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Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal,chondrogenic,and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However,there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study,we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently,a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media,MesenCult™-ACF (ACF),and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology,surface markers,senescence status,differentiation capacity,and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. View Publication

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines,we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers,while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing. View Publication

Although acute myeloid leukemia (AML) affects hematopoietic stem cell (HSC)-supportive microenvironment,it is largely unknown whether leukemia-modified bone marrow (BM) microenvironment can be remodeled to support normal hematopoiesis after complete remission (CR). As a key element of BM microenvironment,endothelial progenitor cells (EPCs) provide a feasible way to investigate BM microenvironment remodeling. Here,we find reduced and dysfunctional BM EPCs in AML patients,characterized by impaired angiogenesis and high ROS levels,could be partially remodeled after CR and improved by N-acetyl-L-cysteine (NAC). Importantly,HSC-supporting ability of BM EPCs is partially recovered,whereas leukemia-supporting ability is decreased in CR patients. Mechanistically,the transcriptome characteristics of leukemia-modified BM EPCs return to near-normal after CR. In a classic AML mouse and chemotherapy model,BM vasculature and normal hematopoiesis are reversed after CR. In summary,we provide further insights into how leukemia-modified BM microenvironment can be remodeled to support normal hematopoiesis after CR,which can be further improved by NAC. Subject terms: Translational research,Acute myeloid leukaemia View Publication

The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare,naturally occurring,homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here,we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human,mobilized hematopoietic stem progenitor cells (HSPC),resulting in a transplant that undergoes normal hematopoiesis,produces CCR5 null T cells,and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection. Subject terms: HIV infections,CRISPR-Cas9 genome editing,Retrovirus,Translational research View Publication

Growing evidence shows that the reprogramming of fatty acid (FA) metabolism plays a key role in HER2-positive (HER2 +) breast cancer (BC) aggressiveness,therapy resistance and cancer stemness. In particular,HER2 + BC has been defined as a "lipogenic disease" due to the functional and bi-directional crosstalk occurring between HER2-mediated oncogenic signaling and FA biosynthesis via FA synthase activity. In this context,the functional role exerted by the reprogramming of CD36-mediated FA uptake in HER2 + BC poor prognosis and therapy resistance remains unclear. In this study,we aimed to elucidate whether enhanced CD36 in mesenchymal HER2 + cancer stem cells (CSCs) is directly involved in anti-HER2 treatment refractoriness in HER2 + BC and to design future metabolism-based approaches targeting both FA reprogramming and the “root” of cancer. Molecular,biological and functional characterization of CD36-mediated FA uptake was investigated in HER2 + BC patients,cell lines,epithelial and mesenchymal CSCs. Cell proliferation was analyzed by SRB assay upon treatment with lapatinib,CD36 inhibitor,or Wnt antagonist/agonist. Engineered cell models were generated via lentivirus infection and transient silencing. CSC-like properties and tumorigenesis of HER2 + BC cells with or without CD36 depletion were examined by mammosphere forming efficiency assay,flow cytometry,cell sorting,ALDH activity assay and xenograft mouse model. FA uptake was examined by flow cytometry with FA BODIPY FL C16. Intratumor expression of CSC subsets was evaluated via multiplex immunostaining and immunolocalization analysis. Molecular data demonstrated that CD36 is significantly upmodulated on treatment in therapy resistant HER2 + BC patients and its expression levels in BC cells is correlated with FA uptake. We provided evidence of a consistent enrichment of CD36 in HER2 + epithelial-mesenchymal transition (EMT)-like CSCs from all tested resistant cell models that mechanistically occurs via Wnt signaling pathway activation. Consistently,both in vitro and in vivo dual blockade of CD36 and HER2 increased the anti-CSC efficacy of anti-HER2 drugs favoring the transition of the therapy resistant mesenchymal CSCs into therapy-sensitive mesenchymal-epithelial transition (MET)-like epithelial state. In addition,expression of CD36 in intratumor HER2 + mesenchymal CSCs is significantly associated with resistance to trastuzumab in HER2 + BC patients. These results support the metabolo-oncogenic nature of CD36-mediated FA uptake in HER2 + therapy-refractory BC. Our study provides evidence that targeting CD36 might be an effective metabolic therapeutic strategy in the treatment of this malignancy. The online version contains supplementary material available at 10.1186/s13046-025-03276-z. View Publication

Global disparities in cancer prevention,detection,and treatment demand a unified international effort to reduce the disease’s burden and improve outcomes. Despite advances in chemotherapy and radiotherapy,many tumors remain resistant to these treatments. Gold nanoparticles (AuNPs) have shown promise as radiosensitizers,enhancing the effectiveness of low-energy X-rays by emitting Auger electrons that cause localized cellular damage. In this study,spherical AuNPs of 5 nm and 10 nm were characterized and tested on various cell lines,including malignant breast cells (MCF-7),non-malignant cells (CHO-K1 and MCF-10A),and human lymphocytes. Cells were treated with AuNPs and irradiated with attenuated 6 megavoltage (MV) X-rays or p(66)/Be neutron radiation to assess DNA double-strand break (DSB) damage,cell viability,and cell cycle progression. The combination of AuNPs and neutron radiation induced higher levels of γ-H2AX foci and micronucleus formation compared to treatments with AuNPs or X-ray radiation alone. AuNPs alone reduced cellular kinetics and increased the accumulation of cells in the G2/M phase,suggesting a block of cell cycle progression. For cell proliferation,significant effects were only observed at the concentration of 50 μg/mL of AuNPs,while lower concentrations had no inhibitory effect. Further research is needed to quantify internalized AuNPs and correlate their concentration with the observed cellular effects to unravel the biological mechanisms of their radioenhancement. View Publication

Myelodysplastic syndrome (MDS) is a malignant hematologic disorder with limited curative options,primarily reliant on hematopoietic stem cell transplantation. Anemia,a prevalent symptom of MDS,has few effective treatment strategies. Realgar,though known for its therapeutic effects on MDS,remains poorly understood in terms of its mechanism of action. In this study,both in vivo and in vitro experiments were conducted using Realgar and its primary active component,As 2 S 2,to examine their impact on mouse erythroblasts at the single-cell level. Realgar treatment significantly altered the transcriptional profiles and cellular composition of bone marrow in mice,both in vivo and in vitro. Differentially expressed genes in erythroblasts regulated by Realgar were identified,unveiling potential regulatory functions and signaling pathways,such as heme biosynthesis,hemoglobin production,oxygen binding,IL-17 signaling,and MAPK pathways. These findings suggest that Realgar enhances the differentiation of erythroblasts in mouse bone marrow and improves overall blood cell counts. This work offers preliminary insights into Realgar’s mechanisms,expands the understanding of this mineral medicine,and may inform strategies to optimize its therapeutic potential in hematologic diseases. The online version contains supplementary material available at 10.1186/s12935-025-03768-0. View Publication

The in vitro generation of human red blood cells (RBCs) from stem cells,such as induced pluripotent stem cells (iPSCs),holds promise for transfusable RBCs but faces challenges,including RBC maturation,enucleation,and large-scale production. In this study,we evaluated the effect of conditional TAL1 overexpression on in vitro RBC production via hematopoietic cell-forming complex (HCFC) formation from iPSCs because TAL1 is a key regulatory transcription factor essential for erythropoiesis. TAL1 overexpression in iPSCs,either before or after hematopoietic induction,significantly enhanced HCFC formation and hematopoietic differentiation,as evidenced by increased hematopoiesis-related gene expression,a higher yield of glycophorin A (GPA)+/CD71+ cells,and elevated gamma hemoglobin levels. These findings highlight the potential of TAL1 as a powerful regulator of erythropoiesis in vitro and offer a promising strategy for improving RBC production from stem cells. However,the reduced enucleation efficiency observed after TAL1 overexpression indicates a key challenge that must be addressed to optimize the generation of fully functional,transfusable RBCs. Further research is required to balance the benefits of enhanced differentiation with the need for efficient enucleation,which is critical for the production of mature,viable RBCs. View Publication

The intestinal epithelium of human infants is developmentally immature compared to that of adults. Exactly how this immaturity affects key epithelial functions and their interactions with nearby immune cells remains an understudied area of research,partly due to limited access to non-diseased infant gut tissues. Human intestinal organoids,or “mini guts” generated from tissue stem cells,are promising models for investigating intestinal biology and disease mechanisms. These three-dimensional structures closely mimic their tissue of origin,including cellular physiology and genetics. We have also previously shown that neonatal Th17 cells represent a distinct cell population with a cytokine profile skewed toward IL-22 production rather than IL-17A,as seen in adult Th17 cells. In this study,we sought to model the impact of neonatal-derived Th17 cytokine,namely IL-22 and the intestinal epithelium using infant-derived ileal enteroids. We generated enteroids from ileal biopsies from infants (< 6 months old) and cultured them for seven days with standard organoid growth media,organoid media supplemented with conditioned media from cord-blood-derived Th17 cells,or media supplemented with recombinant IL-22. We assessed morphological changes and conducted transcriptomics profiling via RNAseq. Exposing enteroids to neonatal Th17-cells-derived conditioned media led to enhanced growth,maturation,and differentiation as compared to control media. These effects were ablated when an IL-22 neutralizing antibody was used,while conversely,supplementing with recombinant IL-22 mimicked the Th17 effects,increasing intestinal epithelial cell proliferation and inducing marked differentiation of secretory cells. Our transcriptomic profiling similarly demonstrated significant changes in response to IL-22 with downregulation of Wnt and Notch signaling and upregulation of immune pathways,particularly interferon signaling. The transcriptomic data also suggested that IL-22 treatment led to changes in cell type composition with an increase in stem- and progenitor cells at the expense of enterocytes. Taken together,our data suggests that early-life intestinal development is likely influenced by IL-22-dependent crosstalk between the infant epithelium and exposure to neighboring Th17 cells. This promotes epithelial cell maturation and immune readiness,reflected at both the morphological and molecular levels. Our work also provides a relevant framework for studying healthy infant gut development,which can be further leveraged to examine early-life gastrointestinal disorders,model complex human disease,and therapeutic testing while reducing reliance on animal models. View Publication

Prenatal alcohol exposure (PAE) affects embryonic development,causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation,we used an in vitro model with subchronic moderate (20 mM) and severe (70 mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analyzed genome-wide gene expression (mRNA sequencing),DNA methylation (EPIC Illumina microarrays) and metabolome (non-targeted LC-MS) of the endodermal,mesodermal and ectodermal cells. The largest number of ethanol-induced alterations were observed in endodermal cells,whereas the most prominent changes were in ectodermal cells. Methionine metabolism and genes of the main signaling pathways involved in gastrulation and body patterning were affected by ethanol in all germ layers. Many of the altered genes,including BMP4,FGF8,SIX3 and LHX2,have previously been associated with PAE and phenotypes of FASD,like defects in heart and corpus callosum development as well as holoprosencephaly. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD. View Publication

Influenza virus particularly affects those with chronic lung conditions such as Chronic Obstructive Pulmonary Disease (COPD). Airway epithelial cells are the first line of defense and primary target of influenza infection and release extracellular vesicles (EVs). EVs can transfer of biological molecules such as microRNAs (miRNAs) that can modulate the immune response to viruses through control of the innate and adaptive immune systems. The aim of this work was to profile the EV miRNAs released from bronchial epithelial cells in response to influenza infection and discover if EV miRNA expression was altered in COPD. Influenza infection of air-liquid interface (ALI) differentiated BCi-NS1.1 epithelial cells were characterized by analyzing the expression of antiviral genes,cell barrier permeability and cell death. EVs were isolated by filtration and size exclusion chromatography from the apical surface wash of ALI cultured bronchial epithelial cells. The EV miRNA cargo was sequenced and reads mapped to miRBase. The BCi sequencing results were further investigated by RT-qPCR and by using healthy and COPD primary epithelial cells. Infection of ALI cultured BCi cells with IAV at 3.6 x 10 6 IU/ml for 24 h led to significant upregulation of anti-viral genes without high levels of cell death. EV release from ALI-cultured BCi cells was confirmed using electron microscopy and detection of known tetraspanin EV markers using western blot and the ExoView R100 platform. Differential expression analyses identified 5 miRNA that had a fold change of >0.6: miR-155-5p,miR-122-5p,miR-378a-3p,miR-7-5p and miR-146a-5p (FDR<0.05). Differences between EV,non-EV and cellular levels of these miRNA were detected. Primary epithelial cell release of EV and their miRNA cargo was similar to that observed for BCi. Intriguingly,miR-155 expression was decreased in EVs derived from COPD patients compared to EVs from control samples. Epithelial EV miRNA release may be a key mechanism in modulating the response to IAV in the lungs. Furthermore,changes in EV miRNA expression may play a dysfunctional role in influenza-induced exacerbations of COPD. However,further work to fully characterize the function of EV miRNA in response to IAV in both health and COPD is required. View Publication

Ageing-dependent low-grade inflammation is a hallmark of central nervous system (CNS) diseases. Vascular and immune abnormalities are implicated in the progression of gliomas and occur in the early stages of Alzheimer's disease (AD); however,the mechanisms by which these alterations manifest in the brain parenchyma remain unclear. Using RNAseq,scRNAseq,bioinformatics tools and a cohort of patients with glioma and Alzheimer's disease for validation of results,we have established an analysis of blood–brain barrier (BBB) dysfunction and neuron loss. A mouse model for glioblastoma pathology was also used that reversed BBB disruption and neuron loss,with the incorporation of the IDH mutation. Finally,we established a characterization of the relevant immune populations with an IHC analysis and transcriptional profile. In this study,molecular analyses of the brain ecosystem revealed that blood–brain barrier dysfunction and neuronal synapse integrity exhibit significant threshold-dependent changes that correlate directly and inversely,respectively,with brain ageing (significant changes at 57 years) and the progression of AD and gliomas (survival of 1525 vs 4084 days for patients with High vs Low BBB dysfunction). Using human samples and mouse models,we identified immunoageing processes characterized by an imbalance between pro-inflammatory and anti-inflammatory signals. This dysregulation promotes the extravasation of monocyte-derived macrophages (85% increase of cells),particularly those with a suppressive phenotype,alongside an increase in inflammatory cytokine levels. Notably,our data show that vascular normalization in a glioma model can reverse neuronal loss and attenuate the aggressiveness of the tumours. Finally,tumour development can be prevented by reactivating the ageing immune system. We propose that the ageing brain represents a common,BBB dysfunction-associated process driving chronic inflammation. This inflammation is regulated by TREM2+/TIM3+ suppressive myeloid cells,which play a central role in disease progression. Our findings suggest that targeting these pathways could offer therapeutic strategies to mitigate CNS pathologies linked to ageing,characterized by toxic neuroinflammation and myeloid dysfunction. This study was funded by ISCIII and co-funded by the European Union. View Publication