技术资料

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

Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of ?-Synuclein (?-Syn) protein. Currently,no treatment can slow nor halt the progression of PD. Multiplications and mutations of the ?-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress ?-Syn replicate several features of PD. Decreasing total ?-Syn levels,therefore,is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of ?-Syn levels and identified CDK14,a kinase of largely unknown function as a regulator of ?-Syn. To test the potential therapeutic effects of CDK14 reduction in PD,we ablated Cdk14 in the ?-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical ?-Syn pathology,indicated by reduced numbers of pS129 ?-Syn-containing cells. In primary neurons,we found that Cdk14 depletion protects against the propagation of toxic ?-Syn species. We further validated these findings on pS129 ?-Syn levels in PD patient neurons. Finally,we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated ?-Syn in human neurons,in PFF-challenged rat neurons and in the brains of ?-Syn-humanized mice. In summary,we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy. View Publication

SummaryHuman cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome,and disease severity varies depending on the location and type of mutation. Here,we focused on neuronal activity in Rett syndrome patient-derived organoids,analyzing two types of MeCP2 mutations – a missense mutation (R306C) and a truncating mutation (V247X) - using calcium imaging with three-photon microscopy. Compared to isogenic controls,we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses,with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment. View Publication

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication