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ABSTRACTPatched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling,an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development,molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here,we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling,and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models. Summary: Cerebellar organoids recapitulate in vivo processes of regionalisation and SHH signalling,and offer new insight into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models. View Publication

SummaryCell size is a crucial physical property that significantly impacts cellular physiology and function. However,the influence of cell size on stem cell specification remains largely unknown. Here,we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly,cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore,the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements,we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically,the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation,which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together,our study has established a novel connection between cell size diminution and DE differentiation,which is mediated by AMOT nuclear translocation. Additionally,our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation. Graphical abstract Highlights•Cell size decreases during the differentiation of human pluripotent stem cells into endoderm•Hypertonic pressure is conducive to the differentiation of human definitive endoderm•Actomyosin contributes to both size diminution and endoderm promotion under hypertonic pressure•Cell size diminution represses YAP activity via promoting AMOT nuclear translocation Jiang and colleagues show that cell size exhibits a gradual decrease during human endoderm differentiation. The application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced endoderm differentiation. This enhancement is reliant on actomyosin activity and achieved by promoting the nuclear translocation of AMOT,thereby repressing YAP activity. View Publication

Diabetes mellitus,a chronic and non-transmissible disease,triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ?-like cells (P?LCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways,directing the differentiation of iPSCs into pancreatic ? cells. Despite significant progress and improved protocols,the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here,we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into P?LCs. Our protocol has shown to be effective,with the resulting cells exhibiting key functional properties of pancreatic ? cells,including the expression of crucial molecular markers (pdx1,nkx6.1,ngn3) and the capability to secrete insulin in response to glucose. Furthermore,the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ? cell genes. View Publication

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS),a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease,evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues,and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations,we performed whole-transcriptome profiling of motor neurons (MNs) differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database,we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation,the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity,highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12147-8. View Publication

Neurotropic viruses are the most common cause of infectious encephalitis and highly target neurons for infection. Our understanding of the intrinsic capacity of neuronal innate immune responses to mediate protective antiviral responses remains incomplete. Here,we evaluated the role of intercellular crosstalk in mediating intrinsic neuronal immunity and its contribution to limiting viral infection. We found that in the absence of viral antagonism,neurons transcriptionally induce robust interferon signaling and can effectively signal to uninfected bystander neurons. Yet,in two-dimensional cultures,this dynamic response did not restrict viral spread. Interestingly,this differed in the context of viral infection in three-dimensional forebrain organoids with complex neuronal subtypes and cellular organization,where we observed protective capacity. We showed antiviral crosstalk between infected neurons and bystander neural progenitors is mediated by type I interferon signaling. Using spatial transcriptomics,we then uncovered regions containing bystander neural progenitors that expressed distinct antiviral genes,revealing critical underpinnings of protective antiviral responses among neuronal subtypes. These findings underscore the importance of interneuronal communication in protective antiviral immunity in the brain and implicate key contributions to protective antiviral signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03381-y. View Publication

Friedreich ataxia (FRDA) is a multisystemic,autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently,there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice,and rescue was mediated by FXN transfer from tissue engrafted,HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation,we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease,such as non-homogenous microtubule staining in neurites,increased caspase-3 expression,mitochondrial superoxide levels,mitochondrial fragmentation,and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release,normalization of ER stress response gene,XBP-1,and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles,as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons,that was undetected in gene edited neurons. Taken together,these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA,validate the efficacy profile of our FXN gene editing approach in a disease relevant model,and support our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia. View Publication

IntroductionGlucose Transporter 1-Deficiency Syndrome (GLUT1-DS) is a rare genetic disorder caused by mutations in the gene encoding for GLUT1 and characterized by impaired glucose uptake in the brain. This leads to brain hypometabolism and the development of symptoms that include epilepsy,motor dysfunctions and cognitive impairment. The development of patient-specific in vitro models is a valuable tool for understanding the pathophysiology of rare genetic disorders and testing new therapeutic interventions.MethodsIn this study,we generated brain organoids from induced pluripotent stem cells (iPSCs) derived either from a GLUT1-DS patient or a healthy individual. The functional organoids were analyzed for cellular composition,maturity,and electrophysiological activity using a custom-made microelectrode array (MEA) platform,which allowed for the detection of spikes,burst patterns,and epileptiform discharges.ResultsImmunostaining revealed a similar distribution of neurons and astrocytes in both healthy and GLUT1-DS brain organoids,though GLUT1-DS brain organoids exhibited reduced cellular density and smaller overall size. Electrophysiological recordings demonstrated functional spike profiles in both organoid types. Notably,our study demonstrates that brain organoids derived from a GLUT1-DS patient exhibit distinct epileptiform activity and heightened sensitivity to glucose deprivation,reflecting key features of the disorder.DiscussionThese findings validate the use of brain organoids as a model for studying GLUT1-DS and highlight their potential for testing novel therapeutic strategies aimed at improving glucose metabolism and managing epilepsy in patients. View Publication

Recent studies demonstrate that brain infiltration of peripheral immune cells and their interaction with brain-resident cells contribute to Parkinson’s disease (PD). However,mechanisms of T cell-brain cell communication are not fully elucidated and models allowing investigation of interaction between T cells and brain-resident cells are required. In this study,we developed a three-dimensional (3D) model composed of stem cell-derived human midbrain organoids (hMO) and peripheral blood T cells. We demonstrated that organoids consist of multiple midbrain-specific cell types,allowing to study T cell motility and interactions with midbrain tissue in a spatially organized microenvironment. We optimized co-culture conditions and demonstrated that T cells infiltrate hMO tissue,leading to neural cell loss. Our work establishes a novel 3D cell co-culture model as a promising tool to investigate the effect of the adaptive immune system on the midbrain and can be used in future studies to address these processes in the context of PD. View Publication

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study,we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling,including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover,we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart,including 551 genes,504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall,our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease. Ruiz-Orera et al. used comparative transcriptomics and translatomics to analyze the cardiac evolution in primates and discovered species-specific and lineage-specific genomic innovations that might contribute to cardiac development and disease. View Publication

An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits,we investigated an in vitro neural tissue model for inter-regional connections,in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids,suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition,optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro. Connecting cerebral organoids with an axon bundle models inter-regional projections and enhances neural activity. Optogenetic stimulation induces short-term plasticity,offering insights into macroscopic circuit development and functionality. View Publication

Neutrophils are critical for host defense against fungi. However,the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here,we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation,antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan,including an upregulation of the pentose phosphate pathway (PPP),similar to primary human neutrophils in vitro. Furthermore,we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively,these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections,and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation,limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus,demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively,these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity. View Publication

SUMMARY During cell fate transitions,cells remodel their transcriptome,chromatin,and epigenome; however,it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here,we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that,following heterokaryon formation,the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG,conversely,has significant nascent RNA transcription only at 48 h after cell fusion but,strikingly,exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent. In brief Martinez-Sarmiento et al. demonstrate that,during heterokaryon reprogramming,global chromatin decondensation and loss of repressive histone modifications occur at late stages after cell fusion. Activation of OCT4 precedes global chromatin decompaction and does not require the opening of its local genomic region. Conversely,NANOG activation occurs after OCT4 activation,and the NANOG locus undergoes opening prior to its transcriptional activation. Graphical Abstract View Publication