技术资料

During the metastatic cascade,cancer cells travel through the bloodstream as circulating tumor cells (CTCs) to a secondary site. Clustered CTCs have greater shear stress and treatment resistance,yet their biology remains poorly understood. We therefore engineered a tunable superhydrophobic array device (SHArD). The SHArD-C was applied to culture a clinically relevant model of CTC clusters. Using our device,we cultured a model of cancer cell aggregates of various sizes with immortalized cancer cell lines. These exhibited higher E-cadherin expression and are significantly more capable of surviving high fluid shear stress-related forces compared to single cells and model clusters grown using the control method,helping to explain why clustering may provide a metastatic advantage. Additionally,the SHArD-S,when compared with the AggreWell 800 method,provides a more consistent spheroid-forming device culturing reproducible sizes of spheroids for multiple cancer cell lines. Overall,we designed,fabricated,and validated an easily tunable engineered device which grows physiologically relevant three-dimensional (3D) cancer models containing tens to thousands of cells. View Publication

More than 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ∼1000 genes that may contribute. The new challenge is to investigate the molecular and cellular functions of these genes during neural and brain development,and then even more challenging,to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study,we used single-cell RNA-seq analysis to study one of the top risk genes,CHD8,in cerebral organoids,which models early neural development. We identified 21 cell clusters in the organoid samples,representing non-neuronal cells,neural progenitors,and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of a CHD8 knockout allele,generated by CRISPR/Cas9 editing,and their isogenic controls uncovered thousands of differentially expressed genes,which were enriched with functions related to neural and brain development,cilium organization,and extracellular matrix organization. The affected genes were also enriched with genes and pathways previously implicated in ASD,but surprisingly not for schizophrenia and intellectual disability risk genes. The comparisons also uncovered cell composition changes,indicating potentially altered neural differential trajectories upon CHD8 reduction. Moreover,we found that cell-cell communications were affected in the CHD8 knockout organoids,including the interactions between neural and glial cells. Taken together,our results provide new data and information for understanding CHD8 functions in the early stages of neural lineage development and interaction. View Publication

Besides neutralizing antibodies,which are considered an important measure for vaccine immunogenicity,Fc-mediated antibody functions can contribute to antibody-mediated protection. They are strongly influenced by structural antibody properties such as subclass and Fc glycan composition. We here applied a systems serology approach to dissect humoral immune responses induced by MVA-MERS-S,an MVA-vectored vaccine against the Middle East respiratory syndrome coronavirus (MERS-CoV). Building on preceding studies reporting the safety and immunogenicity of MVA-MERS-S,our study highlights the potential of a late boost,administered one year after prime,to enhance both neutralizing and Fc-mediated antibody functionality compared to the primary vaccination series. Distinct characteristics were observed for antibodies specific to the MERS-CoV spike protein S1 and S2 subunits,regarding subclass and glycan compositions as well as Fc functionality. These findings highlight the benefit of a late homologous booster vaccination with MVA-MERS-S and may be of interest for the design of future coronavirus vaccines. Subject areas: Cell biology,Immune response,Immunology,Virology View Publication

Proper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance,the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately,the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation. We utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4,a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis,and confirmed the targeting by dual-luciferase reporter assay,western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance. In this study,overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation,respectively. Accordingly,we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression,as well as improved megakaryocytic differentiation,similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p. miR-1915-3p acts as a negative regulator of erythropoiesis,and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs. The online version contains supplementary material available at 10.1186/s12959-024-00615-6. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters,RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide),which are involved in different redox reactions,including the energetic metabolism processes occurring in mitochondria. To date,human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons,which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far,no attention has been dedicated to astrocytes. Here,we demonstrate that in vitro differentiation of astrocytes,which guarantee trophic and metabolic support to neurons,from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons,RTD2 does not compromise the morpho-functional features of astrocytes. View Publication

PU.1 ( SPI1 ) is pivotal in hematopoiesis,yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis,we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells,respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically,KLF1 overexpression restores myeloid lineage potential,while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together,our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT,potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences,Molecular biology,Molecular interaction,Cell biology; View Publication

Most studies used animal serum-containing medium for bioengineered-root regeneration,but ethical and safety issues raised by animal serum are a potentially significant risk for clinical use. Thus,this study aimed to find a safer method for bioengineered-root regeneration. The biological properties of human dental pulp stem cells (hDPSCs) cultured in animal component-free (ACF) medium or serum-containing medium (5%,10% serum-containing medium,SCM) were compared in vitro . hDPSCs were cultured in a three-dimensional (3D) environment with human-treated dentin matrix (hTDM). The capacity for odontogenesis was compared using quantitative real-time PCR (qPCR) and Western blot. Subsequently,the hDPSCs/hTDM complexes were transplanted into nude mice subcutaneously. Histological staining was then used to verify the regeneration effect in vivo . ACF medium promoted the migration of hDPSCs,but slightly inhibited the proliferation of hDPSCs in the first three days of culture compared to SCM. However,it had no significant effect on cell aging and apoptosis. After 7 days of 3D culture in ACF medium with hTDM,qPCR showed that DMP1,DSPP,OCN,RUNX2,and β-tubulin III were highly expressed in hDPSCs. In addition,3D cultured hDPSCs/hTDM complexes in ACF medium regenerated dentin,pulp,and periodontal ligament-like tissues similar to SCM groups in vivo . ACF medium was proved to be an alternative medium for bioengineered-root regeneration. The strategy of using ACF medium to regenerate bioengineered-root can improve clinical safety for tooth tissue engineering. View Publication

Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. In this study,we investigated the role of HES1,a member of HES family,in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry,histopathology analysis,and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers,decreases hematopoietic stem progenitor cell (HSPC) pool,results in defective multi-lineage differentiation. Functionally,fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1 fl/fl Flt3Cre embryos are resulted from decreased proliferation and elevated apoptosis,associated with de-repressed HES1 targets,p27 and PTEN in Hes1 -KO fetal HSPCs. Finally,pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. Together,our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis,and provide new insight into the differences between fetal and adult HSC maintenance. The online version contains supplementary material available at 10.1186/s13287-024-03836-8. View Publication

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii,a parasite that naturally travels from the human gut to the central nervous system (CNS),can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems,the rhoptries and dense granules,to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells,brain organoids and in vivo,and probe protein activity using imaging,pull-down assays,scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept,we demonstrate GRA16-mediated brain delivery of the MeCP2 protein,a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system,we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology,Biologics,Synthetic biology View Publication

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier,where the cells play essential roles in the digestion of food,absorption of nutrients and water,protection from microbial infections,and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling,which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells,indeed,is linked to gastrointestinal disorders such as irritable bowel syndrome,inflammatory bowel disease,and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and,therefore,homeostasis. Here,we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol,which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover,analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn’s Disease revealed that the dysregulation of peroxisome number,excessive cellular cholesterol,and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn’s Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis. Subject terms: Cell biology,Medical research View Publication

Previous studies have shown that aggregated alpha-synuclein (α-s) protein,a key pathological marker of Parkinson’s disease (PD),can propagate between cells,thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models,including rodent and human cell cultures. In this study,our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here,we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore,aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells,such as in presynaptic proteins,mitochondrial motility,calcium oscillations and neuronal activity. The model enabled an assessment of the early,middle,and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates,further investigation is required to elucidate the underlying mechanisms. Taken together,this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms. Subject terms: Parkinson's disease,Neurological models View Publication