseases such as autoimmune disorders and HIV. Current CAR T cell therapies predominantly use viral transduction methods which,despite their efficacy,raise safety concerns related to genomic integration and potentially associated malignancies as well as labor- and cost-intensive manufacturing. Therefore,non-viral gene transfer methods,especially mRNA-based approaches,have attracted research interest due to their transient modification and enhanced safety profile. In this study,the optimization of CAR-mRNA for T cell applications is investigated,focusing on the impact of mRNA modifications,in vitro transcription protocols,and purification techniques on the translation efficiency and immunogenicity of mRNA. Furthermore,the refined CAR-mRNA was used to generate transient CAR T cells from acute myeloid leukemia patient samples,demonstrating efficacy in vitro and proof-of-concept for clinically relevant settings. These results highlight the potential of optimized mRNA to produce transient and safe CAR T cells. View Publication

Adipose-derived stromal cells (ASCs) possess significant regenerative potential,playing a key role in tissue repair and angiogenesis. During wound healing,ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs,which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior,with a focus on angiogenesis. To mimic the wound-healing environment,ASC were cultured in a porcine gelatin sponge,an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation,angiogenesis,and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors,including IL-1,IL-6,IL-8,and VEGF,were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration,tubulogenesis,and reduced endothelial permeability,all critical processes in angiogenesis. Notably,ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects,underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms,offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies. View Publication

Macrophages are the major source of WNT ligands. However,the regulation of WNT expression in macrophages has not been studied. In the present study,we have discovered that activation of canonical β-Catenin signaling suppresses WNT expression in macrophages. EVs from these pre-conditioned macrophages promoted intestinal stem cell regeneration and mitigated intestinal injury. ChIP-seq analysis and validation studies using recombinant DNA construct expressing Luciferase reporter under WNT promoter (e.g. WNT5a and WNT9b) were conducted to demonstrate the involvement of β-Catenin in the transcriptional regulation of WNT expression. The regulatory role of β-Catenin in WNT expression in macrophages was examined by treating these cells with a Tankyrase inhibitor. In addition,the gene expressing β-Catenin was deleted in macrophages using Csf1r.iCre; Ctnnb1 fl/fl mice model. Both pharmacological and genetically modulated macrophages were examined for WNT expression and activity by qPCR and TCF/LEF luciferase assay respectively. Additionally,Csf1r.iCre; Ctnnb1 fl/fl mice were exposed to irradiation to compare the radiosensitivity with their wildtype littermate. Extracellular vesicles (EVs) were isolated from pre-conditioned WNT-enriched macrophages and infused in irradiated C57BL/6 and Lgr5/eGFP-IRES-Cre-ERT2 ; R26-ACTB-tdTomato-EGFP mice to determine the regenerative response of intestinal stem cell (ISC) and epithelial repair. Regenerative effects of EVs were also examined in mice model DSS induced colitis. ChIP-seq analysis and subsequent validation study suggested physical association of β-Catenin with WNT promoters to suppress WNT expression. Macrophage specific deletion of gene expressing β-Catenin or pharmacological inhibition of Tankyrase improves the WNT expression in macrophages several folds compared to control. Transfusion of these preconditioned macrophages or EVs from these cells delivers optimum level of morphogenic WNT to injured epithelium,activates ISC regeneration and mitigated radiation induced intestinal injury. Intestinal epithelium in Csf1r.iCre; Ctnnb1 fl/fl mice also showed radioresistance compared to wild type littermate. Moreover,EVs derived from WNT enriched macrophages can mitigate intestinal injury in mice model of DSS induced acute colitis. The study provides substantial evidence that macrophage-targeted modulation of canonical WNT signaling induces WNT expression in macrophages. Treatment with preconditioned macrophage derived WNT-enriched EVs can be a promising therapeutic approach against intestinal injury. The online version contains supplementary material available at 10.1186/s12964-025-02065-7. View Publication

Lactate dehydrogenase A (LDHA) can regulate tumorigenesis and cancer progression. Nevertheless,whether the regulation of LDHA is involved in the development of gemcitabine resistance in PDAC has not yet been fully elucidated. Increasing studies have shown that cancer acquired drug resistance led to treatment failure is highly attributed to the cancer stem cell (CSC) properties. Therefore,we aim to demonstrate the functions and regulatory mechanisms of LDHA on cancer stem cell (CSC) properties and gemcitabine resistance in PDAC. We investigate the metabolite profiles by liquid chromatography-mass spectrometry between gemcitabine–resistant PDAC and parental PDAC cells. Additionally,gain-of-function and loss-of-function experiments were conducted to examine the roles of LDHA on CSC properties and gemcitabine resistance in the gemcitabine–resistant PDAC and parental PDAC cells. To investigate regulators involved in LDHA-mediated gemcitabine resistance and CSC of pancreatic cancer cells,we further used a combination of the miRNA microarray results and software predictions and confirmed that miR-4259 is a direct target of LDHA by luciferase assay. Furthermore,we constructed serial miR-4259 promoter reporters and searched for response elements using the TESS 2.0/TFSEARCH software to find the transcription factor binding site in the promoter region of miR-4259. We observed that elevated LDHA expression significantly correlates with recurrent pancreatic cancer patients following gemcitabine treatment and with CSC properties. We further identify that FOXO3a-induced miR-4259 directly targets the 3’untranslated region of LDHA and reduced LDHA expression,leading to decreased gemcitabine resistance and a reduction in the CSC phenotypes of pancreatic cancer. Our results demonstrated that LDHA plays a critical role in cancer stemness and gemcitabine resistance of pancreatic cancer,and indicate that targeting the FOXO3a/miR-4259/LDHA pathway might serve as a new treatment for pancreatic cancer patients with a poor response to gemcitabine chemotherapy. The online version contains supplementary material available at 10.1186/s40170-025-00377-3. View Publication

Cerebral organoids (COs) are valuable tools for studying the intricate interplay between glial cells and neurons in brain development and disease,including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and inducing pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly with the neuronal progenitors. Compared with conventional COs,CO-iMs were more efficient at generating CD45 + /CD11b + /Iba-1 + microglia and presented a physiologically relevant proportion of microglia (~ 7%). CO-iMs presented substantially increased expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs are susceptible to HIV infection,resulting in a significant increase in several pro-inflammatory cytokines/chemokines,which are abrogated by the addition of antiretrovirals. Thus,CO-iM is a robust model for deciphering neuropathogenesis,neuroinflammation,and viral infections of brain cells in a 3D culture system. The online version contains supplementary material available at 10.1186/s12974-025-03353-2. View Publication