技术资料

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TSCV) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TSCT) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development. View Publication

ETV6::RUNX1,the most common oncogenic fusion in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL),induces a clinically silent preleukemic state that can persist in carriers for over a decade and may progress to overt leukemia upon acquisition of secondary lesions. The mechanisms contributing to quiescence of ETV6::RUNX1+ preleukemic cells still remain elusive. In this study,we identify linker histone H1-0 as a critical mediator of the ETV6::RUNX1+ preleukemic state by employing human -induced pluripotent stem cell (hiPSC) models engineered by using CRISPR/Cas9 gene editing. Global gene expression analysis revealed upregulation of H1-0 in ETV6::RUNX1+ hiPSCs that was preserved upon hematopoietic differentiation. Moreover,whole transcriptome data of 1,727 leukemia patient samples showed significantly elevated H1-0 levels in ETV6::RUNX1+ BCP-ALL compared to other leukemia entities. Using dual-luciferase promoter assays,we show that ETV6::RUNX1 induces H1-0 promoter activity. We further demonstrate that depletion of H1-0 specifically inhibits ETV6::RUNX1 signature genes,including RAG1 and EPOR. Single-cell sequencing showed that H1-0 is highly expressed in quiescent hematopoietic cells. Importantly,H1-0 protein levels correspond to susceptibility of BCP-ALL cells towards histone deacetylase inhibitors (HDACis) and combinatorial treatment using the H1-0-inducing HDACi Quisinostat showed promising synergism with established chemotherapeutic drugs. Taken together,our data identify H1-0 as a key regulator of the ETV6::RUNX1+ transcriptome and indicate that the addition of Quisinostat may be beneficial to target non-responsive or relapsing ETV6::RUNX1+ BCP-ALL. View Publication

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases,including those with genetic origins. Currently,primary ECs are the main source for disease modelling in this field. However,they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level,we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor,known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level,which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly,the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability,while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary,we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09925-9. View Publication

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

Aging is one of the most prominent risk factors for neurodegeneration,yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging,we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons,which retained their aging hallmarks,and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins,especially spliceosome components. Intriguingly,splicing proteins—like the dementia- and ALS-associated protein TDP-43—mislocalize to the cytoplasm in aged neurons,which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules,but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery,poor HSP90? chaperone activity and the failure to respond to new stress events. Together,our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons. Rhine et al. find that neuronal aging leads to widespread dysregulation of RNA biology,including mislocalization of splicing proteins like TDP-43,chronic cellular stress and reduced resiliency. 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

SUMMARY Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19,HIV,and apolipoprotein-L1 (APOL1) nephropathy,but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids,primary endothelial cells,and patient samples,we demonstrate that IFN-? induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing,immunoblotting,and quantitative fluorescence-based assays reveal that IFN-?-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-? signaling inhibits APOL1 expression,prevents upregulation of pyroptosis-associated genes,and rescues vascular networks. Multiomic analyses in patients with COVID-19,proteinuric kidney disease,and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-? signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis,suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. In brief Juliar et al. address interferon signaling in kidney disease. Organoids,primary cells,and clinical datasets reveal that interferon signaling simultaneously induces APOL1 expression and endothelial cell pyroptosis. This suggests a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. The findings may also be relevant in other organs. Graphical Abstract 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