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BackgroundThe causal relationship between the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the preservation of SERCA2a function in mitigating myocardial ischemia–reperfusion (mI/R) injury,along with the associated regulatory mechanisms,remains incompletely understood. This study aims to unravel how NRF2 directly or indirectly influences SERCA2a function and its regulators,phospholamban (PLN) and Dwarf Open Reading Frame (DWORF),by testing the pharmacological repositioning of AEOL-10150 (AEOL) in the context of mI/R injury.MethodsC57BL6/J,Nrf2 knockout (Nrf2?/?),and wild-type (Nrf2+/+) mice,as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) were subjected to I/R injury. Gain/loss of function techniques,RT-qPCR,western blotting,LC/MS/MS,and fluorescence spectroscopy were utilized. Cardiac dimensions and function were assessed by echocardiography.ResultsIn the early stages of mI/R injury,AEOL administration reduced mitochondrial ROS production,decreased myocardial infarct size,and improved cardiac function. These effects were due to NRF2 activation,leading to the overexpression of the micro-peptide DWORF,consequently enhancing SERCA2a activity. The cardioprotective effect induced by AEOL was diminished in Nrf2?/? mice and in Nrf2/Dworf knockdown models in hiPSCMs subjected to simulated I/R injury. Our data show that AEOL-induced NRF2-mediated upregulation of DWORF disrupts the phospholamban-SERCA2a interaction,leading to enhanced SERCA2a activation and improved cardiac function.ConclusionsTaken together,our study reveals that AEOL-induced NRF2-mediated overexpression of DWORF enhances myocardial function through the activation of the SERCA2a offering promising therapeutic avenues for mI/R injury.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01242-1. Highlights• Novel AEOL-10150 therapeutic potential. AEOL-10150 demonstrates promise in activating NRF2 and mitigating myocardial ischemia-reperfusion injury.• DWORF overexpression breakthrough. Overexpression of DWORF significantly contributes to preserving cardiac function and reducing myocardial injury through the NRF2-DWORF pathway.• Enhanced cardiac protection mechanisms. The study highlights the dual role of AEOL-10150 and DWORF in enhancing cardiac protection and preventing heart failure.• Future research directions. Additional studies are required to validate the long-term efficacy of AEOL-10150 and the regulatory effects of NRF2-DWORF axis in clinical applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01242-1. View Publication

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart,with several clinical trials now in planning or recruitment stages. However,because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs,it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here,we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically,we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures,signal regulatory protein ? (SIRPA)+CD90?CD200+ and SIRPA+CD90?CD200?,identify arrhythmogenic and non-arrhythmogenic cardiomyocytes,respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias. Selvakumar,Clayton et al. use a porcine model of myocardial infarction and PSC-CM transplantation and identify atrial and pacemaker-like cardiomyocytes as the cause of engraftment arrhythmias and surface marker signatures to distinguish between arrhythmogenic and non-arrhythmogenic cardiomyocytes. View Publication

SUMMARY Individuals with Williams syndrome (WS),a neurodevelopmental disorder caused by hemizygous loss of 26–28 genes at 7q11.23,characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes,GTF2I,are associated with altered sociality and are proposed to underlie hypersociality in WS. However,the contribution of GTF2I to human neurodevelopment remains poorly understood. Here,human cellular models of neurodevelopment,including neural progenitors,neurons,and three-dimensional cortical organoids,are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation,including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality. Graphical Abstract In brief GTF2I is thought to influence the phenotypic expression of human sociality and is implicated in neurodevelopmental disease. Adams et al. use hiPSC-derived cell platforms to investigate the role of GTF2I in human neurodevelopment. Loss of GTF2I promotes increased cell death,reduced synaptic integrity,and decreased electrical activity of cortical organoids. View Publication

N-terminal acetyltransferases including NAA10 catalyze N-terminal acetylation,an evolutionarily conserved co- and post-translational modification. However,little is known about the role of N-terminal acetylation in cardiac homeostasis. To gain insight into cardiac-dependent NAA10 function,we studied a previously unidentified NAA10 variant p.(Arg4Ser) segregating with QT-prolongation,cardiomyopathy,and developmental delay in a large kindred. Here,we show that the NAA10R4S variant reduced enzymatic activity,decreased NAA10-NAA15 complex formation,and destabilized the enzymatic complex N-terminal acetyltransferase A. In NAA10R4S/Y-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs),dysregulation of the late sodium and slow delayed rectifier potassium currents caused severe repolarization abnormalities,consistent with clinical QT prolongation. Engineered heart tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased contractile force and sarcomeric disorganization,consistent with the pedigree’s cardiomyopathic phenotype. Proteomic studies revealed dysregulation of metabolic pathways and cardiac structural proteins. We identified small molecule and genetic therapies that normalized the phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines the roles of N-terminal acetylation in cardiac regulation and delineates mechanisms underlying QT prolongation,arrhythmia,and cardiomyopathy caused by NAA10 dysfunction. N-terminal acetylation dysregulation in the heart causes severe arrhythmia and cardiomyopathy. The authors show that stem cell models demonstrate ion channel trafficking defects and sarcomeric disarray as the underlying mechanisms,with gene therapy reversing both phenotypes View Publication

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle,which are among the most important livestock species for milk and meat production. Here,we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First,we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4),followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist,bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes,including PRDM1/BLIMP1,TFAP2C,SOX17,and NANOS3,were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle. View Publication

SummaryThe ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus,the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay,we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons,a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival. Graphical abstract Highlights•rs7132908 is a causal variant at the chr12q13 obesity locus•rs7132908 regulates nearby effector genes with allele and cell-type specificity•Obesity risk allele decreases generation of neurons that regulate appetite A locus on chr12q13 is strongly associated with childhood obesity by genome-wide associate studies. Littleton et al. identified a causal variant at this locus,which regulates gene expression in neural cell types. The obesity risk allele also decreased the proportion of appetite-regulating hypothalamic neurons generated by stem cell differentiation. View Publication

PIEZO1 is critical to numerous physiological processes,transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1,we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging,our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore,the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands,achieving temporal resolution approaching that of patch clamp electrophysiology. Finally,we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems,with potential for elucidating disease mechanisms and targeted drug screening. PIEZO1 is critical in numerous physiological processes,but monitoring its activity and localization in cells can be challenging. Here,the authors present a chemogenetic platform to visualize endogenous human PIEZO1 localization and activity in native cellular conditions,expanding the knowledge on mechanotransduction across single cells and tissue organoids. View Publication

Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no curative treatment. The blood-brain barrier (BBB),multiple physiological functions formed by mainly specialized brain microvascular endothelial cells (BMECs),serves as a gatekeeper to protect the central nervous system (CNS) from harmful molecules in the blood and aberrant immune cell infiltration. The accumulation of evidence indicating that alterations in the peripheral milieu can contribute to neurodegeneration within the CNS suggests that the BBB may be a previously overlooked factor in the pathogenesis of ALS. Animal models suggest BBB breakdown may precede neurodegeneration and link BBB alteration to the disease progression or even onset. However,the lack of a useful patient-derived model hampers understanding the pathomechanisms of BBB dysfunction and the development of BBB-targeted therapies. In this study,we differentiated BMEC-like cells from human induced pluripotent stem cells (hiPSCs) derived from ALS patients to investigate BMEC functions in ALS patients. TARDBP N345K/+ carrying patient-derived BMEC-like cells exhibited increased permeability to small molecules due to loss of tight junction in the absence of neurodegeneration or neuroinflammation,highlighting that BMEC abnormalities in ALS are not merely secondary consequences of disease progression. Furthermore,they exhibited increased expression of cell surface adhesion molecules like ICAM-1 and VCAM-1,leading to enhanced immune cell adhesion. BMEC-like cells derived from hiPSCs with other types of TARDBP gene mutations (TARDBP K263E/K263E and TARDBP G295S/G295S) introduced by genome editing technology did not show such BMEC dysfunction compared to the isogenic control. Interestingly,transactive response DNA-binding protein 43 (TDP-43) was mislocalized to cytoplasm in TARDBP N345K/+ carrying model. Wnt/?-catenin signaling was downregulated in the ALS patient (TARDBP N345K/+)-derived BMEC-like cells and its activation rescued the leaky barrier phenotype and settled down VCAM-1 expressions. These results indicate that TARDBP N345K/+ carrying model recapitulated BMEC abnormalities reported in brain samples of ALS patients. This novel patient-derived BMEC-like cell is useful for the further analysis of the involvement of vascular barrier dysfunctions in the pathogenesis of ALS and for promoting therapeutic drug discovery targeting BMEC. View Publication

BackgroundGenome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified a plethora of risk loci. However,the disease variants/genes and the underlying mechanisms have not been extensively studied.MethodsBulk ATAC-seq was performed in induced pluripotent stem cells (iPSCs) differentiated various brain cell types to identify allele-specific open chromatin (ASoC) SNPs. CRISPR-Cas9 editing generated isogenic pairs,which were then differentiated into glutamatergic neurons (iGlut). Transcriptomic analysis and functional studies of iGlut co-cultured with mouse astrocytes assessed neuronal excitability and lipid droplet formation.ResultsWe identified a putative causal SNP of CLU that impacted neuronal chromatin accessibility to transcription-factor(s),with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. And,neuronal CLU facilitated neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes caused astrocytes to uptake less glutamate thereby altering neuron excitability.ConclusionsFor a strong AD-associated locus near Clusterin (CLU),we connected an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00840-1. View Publication

SummaryLineage-specific differentiation of human induced pluripotent stem cells (hiPSCs) relies on complex interactions between biochemical and physical cues. Here we investigated the ability of hiPSCs to undergo lineage commitment in response to inductive signals and assessed how this competence is modulated by substrate stiffness. We showed that Activin A-induced hiPSC differentiation into mesendoderm and its derivative,definitive endoderm,is enhanced on gel-based substrates softer than glass. This correlated with changes in tight junction formation and extensive cytoskeletal remodeling. Further,live imaging and biophysical studies suggested changes in cell motility and interfacial contacts underlie hiPSC layer reshaping on soft substrates. Finally,we repurposed an ultra-soft silicone gel,which may provide a suitable substrate for culturing hiPSCs at physiological stiffnesses. Our results provide mechanistic insight into how epithelial mechanics dictate the hiPSC response to chemical signals and provide a tool for their efficient differentiation in emerging stem cell therapies. Graphical abstract Highlights•Tuning of substrate stiffness can enhance mesendoderm/endoderm hiPSC differentiation•Altered tight junction formation drives increased differentiation on soft substrates•Changes in cell motility and interfacial contacts underlie hiPSC layer remodeling Mechanobiology; Stem cells research; Biophysics View Publication

BackgroundOsteoarthritis (OA) is a complex,age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility,joint stiffness,pain,and a significant decrease in quality of life. Among other risk factors,such as genetics and age,hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815–823,2011). It has been shown that post-mitotic cells,such as articular chondrocytes,heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However,these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes,resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state.ResultsWe showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5,CAV1,and CD44,among other genes,which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover,by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs),we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity.ConclusionOur findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes,concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1,ITGA5,and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes,we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting,detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-024-01676-0. View Publication

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here,we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels,we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis,phospholipid composition,and inner leaflet surface charge,thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together,our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration. The biomechanical mechanisms enabling the invasive growth of brain tumors remain opaque. Here,Junqueira Alves et al. reveal that the guidance receptor Plexin-B2 controls membrane tension,facilitating confined migration of brain tumor cells. View Publication