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Background & aimsHumans with WNT2B deficiency have severe intestinal disease,including significant inflammatory injury,highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.MethodsWe investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon,and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue.ResultsMice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased,epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6,and biopsy tissue from humans showed increased neutrophils.ConclusionsWNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation,particularly in the colon. Graphical abstract View Publication

To comprehend the volumetric neural connectivity of a brain organoid,it is crucial to monitor the spatiotemporal electrophysiological signals within the organoid,known as intra-organoid signals. However,previous methods risked damaging the three-dimensional (3D) cytoarchitecture of organoids,either through sectioning or inserting rigid needle-like electrodes. Also,the limited numbers of electrodes in fixed positions with non-adjustable electrode shapes were insufficient for examining the complex neural activity throughout the organoid. Herein,we present a magnetically reshapable 3D multi-electrode array (MEA) using direct printing of liquid metals for electrophysiological analysis of brain organoids. The adaptable distribution and the softness of these printed electrodes facilitate the spatiotemporal recording of intra-organoid signals. Furthermore,the unique capability to reshape these soft electrodes within the organoid using magnetic fields allows a single electrode in the MEA to record from multiple points,effectively increasing the recording site density without the need for additional electrodes. Conventional platforms for electrophysiological recording of organoids have limited recording site density. Here,the authors present the magnetically reshapable 3D liquid metal-based electrode array for high-resolution analysis on neural activities of brain organoids. 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

BackgroundAtrial fibrillation has an estimated prevalence of 1.5–2%,making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic,as well as local,inflammatory processes has been reported. However,the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology,a direct,causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes,to propose a mechanistic link between inflammation and atrial fibrillation.MethodsTwo hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic,pacing-free,atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity,electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes.ResultsMulti electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A,KCNA5,ATP1A1,and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes,including reduced beat irregularity,improved conduction,and reversed RNA expression profiles.ConclusionsThis study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia,documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further,beat rate irregularity could be alleviated using glucocorticoids. Overall,these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03814-0. View Publication

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context,the roles of microRNAs have received less attention. In the present study,we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients,showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1),which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia. Synopsis The involvement of microRNAs in the pathogenesis of schizophrenia is not well-understood. This study shows that miR-99b-5p regulates Z-DNA binding protein 1 (Zbp1) to control inflammatory responses in microglia and the development of schizophrenia-like symptoms in mice. miR-99b-5p is downregulated in the blood and brains of schizophrenia patients.miR-99b-5p inhibition induces schizophrenia-like phenotypes in mice and microglial inflammation.Zbp1 is a novel miR-99b-5p target in microglia.Zbp1 antisense oligos ameliorate the pathological outcomes of decreased miR-99b-5p levels. Dysregulation of a novel miR-99b-5p-Zbp1 (Z-DNA binding protein 1) pathway in microglia induces inflammatory responses and schizophrenia-like phenotypes in mice. 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

Mutations in the microtubule binding motor protein,kinesin family member 5A (KIF5A),cause the fatal motor neuron disease,Amyotrophic Lateral Sclerosis. While KIF5 family members transport a variety of cargos along axons,it is still unclear which cargos are affected by KIF5A mutations. We generated KIF5A null mutant human motor neurons to investigate the impact of KIF5A loss on the transport of various cargoes and its effect on motor neuron function at two different timepoints in vitro. The absence of KIF5A resulted in reduced neurite complexity in young motor neurons (DIV14) and significant defects in axonal regeneration capacity at all developmental stages. KIF5A loss did not affect neurofilament transport but resulted in decreased mitochondria motility and anterograde speed at DIV42. More prominently,KIF5A depletion strongly reduced anterograde transport of SFPQ-associated RNA granules in DIV42 motor neuron axons. We conclude that KIF5A most prominently functions in human motor neurons to promote axonal regrowth after injury as well as to anterogradely transport mitochondria and,to a larger extent,SFPQ-associated RNA granules in a time-dependent manner. View Publication

Oropouche fever is a re-emerging global viral threat caused by infection with Oropouche orthobunyavirus (OROV). While disease is generally self-limiting,historical and recent reports of neurologic involvement highlight the importance of understanding the neuropathogenesis of OROV. In this study,we characterize viral replication kinetics in neurons and microglia derived from immortalized,primary,and induced pluripotent stem cell-derived cells,which are all permissive to infection. We demonstrate that ex vivo rat brain slice cultures can be infected by OROV and produce antiviral cytokines and chemokines,including IL-6,TNF-? and IFN-?,which introduces an additional model to study viral kinetics in the central nervous system. These findings provide additional insight into OROV neuropathogenesis and in vitro modeling strategies for a newly re-emerging arbovirus. View Publication

Microglia are the immune cells in the central nervous system (CNS) and become pro-inflammatory/activated in Alzheimer’s disease (AD). Cell surface glycosylation plays an important role in immune cells; however,the N-glycosylation and glycosphingolipid (GSL) signatures of activated microglia are poorly understood. Here,we study comprehensively combined transcriptomic and glycomic profiles using human induced pluripotent stem cells-derived microglia (hiMG). Distinct changes in N-glycosylation patterns in amyloid-? oligomer (A?O) and LPS-treated hiMG were observed. In A?O-treated cells,the relative abundance of bisecting N-acetylglucosamine (GlcNAc) N-glycans decreased,corresponding with a downregulation of MGAT3. The sialylation of N-glycans increased in response to A?O,accompanied by an upregulation of genes involved in N-glycan sialylation (ST3GAL4 and 6). Unlike A?O-induced hiMG,LPS-induced hiMG exhibited a decreased abundance of complex-type N-glycans,aligned with downregulation of mannosidase genes (MAN1A1,MAN2A2,and MAN1C1) and upregulation of ER degradation related-mannosidases (EDEM1-3). Fucosylation increased in LPS-induced hiMG,aligned with upregulated fucosyltransferase 4 (FUT4) and downregulated alpha-L-fucosidase 1 (FUCA1) gene expression,while sialofucosylation decreased,aligned with upregulated neuraminidase 4 (NEU4). Inhibition of sialylation and fucosylation in A?O- and LPS-induced hiMG alleviated pro-inflammatory responses. However,the GSL profile did not exhibit significant changes in response to A?O or LPS activation,at least in the 24-hour stimulation timeframe. A?O- and LPS- specific glycosylation changes could contribute to impaired microglia function,highlighting glycosylation pathways as potential therapeutic targets for AD.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-96596-1. View Publication

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here,we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules,retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR),resulted in rapid,synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model,and they possess all the features necessary to be considered valid. Collectively,our data demonstrate a facile,scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta. View Publication