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BackgroundEmbryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs,which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation. How this primed enhancer state is established and maintained and how it affects the regulation of developmental gene networks remains poorly understood.ResultsHere,we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of postgastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation,marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases,lineage-specific enhancers are epigenetically marked already in the zygote,weeks before their activation during lineage specification. Moreover,we outline a generalizable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function.ConclusionsOur findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03658-8. View Publication

SUMMARY GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology,we deleted EXOC2,which encodes an essential exocyst subunit,in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2,suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to,surprisingly,the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA,making it a potential therapeutic target in C9ORF72-ALS/FTD. In brief Halim et al. deleted the gene EXOC2 from patient stem cells and then differentiated them into motor neurons. They found that several amyotrophic lateral sclerosis-related phenotypes were rescued in patient neurons when EXOC2 was deleted or knocked down by a drug. This study identifies EXOC2 as a potential therapeutic target. Graphical Abstract View Publication

Cathepsin B (CatB),a protease in endosomal and lysosomal compartments,plays a key role in neuronal protein processing and degradation,but its function in brain development remains unclear. In this study,we found that CatB is highly expressed in the cortex of E12.5–E16.5 mice. Morphological analysis revealed significant defects in cortical development in CatB knockout (KO) mice,particularly in layer 6. In vitro experiments showed that CatB deficiency notably impaired neuronal migration and development. Behaviorally,CatB KO mice displayed prominent depressive-like behaviors,and electrophysiological recordings demonstrated significantly reduced neuronal activity in layer 6 of the medial prefrontal cortex. Mechanistically,proteomics analysis revealed that CatB KO affected neuronal migration and axonal growth,and decreased the expression of key transcription factors involved in neuronal development,particularly PEG3. Deficiency of PEG3 also significantly impaired neuronal migration and development. Our findings uncover a role for CatB in cortical development and suggest a mechanism linking CatB deficiency with depression and developmental defects through the destabilization of PEG3. Cathepsin B (CatB) is essential for cortical development. Its deficiency impairs neuronal migration,reduces PEG3 expression,and leads to layer 6 defects and depression-like behaviors,revealing a novel link between CatB and brain development. View Publication

BackgroundELMSAN1 (ELM2?SANT domain?containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex),playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However,the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage,we employed human?induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1.Methods and ResultsWe generated ELMSAN1?deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation,ELMSAN1 depletion inhibited pluripotency deactivation,decreased the expression of cardiac?specific markers,and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure,calcium handling,and ion channels was also noted in ELMSAN1?deficient cardiomyocytes derived from hiPSCs. Additionally,through a series of structural and functional assessments,we found that ELMSAN1?null hiPSC cardiomyocytes are immature,exhibiting incomplete sarcomere Z?line structure,decreased calcium handling,and impaired electrophysiological properties. Of note,we found that the cardiac?specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights,indicating maturation reduction with the energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes.ConclusionsIn this study,we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation,including cardiac differentiation,sarcomere formation,calcium handling,electrophysiological maturation,and proliferation. View Publication

Pluripotent stem cells possess a unique nuclear architecture characterized by a larger nucleus and more open chromatin,which underpins their ability to self-renew and differentiate. Here,we show that the nucleolus-specific RNA helicase DDX18 is essential for maintaining the pluripotency of human embryonic stem cells. Using techniques such as Hi-C,DNA/RNA-FISH,and biomolecular condensate analysis,we demonstrate that DDX18 regulates nucleolus phase separation and nuclear organization by interacting with NPM1 in the granular nucleolar component,driven by specific nucleolar RNAs. Loss of DDX18 disrupts nucleolar substructures,impairing centromere clustering and perinucleolar heterochromatin (PNH) formation. To probe this further,we develop NoCasDrop,a tool enabling precise nucleolar targeting and controlled liquid condensation,which restores centromere clustering and PNH integrity while modulating developmental gene expression. This study reveals how nucleolar phase separation dynamics govern chromatin organization and cell fate,offering fresh insights into the molecular regulation of stem cell pluripotency. Pluripotent stem cells depend on specialized nuclear organization for their function. Here,the authors show that DDX18 regulates nucleolar phase separation and chromatin architecture to preserve human embryonic stem cell pluripotency. View Publication

BackgroundChronic ischemic limb disease often leads to amputation,which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases,but studies with primary human SMCs have been limited by a lack of availability. Here,we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers,or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D?+?2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D?+?2D shaker protocol) or in a PBS bioreactor (the 3D?+?2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA],smooth muscle protein 22 [SM22],and Calponin-1),and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay),contraction in response to the treatment with a prostaglandin H2 analog (U46619),and tube formation on Geltrex,as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D?+?2D protocols yielded?>?5.6?×?107 hiPSC-SMCs/differentiation,which was?~?nine-fold more than that produced via 2D differentiation,and flow cytometry analyses confirmed that?>?98% of the 3D?+?2D-differentiated hiPSC-SMCs expressed SMA,?>?81% expressed SM22,and?>?89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D?+?2D shaker protocol also displayed typical SMC-like migratory,contraction,and tube-formation activity in-vitro and significantly improved measurements of perfusion,vessel density,and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D?+?2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice. View Publication

BackgroundRNA-binding proteins (RBPs) are essential in cardiac development. However,a large of them have not been characterized during the process.MethodsWe applied the human embryonic stem cells (hESCs) differentiated into cardiomyocytes model and constructed SAMD4A-knockdown/overexpression hESCs to investigate the role of SAMD4A in cardiomyocyte lineage specification.ResultsSAMD4A,an RBP,exhibits increased expression during early heart development. Suppression of SAMD4A inhibits the proliferation of hESCs,impedes cardiac mesoderm differentiation,and impairs the function of hESC-derived cardiomyocytes. Correspondingly,forced expression of SAMD4A enhances proliferation and promotes cardiomyogenesis. Mechanistically,SAMD4A specifically binds to FGF2 via a specific CNGG/CNGGN motif,stabilizing its mRNA and enhancing translation,thereby upregulating FGF2 expression,which subsequently modulates the AKT signaling pathway and regulates cardiomyocyte lineage differentiation. Additionally,supplementation of FGF2 can rescue the proliferation defect of hESCs in the absence of SAMD4A.ConclusionsOur study demonstrates that SAMD4A orchestrates cardiomyocyte lineage commitment through the post-transcriptional regulation of FGF2 and modulation of AKT signaling. These findings not only underscore the essential role of SAMD4A in cardiac organogenesis,but also provide critical insights into the molecular mechanisms underlying heart development,thereby informing potential therapeutic strategies for congenital heart disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04269-7. View Publication

Organoids are self-assembled 3D cellular structures that resemble organs structurally and functionally,providing in vitro platforms for molecular and therapeutic studies. Generation of organoids from human cells often requires long and costly procedures with arguably low efficiency. Prediction and selection of cellular aggregates that result in healthy and functional organoids can be achieved by using artificial intelligence-based tools. Transforming images of 3D cellular constructs into digitally processable data sets for training deep learning models requires labeling of morphological boundaries,which often is performed manually. Here,we report an application named OrganoLabeler,which can create large image-based data sets in a consistent,reliable,fast,and user-friendly manner. OrganoLabeler can create segmented versions of images with combinations of contrast adjusting,K-means clustering,CLAHE,binary,and Otsu thresholding methods. We created embryoid body and brain organoid data sets,of which segmented images were manually created by human researchers and compared with OrganoLabeler. Validation is performed by training U-Net models,which are deep learning models specialized in image segmentation. U-Net models,which are trained with images segmented by OrganoLabeler,achieved similar or better segmentation accuracies than the ones trained with manually labeled reference images. OrganoLabeler can replace manual labeling,providing faster and more accurate results for organoid research free of charge. View Publication

The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer,known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation,promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/?-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells,as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity. 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

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.Keypoints OXA1L gene bi?allelic variants cause mitochondrial myopathy.OXA1L deficiency results in combined mitochondrial respiratory chain defects and OXPHOS impairments.OXA1L deficiency leads to elevated ROS production,which may activate the NF??B signalling pathway,disturbing myogenic gene expression and triggering cell apoptosis. OXA1L gene bi?allelic variants cause mitochondrial myopathy.OXA1L deficiency results in combined mitochondrial respiratory chain defects and OXPHOS impairments.OXA1L deficiency leads to elevated ROS production,which may activate the NF??B signalling pathway,disturbing myogenic gene expression and triggering cell apoptosis. View Publication

Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases,thereby contributing to cognitive impairment. Among other factors,DNA damage strongly promotes cellular aging,however,the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro,ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype expression,reduced BBB integrity,and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line,EC-KO mice showed more P21+ cells,augmented expression of angiogenic markers,and a concomitant increase in the number of brain ECs and pericytes. Moreover,EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter,suggesting a regional susceptibility. Collectively,our results underline the role of endothelial aging as a driver of impaired BBB function,endothelial sprouting,and increased immune cell migration into the brain,thereby contributing to impaired brain homeostasis as observed during the aging process. View Publication