技术资料

Genotoxic stress-induced stem cell maldifferentiation (GSMD) integrates DNA damage responses with loss of stemness and lineage-specific differentiation to prevent damaged stem cell propagation. However,molecular mechanisms governing GSMD remain unclear. Here,we identify the p53-induced long non-coding RNA LOC644656 as a key regulator of GSMD in human embryonic stem cells. LOC644656 accumulates in the nucleus upon DNA damage,disrupting pluripotency by interacting directly with POU5F1 and KDM1A/LSD1-NuRD complexes,repressing stemness genes,and activating TGF-β signaling. Additionally,LOC644656 mitigates DNA damage by binding DNA-PKcs and modulating the DNA damage response. In cancer,elevated LOC644656 correlates with poor patient survival and enhanced chemoresistance. Our findings demonstrate that LOC644656 mediates stemness suppression and resistance to genotoxic stress by coordinating DNA damage signaling and differentiation pathways. Thus,LOC644656 represents a potential therapeutic target for overcoming chemoresistance and advancing stem cell biology. The tumor suppressor p53 prevents malignant transformation and regulates stem cell function and differentiation. Here,the authors discover a p53-induced lncRNA,LOC644656,that regulates genotoxic stress-induced stem cell maldifferentiation and chemoresistance in cancer cells. View Publication

Mechanical forces of the tumor microenvironment change dynamically during key events of tumorigenesis such as invasion and metastasis. These changes in compressive forces often affect the breast cancer cell phenotype. However,it is lesser known how these dynamic mechanical forces in the tumor microenvironment affect the phenotypes of tumor infiltrated leukocytes (TIL) and their subsequent anticancer activities. Here we find,in primary patient-derived explant cultures (PDEC) containing resident TILs,that low compression promotes a change in the original identity of breast cancer cells from luminal to a more mesenchymal and undifferentiated state. These altered tumor cells induce an upregulation of immunosuppressive cytokines such as interleukin-10 (IL-10) and Transforming Growth Factor Beta (TGF-β),as well as polarization of macrophages towards pro-tumor M2(Gc)-type and depletion of CD8+ effector memory T-cells. These immunosuppressive events are mediated by tumor cell derived fibroblast growth factor 2 (FGF2) and prostaglandin E2 (PGE2). We also find that FGF2 rich areas in primary tumors show enrichment in M2-like-macrophages and diminished numbers of CD8 + T and B-cells. Our results suggest that low compressive forces in the tumor microenvironment induce local immunosuppression via FGF2 secretion arising from phenotypic plasticity of tumor cells. Mechanical forces modulate tumor cell phenotype,which in turn shapes the tumor microenvironment and contributes to downstream processes such as invasion and metastasis. Here authors show in primary patient-derived breast cancer explant cultures that low compression changes the tumour cells to a less differentiated state,leading to secretion of immunosuppressive cytokines,which impacts the tumor resident immune cells. View Publication

Acute radiation syndrome (ARS) results from high-dose ionizing radiation (IR) exposure,with bone marrow (BM) being highly susceptible due to its proliferative activity. BM injury causes pancytopenia,leading to infections,anemia,and bleeding. Mesenchymal stem cells (MSCs) hold promise for ARS treatment because of their immunomodulatory,anti-inflammatory,and regenerative properties. However,challenges such as replicative senescence,poor survival,and engraftment in irradiated microenvironments limit their efficacy. This study evaluated rapamycin-preconditioned placenta-derived MSCs (rPD-MSCs) in a mouse ARS model. Rapamycin was selected for preconditioning due to its ability to induce autophagy and modulate cytokine secretion. We assessed rapamycin-dependent modulation of autophagy-related genes and proteins,as well as hematopoietic cytokines secretion in PD-MSCs,and evaluated morphological changes in blood and BM at 7 and 21 days post-irradiation in ICR/CD1 mice. Preconditioning with rapamycin alters the secretion of granulocyte colony-stimulating factor (G-CSF),stem cell factor (SCF),and Fms-related tyrosine kinase 3 ligand (Flt3LG) in PD-MSCs without affecting cell viability. rPD-MSCs better enhance hematopoietic recovery,restore bone marrow cellularity,and increase peripheral blood cell counts by elevating the secretion of hematopoietic cytokines compared to non-preconditioned cells. These results highlight rapamycin preconditioning as a promising strategy to enhance MSCs therapeutic potential for ARS,supporting further preclinical and clinical exploration. View Publication

Th17 lymphocytes are a distinct subpopulation of T cells that are characterized by the production of interleukins IL-17,IL-21,IL-22,and IL-26,and high expression of RORγt. These cells play an important role in inflammation and autoimmune diseases. Recent studies using rodent and human models have also highlighted their promising properties as agents in cellular immunotherapy for cancer. However,much less is known about the properties of canine Th17 lymphocytes,despite the domestic dog being an important model used in comparative medicine. In this study,we developed methods of activation and differentiation of canine CD4+ T lymphocytes towards the Th17 phenotype. Additionally,we targeted the Wnt/β-catenin signaling pathway to modulate the efficiency of Th17 cells differentiation. CD4+ T cells were successfully activated with magnetic EpoxyBeads,and in combination with the appropriate programming medium,they acquired the Th17 phenotype. Furthermore,indomethacin,an inhibitor of the Wnt/β-catenin pathway,significantly increased the efficiency of differentiation,causing elevated production of IL-17 and changed T cell metabolism by promoting oxidative phosphorylation. The protocol elaborated in our study provides an efficient method of canine Th17 lymphocyte differentiation. Our findings also suggested that the modification of the Wnt/β-catenin signaling pathway could be a valuable strategy for optimizing canine Th17 cell differentiation and advancing cell-based immunotherapy. View Publication

The intestinal mucosa undergoes a dynamic process of continual proliferation,differentiation,and apoptosis. Delineating the mechanisms involved in intestinal epithelial cell (IEC) differentiation is crucial to our understanding of not only normal gut adaptation but also aberrant intestinal growth. Bone morphogenetic protein (BMP) signaling is a pivotal regulator of intestinal proliferation and differentiation. However,the molecular underpinnings of the BMP pathway in this context are not entirely known. Here,we show a key role for the BMP4/microRNA (miR)-181/glycolysis signaling pathway in the maintenance of intestinal epithelial cell proliferation and differentiation. Treatment with BMP4 increased the expression of enterocyte markers and decreased proliferation of IECs,and importantly,decreased the expression of miR-181a-5p in mouse and human intestinal organoids. miR-181a-5p is a member of the miR-181 family with the highest expression in IECs. Treatment with locked nucleic acid (LNA) miR-181a-5p inhibitor significantly increased enterocyte differentiation as noted by increased expression of enterocyte markers in human and mouse intestinal organoids. In addition,LNA miR-181a-5p inhibitor repressed intestinal stem cell self-renewal as noted by the decreased organoid forming efficiency and expression of Ki67,cyclin D1,OLFM4 in human and mouse intestinal organoids. Moreover,in vivo administration of LNA miR-181a-5p inhibitor enhanced increased intestinal enterocyte differentiation and repressed intestinal cell proliferation. In contrast,overexpression of miR-181a-5p mimic decreased basal and BMP4-induced expression of enterocyte markers. Moreover,BMP4 treatment or inhibition of miR-181a-5p repressed hexokinase (HK) 1 expression and inhibited glycolysis. Consistently,knockdown of HK1 or inhibition of glycolysis using 2-deoxyglucose (2-DG) promoted enterocyte maturation and inhibited proliferation of IECs. Together,we provide evidence showing that miR-181a-5p inhibits intestinal enterocyte differentiation and promotes IEC proliferation through HK1-dependent glycolysis. Importantly,our findings identify miR-181a-5p as downstream in mediating BMP4 induction of enterocyte differentiation and inhibition of proliferation in IECs. View Publication

Corneal keratocytes (CK) and endothelial cells (CEnC) maintain corneal stromal transparency. Damage to these cell types can lead to visual impairment. Although corneal transplantation is effective,donor shortages limit its availability. Human pluripotent stem cells (hPSC) offer a promising alternative for generating corneal cell types. While hPSC-derived epithelial cells and CEnC are well-studied,attempts to differentiate CK from hPSC have received less attention. This study investigates the differentiation of hPSC-CK using defined culture conditions and approaches to enrich both CK and CEnC. hPSC were cultured on laminin 521 (LN-521) coatings and differentiated using transforming growth factor β (TGFβ) and glycogen synthase kinase-3 (GSK-3) inhibitors,along with retinoic acid (RA) with (mEn protocol) or without (En protocol) fibroblast growth factor-2 (FGF2). Differentiated cells were characterized using qPCR and immunofluorescence on days (D) 8,10,and 13. CK enrichment utilized collagen-1-coated plates with keratocyte-specific media,while CEnC were purified through metabolic starvation. Results showed the formation of heterogenous cultures containing both CK and CEnC. CK-like cells expressed keratocan (KERA),lumican (LUM),paired box 6 (PAX6) and actin α2 (ACTA2) genes,with proteoglycan expression (lumican (LUM) and decorin (DCN)) increasing over time. En protocol however maintained a stable CK phenotype. Furthermore,enriched hPSC-CK were LUM+/DCN+/PAX6−/CD166− and hPSC-CEnC were CD166+/ZO-1+. Using xeno-free,defined conditions,we differentiated both CK and CEnC from hPSC using a single protocol with further optimized enrichment methods for hPSC-CK and hPSC-CEnC purification,advancing cell differentiation techniques.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-03509-3. View Publication

Autophagy is a ‘waste-disposal’ pathway that protects against age-related pathology. It is widely accepted that autophagy declines with age,yet the role that sex and diet-related obesity play during aging remain unknown. Here,we present the most comprehensive in vivo study of autophagic flux to date. We employed transgenic mice overexpressing tandem-florescent LC3B (RFP-GFP-LC3B) to measure autophagic flux in the blood (PBMCs),heart,and motor cortex neurons of aging mice that were fed regular chow or a high-fat diet for 6-,12- or 18-months. In male mice,aging decreased autophagic flux in the heart,increased it in the blood,and had no effect in motor cortex neurons. Age-dependent changes autophagic flux were less pronounced in female mice. High-fat diet influenced autophagic flux in the blood and heart of male but not female mice. Overall,we uncovered sexual dimorphisms that underpin how autophagy changes with age across different tissues and in response to a high-fat diet. View Publication

Simple SummaryThis study aimed to classify and investigate the distribution of gene variants in 13 clinically relevant genes of 5923 Danish patients with suspected hereditary predisposition to breast and/or ovarian cancer,all of whom were tested with the same gene panel. The growth in genetic analysis over the last 25 years has generated an increasing number of variants of unknown significance (VUSs). These present challenges for daily clinical counselling and decision-making about whether a carrier should be offered inclusion in a surveillance program or risk-reducing surgery. We examined VUSs using two methods: an association analysis comparing the case group to a Swedish control group,and splice analysis using RNA sequencing. AbstractBackground: Gene variants of unknown significance (VUSs) present a challenge in genetic counselling. The primary aim of this study was to describe the spectrum of genetic findings in a cohort of 5923 Danish patients with suspected predisposition to hereditary breast and/or ovarian cancer,with a focus on classifying gene variants and investigating their distribution. Methods: The gene variants were classified using the American College of Medical Genetics (ACMG) guidelines as well as gene-specific guidelines where applicable. The identified VUSs were further examined through association analysis,comparison of the frequencies in this Danish population to those in the Swedish population using gnomAD 2.1,and splice analysis using RNA sequencing. Results: Of 167 variants that were clinically classified as VUSs prior to this research study,38 (22.8%) were either up- or downgraded based on the guidelines that were used. We found that 630 patients (10.6%) carried a likely pathogenic or pathogenic variant,mainly in BRCA1 (31.9%) and BRCA2 (26.0%). VUSs were carried by 1606 (27.1%) patients,mainly in BARD1 (27.6%) and ATM (19.3%). Our association study assigned criteria for 10 gene variants,while our splice analysis assigned criteria for 3 gene variants but did not reclassify the variants. Conclusions: A total of 22.8% of the 167 variants that were observed in this study and which were previously classified as VUSs in a clinical setting were reclassified in this study. In total,10.6% of the patients with a suspected predisposition to hereditary breast and/or ovarian cancer carried a likely pathogenic or pathogenic variant. The high incidence of VUSs observed in this study reflects the challenges faced in the daily clinical setting. View Publication

Despite the significant progress made in the development of epigenetic age (eAge) clocks designed to estimate the various aspects of aging,currently available models,generated using large DNA methylation microarray datasets,still cannot fully address the issues of batch effects and technical variation. This hinders the use of the publicly available eAge clocks in routine laboratory practice,and it motivates the development of cost-effective,custom epigenetic clocks that are tailored to the given biological subjects and research methods. In this study,we analyzed the local DNA methylation of mesenchymal stem cell samples during culture expansion using high-throughput targeted bisulfite sequencing (BS-seq). Using the obtained data,we trained a minimized eAge model based on a Random Forest Regression with Leave-One-Out Cross-Validation,which determines cell passage with good performance (MAE 1.094 and R2 0.897) and which is comparable to previous solutions. Using the advantage of BS-seq to analyze consecutive CpGs methylation patterns,we demonstrated that combining the analysis of average DNA methylation levels with local methylation heterogeneity scores—thereby reflecting stochastic DNA methylation dynamics—can improve the quality of the epigenetic clock models. Therefore,we propose a research strategy for creating customized epigenetic clocks using targeted BS-seq and provide a mechanistic conceptualization of how information on longitudinal changes in DNA methylation patterns can potentially be used for the assessment of specific aging aspects. View Publication

Pre-clinical studies have shown that exercise can decrease tumour growth through mobilisation,activation,and increased tumour infiltration of natural killer (NK) and CD8+ T cells. It is currently unclear whether this can be extrapolated to patients. Therefore,a pilot study was set up to examine the feasibility of obtaining an additional study biopsy and to generate preliminary data on the potential effects of exercise on peripheral immune cell function and tumour immune infiltration. Twenty patients with stage I-III breast cancer receiving neoadjuvant chemotherapy were included (participation rate: 27%). Patients were randomised into the intervention group receiving a six-week supervised aerobic and resistance exercise program or the control group. Blood samples and tumour biopsies were collected before randomisation and after six weeks of chemotherapy. For 8 of 20 (40%) patients,we were able to obtain and analyse biopsies at diagnosis and six-week follow-up. This showed a decrease in CD56+ cells/mm2 tumour tissue in the three patients of the control group,while it remained stable in most patients of the exercise group. Upon co-culture of peripheral blood mononuclear cells with K562 tumour cells,the exercise group showed increased expression of the degranulation marker CD107a on NK cells (β = 1038.5,95%CI = 56.9; 2020.2,p = 0.04),and a trend towards increased tumour cell lysis in vitro (β = 18.8%,95%CI = −3.9; 41.5,p = 0.10) compared to the control group. In conclusion,the study design was feasible with regard to the participation rate,however,revision is needed with regard to the use of a study-related biopsy prior to a sufficiently powered randomised controlled trial. Highlights•A 6-week exercise intervention increased the degranulation capacity of NK cells.•Exercise caused a trend towards increased in vitro tumour cell lysis.•Exercise during chemotherapy may enhance peripheral NK cell function.•A study-related biopsy is challenging due to missings and insufficient quality. View Publication

BackgroundReindeer (Rangifer tarandus tarandus) herding is based on access to seasonal pastures. Pastureland is,however,being lost and fragmented due to e.g. climate change,human activities,and predators,creating an increasing need for feeding and fencing. This alters disease occurrence,leading to a greater need for disease investigation tools. Knowledge of the activation of immune pathways during disease can be obtained by measuring cytokines,but no commercial methods are currently available for reindeer. This study investigated whether the MILLIPLEX® Bovine Cytokine Magnetic Bead assay could be used to detect interleukin (IL)-6,IL-8,IL-10,IL-17,tumor necrosis factor-alpha (TNF-α),and interferon-gamma (IFN-γ) in reindeer cell supernatants and serum. Peripheral blood mononuclear cells (PBMCs) from reindeer (n = 4) and cattle (Bos taurus,n = 3) were stimulated with mitogens for 6 and 24 h (h) and the quantity of cytokines in cell supernatants was measured. Serum from experimental viral infections in reindeer (Orf virus; ORFV and Varicellovirus cervidalpha2; CvHV2) was also analysed. Additionally,primers were designed to measure cytokine gene expression in response to mitogens by real-time polymerase chain reaction (qPCR). ResultsThe bovine bead-based multiplex immunoassay detected five of six cytokines (IL-8,IL-10,IL-17,TNF-α,IFN-γ) in reindeer PBMC supernatants after stimulation. All cytokines were detected in bovine samples. Although cytokine concentrations were generally higher in bovine samples,analysis of reindeer supernatants demonstrated significantly increased IL-10,IL-17,TNF-α and IFN-γ concentrations in supernatants from stimulated compared to unstimulated PBMCs. Neither reindeer nor cattle samples showed a significant increase for IL-6,while IL-8 was increased only in bovine samples after 6 h stimulation. Serum from reindeer infected with CvHV2 showed significantly increased IFN-γ levels on days 4 and 7 post inoculation. Gene expression of all cytokines was increased by stimulation of reindeer PBMCs,except IL-6 for which primer design was unsuccessful.ConclusionsThis study shows the potential of the bovine bead-based multiplex immunoassay for measuring IL-10,IL-17,TNF-α,and IFN-γ concentrations in reindeer. The qPCR is suitable for measuring gene expression of these cytokines and IL-8. These methods may be used to characterise immune responses in reindeer,but further testing and validation are warranted.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13028-025-00819-4. View Publication

PurposeThe increasing application of virus-specific T cell therapy for treating BK virus infections in immunocompromised patients highlights the necessity for rapid identification of compatible cell donors with optimal BK-specific T cell response. This study aims to characterize the BK virus-specific T cell response in relation to demographic factors,blood group,serological status,and HLA genotypes using samples from a cell donor registry.MethodsPeripheral blood mononuclear cells from cell donors were stimulated with peptide pools derived from VP1 and LTA proteins,and the IFN-γ production was analyzed using ELISpot and validated by flow cytometry.ResultsOur findings provide an overview of the T cell response to BK virus proteins in healthy donors,revealing associations with demographic characteristics,RhD status,CMV or EBV serological status,and HLA alleles. Remarkably,RhD-negative,CMV-seronegative,and EBV-seronegative donors showed a major T cell response against BK virus proteins. Notably,certain HLA alleles were associated with either enhanced or diminished T cell response. Furthermore,our results suggest that HLA-B leader dimorphism,specifically the presence of threonine at position 2,influences the VP1-specific immune response,resulting in enhanced T cell activation.ConclusionThis study,beyond advancing our understanding of the relationship between donor characteristics and BK virus-specific T cell response,has significant implications for improving the selection of optimal cell donors for patient-specific adoptive therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-025-01901-2. View Publication