技术资料

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However,they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX),Daunorubicin (DNR),Epirubicin (EPI),and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class,or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this,we studied the effects of treatment of DOX,DNR,EPI,MTX,and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum,while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling,a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time,giving rise to four distinct gene expression response signatures,denoted as TOP2i early-acute,early-sustained,and late response genes,and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators,which mediate AC sensitivity across breast cancer patients. However,there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability,we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed,DOX response eQTLs are enriched in genes that respond to all TOP2i. Next,we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i,respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes,including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity. Author summaryAnthracycline drugs such as Doxorubicin are effective treatments for breast cancer; however,they can cause cardiotoxicity in some women. It is unclear whether women would experience the same toxicity for all drugs in this class,or whether specific drugs would be better tolerated in specific individuals. We used an in vitro system of induced pluripotent stem cell-derived cardiomyocytes from six healthy females to test the effects of five breast cancer drugs on cell heath and global gene expression. We identified a strong shared cellular and gene expression response to drugs from the same class. However,there is more variation in gene expression levels between individuals following treatment with each anthracycline compared to untreated cells. We found that many genes in regions previously associated with Doxorubicin-induced cardiotoxicity in cancer patients,respond to at least two drugs in the class. This suggests that drugs in the same class induce similar effects on an individual’s heart. This work contributes to our understanding of how drug response,in the context of off-target effects,varies across individuals. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells,genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however,their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells,here,we aimed to generate human iPSCs overexpressing interferon-beta (IFNB,IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-?. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation,IFNB-iPSCs formed embryoid bodies and upregulated endoderm,mesoderm,and some ectoderm markers. However,an upregulation of key neuroectoderm markers,PAX6 and LHX2,was compromised. A negative effect of IFN-? on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-?. The study describes new IFN-?-producing iPSC lines suitable for the generation of various types of IFN-?-producing cells for future experimental and clinical applications,and it unravels an inhibitory effect of IFN-? on stem cell neuroectoderm differentiation. View Publication

Parkinson’s disease (PD) is a neurodegenerative illness characterized by motor and non-motor features. Hallmarks of the disease include an extensive loss of dopaminergic neurons in the substantia nigra pars compacta,evidence of neuroinflammation,and the accumulation of misfolded proteins leading to the formation of Lewy bodies. While PD etiology is complex and identifying a single disease trigger has been a challenge,accumulating evidence indicates that non-neuronal and peripheral factors may likely contribute to disease onset and progression. The brain is shielded from peripheral factors by the blood-brain barrier (BBB),which tightly controls the entry of systemic molecules and cells from the blood to the brain. The BBB integrates molecular signals originating from the luminal (blood) and abluminal (brain) sides of the endothelial wall,regulating these exchanges. Of particular interest are erythrocytes,which are not only the most abundant cell type in the blood,but they also secrete extracellular vesicles (EVs) that display disease-specific signatures over the course of PD. Erythrocyte-derived EVs (EEVs) could provide a route by which pathological molecular signals travel from the periphery to the central nervous system. The primary objective of this study was to evaluate,in a human-based platform,mechanisms of EEV transport from the blood to the brain under physiological conditions. The secondary objective was to determine the ability of EEVs,generated by erythrocytes of healthy donors or patients,to induce PD-like features. We leveraged two in vitro models of the BBB,the transwell chambers and a microfluidic BBB chip generated using human induced pluripotent stem cells. Our findings suggest that EEVs transcytose from the vascular to the brain compartment of the human BBB model via a caveolin-dependant mechanism. Furthermore,EEVs derived from individuals with PD altered BBB integrity compared to healthy EEV controls,and clinical severity aggravated the loss of barrier integrity and increased EEV extravasation into the brain compartment. PD-derived EEVs reduced ZO-1 and Claudin 5 tight junction levels in BMEC-like cells and induced the selective atrophy of dopaminergic neurons. In contrast,non-dopaminergic neurons were not affected by treatment with PD EEVs. In summary,our data suggest that EEV interactions at the human BBB can be studied using a highly translational human-based brain chip model,and EEV toxicity at the neurovascular unit is exacerbated by disease severity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. HighlightsErythrocytes secrete extracellular vesicles that can transcytose into the brain via a caveolin-dependant mechanism.A microfluidic brain chip can be used to evaluate mechanisms of transcytosis across the blood-brain barrier.The clinical severity of Parkinson’s disease affects how erythrocyte-derived extracellular vesicles interact with cerebral endothelial cells.Erythrocyte-derived extracellular vesicles generated from donors with Parkinson’s disease alter the blood-brain barrier and induce atrophy of dopaminergic neurons.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. View Publication

An estimated 65 million people globally suffer from post-acute sequelae of COVID-19 (PASC),with many experiencing cardiovascular symptoms (PASC-CVS) like chest pain and heart palpitations. This study examines the role of chronic inflammation in PASC-CVS,particularly in individuals with symptoms persisting over a year after infection. Blood samples from three groups—recovered individuals,those with prolonged PASC-CVS and SARS-CoV-2-negative individuals—revealed that those with PASC-CVS had a blood signature linked to inflammation. Trace-level pro-inflammatory cytokines were detected in the plasma from donors with PASC-CVS 18?months post infection using nanotechnology. Importantly,these trace-level cytokines affected the function of primary human cardiomyocytes. Plasma proteomics also demonstrated higher levels of complement and coagulation proteins in the plasma from patients with PASC-CVS. This study highlights chronic inflammation’s role in the symptoms of PASC-CVS. Sinclair et al. explore the contribution of chronic inflammation to cardiovascular symptoms associated with post-acute sequelae of SARS-CoV-2 infection (PASC-CVS). The authors identify trace levels of inflammatory cytokines in individuals with PASC-CVS that impair the function of cardiomyocytes derived from induced pluripotent stem cells. View Publication

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However,it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here,we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration,we developed a DIPG-neural assembloid model,which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model,we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover,laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally,these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01765-4. View Publication

Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development,homeostasis,and hydrostatic organ pressure. Here,we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically,we electrically stimulate hollow,3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ? 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen,generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues. Electrical stimulation of hollow,3D kidney tissues causes these tissues to inflate and change shape. The authors call this process electro-inflation and connect it to electricity driving ions into the center of the tissues,causing water to follow by osmosis. View Publication

SummaryKnowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete,yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress,we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens,aggrecan,perlecan,proteoglycans,and elastic fibers. We identified two populations of chondroprogenitor cells,mesenchyme cells and nascent chondrocytes,and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage,but also play a pivotal autocrine cell signaling role in determining chondrocyte fate. Graphical abstract Highlights•Craniofacial cartilage organoids were grown from human neural crest stem cells•These organoids exhibited elastic cartilage architecture and characteristic markers•Paracrine signaling drove chondrogenesis in mesenchyme cells and nascent chondrocytes•ECM components cemented chondrocyte cell fate through autocrine signaling Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Specialized functions of cells View Publication

Interactions between adipose tissue,liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model,we establish an interconnected microphysiological system (MPS) containing white adipocytes,hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS,we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function,whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance,corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases. In vitro modelling of the adipose tissue-liver axis can advance understanding and therapy of metabolic disease,including by distinguishing effects of obesity and inflammation. Here,authors develop such a system based on isogenic human iPSCs and interconnected microphysiological devices. View Publication

Label-free detection of biological events at single-cell resolution in the brain can non-invasively capture brain status for medical diagnosis and basic neuroscience research. NADH is an universal coenzyme that not only plays a central role in cellular metabolism but may also be used as a biomarker to capture metabolic processes in brain cells and structures. We have developed a new label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H in living cells. The imaging depth of NAD(P)H in tissues with all-optical methods is limited to ~100??m in brain tissue by the strong absorption of the near-ultraviolet fluorescence. Here,acoustic detection of the thermal signature of multi-photon (three-photon) excitation of NAD(P)H,a low quantum yield fluorophore,allows detection at an unprecedented depth while the focused excitation ensures high spatial resolution. We validated the photoacoustic detection of NAD(P)H by monitoring an increase in intracellular NAD(P)H in HEK293T cells and HepG2 cells incubated in NADH solution. We also demonstrated the detection of endogenous NAD(P)H photoacoustic signals in brain slices to 700 ?m depth and in cerebral organoids to 1100 ?m depth. Finally,we developed and demonstrated simultaneous photoacoustic and optical imaging of NAD(P)H in brain cells with a real-time image acquisition and processing pipeline. This approach could open a new door to monitor brain metabolic changes during development and disease,and changes due to neuronal activity,at single-cell level deep in the brains of both humans and animals. Label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H of neurons in brain slices and cerebral organoids. View Publication

BackgroundOP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However,the whole co-culture procedure usually needs 14–18 days,including preparing OP9 cells at least 4 days. Therefore,the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.MethodsIn the experimental group,we set six different densities of OP9 cells and just cultured them for 24 h before co-culture,and in the control group,OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them.ResultsOP9 cells were randomly assigned into two groups. In the experimental group,six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast,in the control group,OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining,and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day,followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group.ConclusionThe peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency. View Publication

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless,a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process,including hypoxic stress,fibrosis,and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions,leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology,demonstrating that the phased combination of TGF? inhibitor SB431542,Rho kinase inhibitor Y27632,and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction,providing a crucial platform for discovering therapeutic targets for ischemic heart disease. Engineered human models of hearts are needed to study pathology and repair. Here,the authors develop a model which replicates the phased remodelling process. The model is then used to study signalling pathway modulators for their therapeutic potential in a mini-repair model. View Publication

SummaryEpigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing,here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay,intellectual disability,hypotonia,and motor issues such as coordination problems,feeding difficulties,and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions,including autism spectrum disorder,and to a lesser extent,seizures,connective tissue disease signs,sleep disturbance,vision problems,and other organ anomalies,were observed in affected individuals. As a molecular biomarker,a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably,while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac,global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless,MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features,a specific blood DNA episignature,and a distinct,MSL2-specific molecular etiology compared to other MSL complex-related disorders. Graphical abstract MSL2 encodes a member of the MSL complex,an epigenetic regulator acetylating histone H4. We identify MSL2 variants leading to a neurodevelopmental disorder with intellectual disability,developmental delay,motor issues,seizures,dysmorphisms,and a specific blood methylation episignature. Patient-derived reprogrammed cells reveal developmental gene dysregulation without altered global H4 acetylation. View Publication