技术资料

Inflammatory cytokines,including interleukin 6 (IL6),are associated with ion channel remodeling and enhance the propensity to alterations in cardiac rhythm generation and propagation,in which the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role. Hence,we investigated the consequences of exposure to IL6 on HCN channels in cell models and human atrial biopsies. In murine atrial HL1 cells and in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs),IL6 elicited STAT3 phosphorylation,a receptor-mediated downstream signaling. Downregulation of HCN1,2,4 by IL6 was observed after 24–48 h; in hiPS-CMs,this effect was reverted by 24 h of application of tocilizumab,a human IL6 receptor antagonist. In parallel,hiPS-CM action potentials (APs) showed a reduced spontaneous frequency. Moreover,we assessed IL6 and HCN expression in dilated left atrial samples from patients with mitral valve disease,an AF-prone condition. IL6 levels were increased in dilated atria compared to controls and positively correlated with echocardiographic atrial dimensions. Interestingly,the highest IL6 transcript levels and the lowest HCN4 and HCN2 expression were in these samples. In conclusion,our data uncovered a novel link between IL6 and cardiac HCN channels,potentially contributing to atrial electrical disturbances and a higher risk of dysrhythmias in conditions with elevated IL6 levels. View Publication

To build in vitro tissues for therapeutic applications,it is essential to replicate the spatial distribution of cells that occurs during morphogenesis in vivo. However,it remains technically challenging to simultaneously regulate the geometric alignment and aggregation of cells during tissue fabrication. Here,we introduce the acoustofluidic bioassembly induced morphogenesis,which is the combination of precise arrangement of cells by the mechanical forces produced by acoustofluidic cues,and the morphological and functional changes of cells in the following in vitro and in vivo cultures. The acoustofluidic bioassembly can be used to create tissues with regulated nano-,micro-,and macro-structures. We demonstrate that the neuromuscular tissue fabricated with the acoustofluidic bioassembly exhibits enhanced contraction dynamics,electrophysiology,and therapeutic efficacy. The potential of the acoustofluidic bioassembly as an in situ application is demonstrated by fabricating artificial tissues at the defect sites of living tissues. The acoustofluidic bioassembly induced morphogenesis can provide a pioneering platform to fabricate tissues for biomedical applications. Tissue engineering is essential for drug screening and regenerative medicine. Here,authors developed an acoustofluidic method that can induce morphogenesis of therapeutic tissues at varied dimensions/scales. View Publication

Monkeypox virus (MPV) is known to inflict injuries and,in some cases,lead to fatalities in humans. However,the underlying mechanisms responsible for its pathogenicity remain poorly understood. We investigated functions of MPV core proteins,H3L,A35R,A29L,and I1L,and discovered that H3L induced transcriptional perturbations and injuries. We substantiated that H3L upregulated IL1A expression. IL1A,in consequence,caused cellular injuries,and this detrimental effect was mitigated when countered with IL1A blockage. We also observed that H3L significantly perturbed the transcriptions of genes in cardiac system. Mechanistically,H3L occupied the promoters of genes governing cellular injury,leading to alterations in the binding patterns of H3K27me3 and H3K4me3 histone marks,ultimately resulting in expression perturbations. In vivo and in vitro models confirmed that H3L induced transcriptional disturbances and cardiac dysfunction,which were ameliorated when IL1A was blocked or repressed. Our study provides valuable insights into comprehensive understanding of MPV pathogenicity,highlights the significant roles of H3L in inducing injuries,and potentially paves the way for the development of therapeutic strategies targeting IL1A. View Publication

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here,we systematically investigated the neutral lipid–synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein,indicating that protein interaction might be required for ZIKV replication. Importantly,inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions,indicating that ZIKV morphogenesis is compromised,likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target. Exploring the role of neutral lipid-synthesizing enzymes in Zika virus infection using different cell culture models,inhibition of cholesterol esterification is found to impair ZIKV morphogenesis. View Publication

Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages,including microglia,to damage of lipid-rich tissues like the brain. Recently,sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues,we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors,BHLHE40/41,in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia,resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing,increased cholesterol efflux and storage,and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues. Factors regulating lipid and lysosomal clearance in microglia and peripheral macrophage are not known. Here,authors nominate and validate transcription factors BHLHE40 and BHLHE41 as regulators of these processes in health and disease. View Publication

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm,leading to death by respiratory failure. Human experimental models to study phMNs are lacking,hindering the understanding of the mechanisms of phMN degeneration in ALS. Here,we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development,phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels,under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine,followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines,offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research,it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest. View Publication

The reconstruction of damaged neural circuits is critical for neurological repair after brain injury. Classical brain-computer interfaces (BCIs) allow direct communication between the brain and external controllers to compensate for lost functions. Importantly,there is increasing potential for generalized BCIs to input information into the brains to restore damage,but their effectiveness is limited when a large injured cavity is caused. Notably,it might be overcome by transplantation of brain organoids into the damaged region. Here,we construct innovative BCIs mediated by implantable organoids,coined as organoid-brain-computer interfaces (OBCIs). We assess the prolonged safety and feasibility of the OBCIs,and explore neuroregulatory strategies. OBCI stimulation promotes progressive differentiation of grafts and enhances structural-functional connections within organoids and the host brain,promising to repair the damaged brain via regenerating and regulating,potentially directing neurons to preselected targets and recovering functional neural networks in the future. Damaged neural circuits could be improved by generalized BCIs via inputting information into the brains,which is restricted when a large injured cavity caused. Here,the authors construct BCIs mediated by organoid grafts to repair the damaged brain View Publication

BackgroundSETBP1 Haploinsufficiency Disorder (SETBP1-HD) is characterised by mild to moderate intellectual disability,speech and language impairment,mild motor developmental delay,behavioural issues,hypotonia,mild facial dysmorphisms,and vision impairment. Despite a clear link between SETBP1 mutations and neurodevelopmental disorders the precise role of SETBP1 in neural development remains elusive. We investigate the functional effects of three SETBP1 genetic variants including two pathogenic mutations p.Glu545Ter and SETBP1 p.Tyr1066Ter,resulting in removal of SKI and/or SET domains,and a point mutation p.Thr1387Met in the SET domain.MethodsGenetic variants were introduced into induced pluripotent stem cells (iPSCs) and subsequently differentiated into neurons to model the disease. We measured changes in cellular differentiation,SETBP1 protein localisation,and gene expression changes.ResultsThe data indicated a change in the WNT pathway,RNA polymerase II pathway and identified GATA2 as a central transcription factor in disease perturbation. In addition,the genetic variants altered the expression of gene sets related to neural forebrain development matching characteristics typical of the SETBP1-HD phenotype.LimitationsThe study investigates changes in cellular function in differentiation of iPSC to neural progenitor cells as a human model of SETBP1 HD disorder. Future studies may provide additional information relevant to disease on further neural cell specification,to derive mature neurons,neural forebrain cells,or brain organoids.ConclusionsWe developed a human SETBP1-HD model and identified perturbations to the WNT and POL2RA pathway,genes regulated by GATA2. Strikingly neural cells for both the SETBP1 truncation mutations and the single nucleotide variant displayed a SETBP1-HD-like phenotype.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13229-024-00625-1. View Publication

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However,our knowledge about the safe usage of anti-inflammatory drugs during pregnancy is still limited. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation,these models commonly lack microglia,which are susceptible to and promote inflammation. Furthermore,microglia are actively involved in neuronal development. Here,we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids. Once the outer plexiform layer forms,these hIPSC-derived microglia (iMG) fully integrate into the retinal organoids. Since the ganglion cell survival declines by this time in 3D-retinal organoids,we adapted the model into 2D and identify that the improved ganglion cell number significantly decreases only with iMG presence. In parallel,we applied the immunostimulant POLY(I:C) to mimic a fetal viral infection. While POLY(I:C) exposure alters the iMG phenotype,it does not hinder their interaction with ganglion cells. Furthermore,iMG significantly enhance the supernatant’s inflammatory secretome and increase retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably,while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG,ibuprofen rescues this effect only if iMG are present. Mechanistically,ibuprofen targets the enzymes cyclooxygenase 1 and 2 (COX1/PTGS1 and COX2/PTGS2) simultaneously,from which iMG mainly express COX1. Selective COX1 blockage fails to restore the calcium peak amplitude upon POLY(I:C) stimulation,suggesting ibuprofen’s beneficial effect depends on the presence and interplay of COX1 and COX2. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03366-x. View Publication

SummaryHeterotaxy is a disorder characterized by severe congenital heart defects (CHDs) and abnormal left-right patterning in other thoracic or abdominal organs. Clinical and research-based genetic testing has previously focused on evaluation of coding variants to identify causes of CHDs,leaving non-coding causes of CHDs largely unknown. Variants in the transcription factor zinc finger of the cerebellum 3 (ZIC3) cause X-linked heterotaxy. We identified an X-linked heterotaxy pedigree without a coding variant in ZIC3. Whole-genome sequencing revealed a deep intronic variant (ZIC3 c.1224+3286A>G) predicted to alter RNA splicing. An in vitro minigene splicing assay confirmed the variant acts as a cryptic splice acceptor. CRISPR-Cas9 served to introduce the ZIC3 c.1224+3286A>G variant into human embryonic stem cells demonstrating pseudoexon inclusion caused by the variant. Surprisingly,Sanger sequencing of the resulting ZIC3 c.1224+3286A>G amplicons revealed several isoforms,many of which bypass the normal coding sequence of the third exon of ZIC3,causing a disruption of a DNA-binding domain and a nuclear localization signal. Short- and long-read mRNA sequencing confirmed these initial results and identified additional splicing patterns. Assessment of four isoforms determined abnormal functions in vitro and in vivo while treatment with a splice-blocking morpholino partially rescued ZIC3. These results demonstrate that pseudoexon inclusion in ZIC3 can cause heterotaxy and provide functional validation of non-coding disease causation. Our results suggest the importance of non-coding variants in heterotaxy and the need for improved methods to identify and classify non-coding variation that may contribute to CHDs. Coding variants in the transcription factor ZIC3 cause X-linked heterotaxy,a laterality defect causing congenital anomalies. Functional genomic analyses of a ZIC3 intronic variant identified in an X-linked heterotaxy pedigree demonstrated pseudoexon inclusion leading to RNA-splicing disruption,highlighting the importance of whole-genome sequencing to identify potential disease-causing variants. View Publication

AbstractBackgroundOXA1L is crucial for mitochondrial protein insertion and assembly into the inner mitochondrial membrane,and its variants have been recently linked to mitochondrial encephalopathy. However,the definitive pathogenic link between OXA1L variants and mitochondrial diseases as well as the underlying pathogenesis remains elusive.MethodsIn this study,we identified bi?allelic variants of c.620G>T,p.(Cys207Phe) and c.1163_1164del,p.(Val388Alafs*15) in OXA1L gene in a mitochondrial myopathy patient using whole exome sequencing. To unravel the genotype–phenotype relationship and underlying pathogenic mechanism between OXA1L variants and mitochondrial diseases,patient?specific human?induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into myotubes,while OXA1L knockout human immortalised skeletal muscle cells (IHSMC) and a conditional skeletal muscle knockout mouse model was generated using clustered regularly interspaced short palindromic repeats/Cas9 genomic editing technology.ResultsBoth patient?specific hiPSC differentiated myotubes and OXA1L knockout IHSMC showed combined mitochondrial respiratory chain defects and oxidative phosphorylation (OXPHOS) impairments. Notably,in OXA1L?knockout IHSMC,transfection of wild?type human OXA1L but not truncated mutant form rescued the respiratory chain defects. Moreover,skeletal muscle conditional Oxa1l knockout mice exhibited OXPHOS deficiencies and skeletal muscle morphofunctional abnormalities,recapitulating the phenotypes of mitochondrial myopathy. Further functional investigations revealed that impaired OXPHOS resulting of OXA1L deficiency led to elevated reactive oxygen species production,which possibly activated the nuclear factor kappa B signalling pathway,triggering cell apoptosis.ConclusionsTogether,our findings reinforce the genotype–phenotype association between OXA1L variations and mitochondrial diseases and further delineate the potential molecular mechanisms of how OXA1L deficiency causes skeletal muscle deficits in mitochondrial myopathy. View Publication

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. Despite the broad and essential role of arterioles in physiology and disease,current knowledge of the functional genomics of arterioles is largely absent. Here,we report extensive maps of chromatin interactions,single-cell expression,and other molecular features in human arterioles and uncover mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries,arterioles exhibited a higher proportion of pericytes which were enriched for blood pressure (BP)-associated genes. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles. We linked BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our data and findings will advance the study of the numerous diseases involving arterioles. Liu et al.,report extensive maps of chromatin interactions,single-cell expression,and other molecular features in human arterioles and uncover mechanisms linking noncoding genetic variants to gene expression and the development of hypertension. View Publication