技术资料

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here,we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels,we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis,phospholipid composition,and inner leaflet surface charge,thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together,our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration. The biomechanical mechanisms enabling the invasive growth of brain tumors remain opaque. Here,Junqueira Alves et al. reveal that the guidance receptor Plexin-B2 controls membrane tension,facilitating confined migration of brain tumor cells. View Publication

SummaryCryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However,tissue processing is time-consuming and costly,limiting routine and diagnostic use. Currently,no commercially available protocols or products exist for multiplexing this process. Here,we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing—cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore,we process up to ?110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor,time,and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings,organism-wide antigen and antibody targeting studies,high-throughput tissue screens,and defined tissue section positioning for,e.g.,spatial transcriptomics experiments. Graphical abstract Highlights•Multiplexed tissue molds (MTMs) drastically upscale cryosectioning procedures•MTMs can simultaneously accommodate up to 19 mouse organs and ?110 cerebral organoids•MTMs reduce analysis costs and processing times of tissues by up to 96%•MTMs could be used to reduce diagnostic costs and for spatial transcriptomics MotivationEfficient cryosectioning remains a critical yet labor- and cost-intensive step for immunohistochemistry and in situ hybridization,limiting routine diagnostic and research applications. The increasing demand for high-throughput tissue analysis—driven by advances in organoid and three-dimensional (3D) culture systems and tissue analysis for diagnostics—necessitates methods capable of processing numerous heterogeneous samples simultaneously. Current protocols lack multiplexing capabilities,leading to variability and extended processing times. Our work introduces multiplexed tissue molds (MTMs),a scalable solution that drastically reduces costs and labor by up to 96% while maintaining tissue integrity and consistency,thereby enabling large-scale (>100 tissues) comparative analyses and enhanced experimental reproducibility as well as access to tissue analysis,where cost is a restrictive factor. Reumann et al. develop multiplexed tissue molds (MTMs),which allow upscaling of tissue processing (up to 19 mouse organs or ?110 cerebral organoids simultaneously) while reducing workload and associated analysis costs by up to 96%. MTMs allow cryosection-based tissue analysis when labor,time,and cost are limiting factors and could be used for patient sample analysis as well as spatial transcriptomics approaches. View Publication

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases,regenerative medicine,and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs,which involves step-by-step differentiation of hiPSCs into definitive endoderm,anterior foregut endoderm,NKX2.1+ lung progenitors,and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs,expressing basal cell markers (NGFR,KRT5,and TP63),could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs,potentially applicable for gene therapy research. View Publication

SummaryDysfunction of the sympathetic nervous system and increased epicardial adipose tissue (EAT) have been independently associated with the occurrence of cardiac arrhythmia. However,their exact roles in triggering arrhythmia remain elusive. Here,using an in vitro coculture system with sympathetic neurons,cardiomyocytes,and adipocytes,we show that adipocyte-derived leptin activates sympathetic neurons and increases the release of neuropeptide Y (NPY),which in turn triggers arrhythmia in cardiomyocytes by interacting with the Y1 receptor (Y1R) and subsequently enhancing the activity of the Na+/Ca2+ exchanger (NCX) and calcium/calmodulin-dependent protein kinase II (CaMKII). The arrhythmic phenotype can be partially blocked by a leptin neutralizing antibody or an inhibitor of Y1R,NCX,or CaMKII. Moreover,increased EAT thickness and leptin/NPY blood levels are detected in atrial fibrillation patients compared with the control group. Our study provides robust evidence that the adipose-neural axis contributes to arrhythmogenesis and represents a potential target for treating arrhythmia. Graphical abstract Highlights•Stem cell-based coculture model can simulate the pathogenesis of cardiac arrhythmia•The adipose-neural axis plays critical roles in cardiac arrhythmias•Leptin,NPY/Y1R,NCX,and CaMKII are potential intervention targets for arrhythmia•Increased EAT thickness and leptin/NPY levels are detected in CS blood of AF patients Fan et al. establish a stem cell-based coculture model to mimic the in vivo cardiac microenvironment and elucidate that the adipose-neural interaction plays a critical role in epicardial adipose tissue-related cardiac arrhythmia through leptin-NPY axis. Their results may provide potential therapeutic targets for treating arrhythmia. View Publication

In this study,Chen et al. describe two independent mechanisms that control ?-catenin levels in neuroglial cells and drive their proliferation. The work provides mechanistic insight into the impact of MEK activation resulting from the biallelic loss of NF1 or BRAF rearrangement in pediatric gliomas. The two major genomic alterations in pediatric pilocytic astrocytoma (PA) are NF1 loss and KIAA1549:BRAF rearrangement. Although these molecular changes result in increased MEK activity and tumor growth,it is not clear exactly how MEK controls human neuroglial cell proliferation. Leveraging human-induced pluripotent stem cells harboring these PA-associated alterations,we used a combination of genetic and pharmacological approaches to demonstrate that MEK-regulated cell growth is mediated by ?-catenin through independent mechanisms involving IRX2 control of CTNNB1 transcription and NPTX1 stabilization of ?-catenin protein levels. These results provide new mechanistic insights into MEK regulation of human brain cell function. View Publication

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations,small insertions,and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA),which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs,especially when the genetic context precludes the screening of multiple pegRNAs,and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system,we generated other isogenic models of inherited retinal diseases (IRDs),including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure,such as plasmid concentrations,PE component ratios,and delivery method settings,showing that our improved workflow increased the hiPSC editing efficiency. View Publication

BackgroundOsteoarthritis (OA) is a complex,age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility,joint stiffness,pain,and a significant decrease in quality of life. Among other risk factors,such as genetics and age,hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815–823,2011). It has been shown that post-mitotic cells,such as articular chondrocytes,heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However,these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes,resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state.ResultsWe showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5,CAV1,and CD44,among other genes,which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover,by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs),we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity.ConclusionOur findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes,concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1,ITGA5,and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes,we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting,detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-024-01676-0. View Publication

Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. We use CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons show reduced velocities of nuclear movements and an increased number of neurites early in neuronal development,consistent with previous findings. Neurite branching is regulated by a host of microtubule-associated proteins,as well as by microtubule polymerization dynamics. However,EB comet dynamics are unchanged in DCX-KO neurons. Rather,we observe a significant reduction in ?-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11,an ?-tubulin glutamylase. Using U2OS cells as an orthogonal model system,we show that DCX and TTLL11 act synergistically to promote polyglutamylation. We propose that Doublecortin acts as a positive regulator of ?-tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code. Lissencephaly is a severe neurodevelopmental disease often caused by mutations in the Dcx gene. Using a human cellular model of lissencephaly,the authors report that DCX restricts neuronal branching by activating tubulin polyglutamylation. View Publication

SummaryVascular complications caused by diabetes mellitus contribute a major threat to increased disability and mortality of diabetic patients,which are characterized by damaged endothelial cells and angiogenesis. Human umbilical cord-derived mesenchymal stem cells (hucMSCs) have been demonstrated to alleviate endothelial cell damage and improve angiogenesis. However,these investigations overlooked the pivotal role of vasculogenesis. In this study,we utilized blood vessel organoids (BVOs) to investigate the impact of high glucose on vasculogenesis and subsequent angiogenesis. We found that BVOs in the vascular lineage induction stage were more sensitive to high glucose and more susceptible to affect endothelial cell differentiation and function. Moreover,hucMSCs can alleviate the high glucose-induced inhibition of endothelial cell differentiation and dysfunction through MAPK signaling pathway downregulation,with the MAPK activator dimethyl fumarate further illustrating the results. Thereby,we demonstrated that high glucose can lead to abnormal vasculogenesis and impact subsequent angiogenesis,and hucMSCs can alleviate this effect. Graphical abstract Highlights•The induction process of BVOs can be divided into vasculogenesis and angiogenesis•The formation of VI-BVOs is more vulnerable to damage from high glucose than MI-BVOs•HucMSCs can improve vasculogenesis through the MAPK signaling pathway Pathophysiology; Stem cells research; Vascular remodeling View Publication

The Y-linked SRY gene initiates mammalian testis-determination. However,how the expression of SRY is regulated remains elusive. Here,we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5’ regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination,including a large multigenerational family,identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing,in a novel in vitro cellular model recapitulating human Sertoli cell formation,resulted in a significant reduction in expression of SRY. Therefore,human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression,a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare,the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process. Disease-causing variants define a conserved and unique NR5A1 responsive enhancer for SRY expression to initiate testis-determination in humans. Modelling regulatory variants causing sex-reversal provides a tool to understand global enhancer activity. View Publication