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The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs,including two-dimensional cell culture models or animal models,have several limitations. Therefore,an appropriate model representative of the human organism is required. Here,we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hM?s) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably,M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore,a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus,the incorporation of hM?s and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies. Author summaryEstablishment of M.tb infection model is imperative to develop new anti-TB drugs based on the pathogenesis of TB. Various animal models,including mice,rats,guinea pigs,non-human primates,rabbits,cattle,and zebrafish,are commonly used in TB research to mimic TB symptoms and study immune responses to M.tb infection. In vitro models,such as agent-based models allow examination of host-pathogen interactions,early granuloma formation and drug screening,providing cellular-level insights. However,these models may not fully represent human immunopathology owing to differences in immune cell distributions. Lung organoids mimic human lung dynamics and functions,providing crucial insights into immune responses to TB. In this study,an M.tb infection model developed using hLOs demonstrated infection of lung epithelial cells and human macrophages,reflecting host-pathogen interactions. This model is attractive for evaluating the efficacy of anti-TB drugs and host-directed therapies. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. View Publication

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer’s disease,we performed single nucleus RNA sequencing in 24 Alzheimer’s disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands,SMAD3,upregulated in Alzheimer’s disease pericytes,has the highest number of ligands including VEGFA,downregulated in Alzheimer’s disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer’s disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here,we detect vast transcriptome changes in Alzheimer’s disease at the gliovascular-unit,prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions,and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer’s disease. Systematic studies are needed to discover molecular determinants of blood brain barrier dysfunction in Alzheimer’s disease. This study identifies perturbed pericytic SMAD3-astrocytic VEGFA interactions as a potential driver of this dysfunction. View Publication

Diabetes involves the death or dysfunction of pancreatic ?-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response,we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types,we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ?-,?-,and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1,which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics. Endoplasmic reticulum and inflammatory stress are associated with diabetes. Maestas et al. use single-cell sequencing to profile primary human islets under stress and identified tissue and cell-type responses. View Publication

SummaryPattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells,we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly,HIF activation leads to control of HSV replication. Mechanistically,HIF activation induces autophagy,which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons,and mice with neuron-specific HIF1/2? deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively,the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation. Graphical abstract Highlights•HSV-1 and -2 disrupt the hypoxia-inducible factor (HIF) network in permissive cells•HIF activation induces autophagy,which exerts anti-HSV activity in neurons•Neuronal HIF activation regulates infection and inflammation in the infected brain Using transcriptome analysis of disrupted transcripts in herpes simplex virus-infected cells,Farahani et al. identify the hypoxia-inducible factor gene network to possess antiviral activity through induction of autophagy. This contributes to antiviral defense and regulation of inflammation during infection in the CNS. View Publication

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