技术资料

Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization,we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo,whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis,thereby modulating its prion-like spreading potential. We suggest that EVs represent a novel post-translational mechanism to regulate the levels of extracellular α-syn and may thus affect the spreading of α-syn pathology. Targeting this proteolytic capacity may offer new therapeutic interventions for mitigating synucleinopathies. Subject terms: Biochemistry,Cell biology,Neuroscience,Pathogenesis View Publication

The cell cycle (CC) underpins diverse cell processes like cell differentiation,cell expansion,and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases,rely on transcriptomic signatures,use imaging modalities limited to adherent cells,or lack high-throughput multiplexing. To solve this,we develop an expanded,Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight,we quantify scCC states across suspension and adherent cell lines,and stimulated primary human T cells. Our approach captures the diversity of scCC states,including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably,primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems,Proteomics,Cell biology,Immunology,Systems biology View Publication

β-cell dysfunction and dedifferentiation towards an α-cell-like phenotype are hallmarks of type 2 diabetes. However,the cell subtypes involved in β-to-α-cell transition are unknown. Using single-cell and single-nucleus RNA-seq,RNA velocity,PAGA/cell trajectory inference,and gene commonality,we interrogated α-β-cell fate switching in human islets. We found five α-cell subclusters with distinct transcriptomes. PAGA analysis showed bifurcating cell trajectories in non-diabetic while unidirectional cell trajectories from β-to-α-cells in type 2 diabetes islets suggesting dedifferentiation towards α-cells. Ten genes comprised the common signature genes in trajectories towards α-cells. Among these,the α-cell gene SMOC1 was expressed in β-cells in type 2 diabetes. Enhanced SMOC1 expression in β-cells decreased insulin expression and secretion and increased β-cell dedifferentiation markers. Collectively,these studies reveal differences in α-β-cell trajectories in non-diabetes and type 2 diabetes human islets,identify signature genes for β-to-α-cell trajectories,and discover SMOC1 as an inducer of β-cell dysfunction and dedifferentiation. Subject terms: Cell signalling,Diabetes,Differentiation View Publication

Alzheimer’s disease (AD) is the leading cause of dementia globally. The accumulation of amyloid and tau proteins,neuronal cell death and neuroinflammation are seen with AD progression,resulting in memory and cognitive impairment. Microglia are crucial for AD progression as they engage with neural cells and protein aggregates to regulate amyloid pathology and neuroinflammation. Recent studies indicate that microglia contribute to the propagation of amyloid beta (Aβ) via their immunomodulatory functions including Aβ phagocytosis and inflammatory cytokine production. Three-dimensional cell culture techniques provide the opportunity to study pathophysiological changes in AD in human-derived samples that are difficult to recapitulate in animal models (e.g.,transgenic mice). However,these models often lack immune cells such as microglia,which play a critical role in AD pathophysiology. In this study,we developed a neuroimmune assembloid model by integrating cerebral organoids (COs) with induced microglia-like cells (iMGs) derived from human induced pluripotent stem cells from familial AD patient with PSEN2 mutation. After 120 days in culture,we found that iMGs were successfully integrated within the COs. Interestingly,our assembloids displayed histological,functional and transcriptional features of the pro-inflammatory environment seen in AD,including amyloid plaque-like and neurofibrillary tangle-like structures,reduced microglial phagocytic capability,and enhanced neuroinflammatory and apoptotic gene expression. In conclusion,our neuroimmune assembloid model effectively replicates the inflammatory phenotype and amyloid pathology seen in AD. The online version contains supplementary material available at 10.1186/s12974-025-03544-x. View Publication

Ovarian cancer remains the most lethal gynaecological malignancy,with tumour recurrence and chemoresistance posing significant therapeutic challenges. Emerging evidence suggests that cancer stem cells (CSCs),a rare subpopulation within tumours with self‐renewal and differentiation capacities,contribute to these hurdles. Therefore,elucidating the mechanisms that sustain CSCs is critical for improving treatment strategies. Mitophagy,a selective process for eliminating damaged mitochondria,plays a key role in maintaining cellular homeostasis,including CSC survival. Our study demonstrates that ovarian CSCs exhibit enhanced mitophagy,accompanied by elevated expression of the mitochondrial outer membrane receptors BNIP3 and BNIP3L. Knockdown of BNIP3 or BNIP3L significantly reduces mitophagy and impairs CSC self‐renewal,indicating that receptor‐mediated mitophagy is essential for CSC maintenance. Mechanistically,we identify that hyperactivated NF‐κB signalling drives the upregulation of BNIP3 and BNIP3L in ovarian CSCs. Inhibition of NF‐κB signalling,either via p65 knockdown or pharmacological inhibitors,effectively suppresses mitophagy. Furthermore,we demonstrate that elevated DNA‐PK expression contributes to the constitutive activation of NF‐κB signalling,thereby promoting mitophagy in ovarian CSCs. In summary,our findings establish that BNIP3/BNIP3L‐mediated mitophagy,driven by DNA‐PK‐dependent NF‐κB hyperactivation,is essential for CSC maintenance. Targeting the DNA‐PK/NF‐κB/BNIP3L‐BNIP3 axis to disrupt mitochondrial quality control in CSCs represents a promising therapeutic strategy to prevent ovarian cancer recurrence and metastasis. View Publication

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study,we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis,bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells,lymphocytes or BL-3 cells. By protein electrophoresis,several protein bands were unique for high-virulence,1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta,IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages. View Publication

Tandemers 2MG,2MG3,3MG and 4MG are derivatives of the potent anti-HIV-1 microbicide candidate griffithsin (GRFT). We compared these compounds anti-HIV-1 activity to GRFT using the viruses CAP206.08 and CAAN5342.A2 that have decreased sensitivity to this lectin. The 2MG and 2MG3 tandemers had similar activity to GRFT against cell-free and cell-associated viruses,while 3MG and 4MG were significantly more potent. Furthermore,the restoration of the 234N or 295N glycan in these viruses,known to increase sensitivity to GRFT,also increased sensitivity to 2MG and 2MG3,and not to 3MG and 4MG. In addition,GRFT resistant viruses generated in-vitro were equally resistant to 2MG and 2MG3 while they had considerably low resistance to 3MG and 4MG. Lastly,all five compounds showed increased inhibitory activity in seminal and vaginal simulants although the effect was more pronounced in the former. These data support further studies of tandemers as potential microbicides. View Publication

Prenatal cannabis exposure (PCE) influences human brain development,but it is challenging to model PCE using animals and current cell culture techniques. Here,we developed a one-stop microfluidic platform to assemble and culture human cerebral organoids from human embryonic stem cells (hESC) to investigate the effect of PCE on early human brain development. By incorporating perfusable culture chambers,air-liquid interface,and one-stop protocol,this microfluidic platform can simplify the fabrication procedure and produce a large number of organoids (169 organoids per 3.5 cm × 3.5 cm device area) without fusion,as compared with conventional fabrication methods. These one-stop microfluidic assembled cerebral organoids not only recapitulate early human brain structure,biology,and electrophysiology but also have minimal size variation and hypoxia. Under on-chip exposure to the psychoactive cannabinoid,$\Delta$-9-tetrahydrocannabinol (THC),cerebral organoids exhibited reduced neuronal maturation,downregulation of cannabinoid receptor type 1 (CB1) receptors,and impaired neurite outgrowth. Moreover,transient on-chip THC treatment also decreased spontaneous firing in these organoids. This one-stop microfluidic technique enables a simple,scalable,and repeatable organoid culture method that can be used not only for human brain organoids but also for many other human organoids including liver,kidney,retina,and tumor organoids. This technology could be widely used in modeling brain and other organ development,developmental disorders,developmental pharmacology and toxicology,and drug screening. View Publication

The recent advances,offered by cell therapy in the regenerative medicine field,offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors,such as myoblasts or satellite cells,possess a marked regenerative capacity,but the exploitation of this potential still encounters significant challenges in clinical application,due to low rate of proliferation in vitro,as well as a reduced self-renewal capacity. In this scenario,induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches,but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs,generated from pericytes and fibroblasts,exploiting skeletal muscle-derived extracellular vesicles (EVs),in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches." View Publication

The incidence of Merkel cell carcinoma (MCC),a rare and highly metastatic skin malignancy,has sharply increased in the last decade. Clinical biomarkers are urgently needed for MCC prognosis,treatment response monitoring,and early diagnosis of relapse. The clinical interest of circulating tumors cells (CTCs) has been validated in many solid cancers. The aim of this study was to compare CTC detection and characterization in blood samples of patients with MCC using the CellSearch System and the RosetteSep -DEPArray workflow,an innovative procedure to enrich,detect and isolate single CTCs. In preliminary experiments (using spiked MCC cell lines) both methods allowed detecting very few MCC cells. In blood samples from 19 patients with MCC at different stages,CellSearch detected MCC CTCs in 26{\%} of patients,and the R-D workflow in 42{\%} of patients. The detection of CTC-positive patients increased to 52{\%} by the cumulative positivity rate of both methodologies. Moreover,Merkel cell polyomavirus DNA,involved in MCC oncogenesis,was detected in tumor biopsies,but not in all single CTCs from the same patient,reflecting the tumor heterogeneity. Our data demonstrate the possibility to detect,isolate and characterize CTCs in patients with MCC using two complementary approaches. View Publication

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age,but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further,we examined the combined and separate effects of an oncogene (c-MYC) and exposure to IL3,GM-CSF and SCF on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized,rapidly fatal and serially transplantable blast population,phenotypically and transcriptionally similar to human AML cells,but only in mice producing IL3,GM-CSF and SCF transgenically,or in regular mice in which the cells were exposed to IL3 or GM-CSF delivered using a co-transduction strategy. In their absence,the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months but,upon transfer to secondary mice producing the human cytokines,the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late (GMPs) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them. View Publication

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. View Publication