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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

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1),a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However,the regulatory network of SCA1 pathology,especially central regulators of the earliest developmental stages and inflammatory events,remains incompletely understood. Here,we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development,and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients. Molecular changes in neurodegeneration occur much earlier than previously expected. In this study,dynamic molecular network analysis of iPSC differentiation uncovers a temporal pathway from histone to ISG15 with the earliest molecular changes of SCA1. 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

IntroductionNeutrophils are highly abundant innate immune cells that are constantly produced from myeloid progenitors in the bone marrow. Differentiated neutrophils can perform an arsenal of effector functions critical for host defense. This study aims to quantitatively understand neutrophil mitochondrial metabolism throughout differentiation and activation,and to elucidate the impact of mitochondrial metabolism on neutrophil functions.MethodsTo study metabolic remodeling throughout neutrophil differentiation,murine ER-Hoxb8 myeloid progenitor-derived neutrophils and human induced pluripotent stem cell-derived neutrophils were assessed as models. To study the metabolic remodeling upon neutrophil activation,differentiated ER-Hoxb8 neutrophils and primary human neutrophils were activated with various stimuli,including ionomycin,monosodium urate crystals,and phorbol 12-myristate 13-acetate. Characterization of cellular metabolism by isotopic tracing,extracellular flux analysis,metabolomics,and fluorescence-lifetime imaging microscopy revealed dynamic changes in mitochondrial metabolism.ResultsAs neutrophils mature,mitochondrial metabolism decreases drastically,energy production is offloaded from oxidative phosphorylation,and glucose oxidation through the TCA cycle is substantially reduced. Nonetheless,mature neutrophils retain the capacity for mitochondrial metabolism. Upon stimulation with certain stimuli,TCA cycle is rapidly activated. Mitochondrial pyruvate carrier inhibitors reduce this re-activation of the TCA cycle and inhibit the release of neutrophil extracellular traps. Treatment with these inhibitors also impacts neutrophil redox status,migration,and apoptosis without significantly changing overall bioenergetics.ConclusionsTogether,these results demonstrate that mitochondrial metabolism is dynamically remodeled and plays a significant role in neutrophils. Furthermore,these findings point to the therapeutic potential of mitochondrial pyruvate carrier inhibitors in a range of conditions where dysregulated neutrophil response drives inflammation and contributes to pathology. 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

Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal genetic disease,with most patients carrying mutations in PKD1. The main feature is the formation of bilateral renal cysts,leading to end stage renal failure in a significant proportion of those affected. Despite recent advances made in understanding ADPKD,there are currently no effective curative therapies. The emergence of human induced pluripotent stem cell (hiPSC)-derived kidney disease models has led to renewed hope that more physiological systems will allow for the development of novel treatments. hiPSC-derived organoid models have been used to recapitulate ADPKD,however they present numerous limitations which remain to be addressed. In the present study,we report an efficient method for generating organoids containing a network of polarised and ciliated epithelial tubules. PKD1 null (PKD1?/?) organoids spontaneously develop dilated tubules,recapitulating early ADPKD cystogenesis. Furthermore,PKD1?/? tubules present primary cilia defects when dilated. Our model could therefore serve as a valuable tool to study early ADPKD cystogenesis and to develop novel therapies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-94855-9. 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

BackgroundLiver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine.MethodsIn this study,we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-? inhibitor (A-83-01,A),GSK3 inhibitor (CHIR99021,C),and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC).ResultsThese HBOs exhibited bile canaliculi of MHs connected to BD structures,mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures,and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues.ConclusionsThis study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases,drug discovery,and personalized medicine.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03877-z. View Publication

Cell fate progression of pluripotent progenitors is strictly regulated,resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study,we use human brain and retina organoid models and present single-cell profiling of H3K27ac,H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage,providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction,resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination. The mechanisms underlying human cell diversity are unclear. Here the authors provide a single-cell epigenome map of human neural organoid development and dissect how epigenetic changes control cell fate specification from pluripotency to distinct cerebral and retina neural types. View Publication

SummaryGamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However,no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages,including adulthood. Here,we showed that such mosaicism is present in wild-type (WT) and down syndrome (DS) neuronal networks,as assessed at increasing scales of complexity (cultures,brain slices,behaving mice). Nevertheless,WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover,we found heterogeneous GABAergic responses in developed control and trisomic human induced-pluripotent-stem-cells-derived neurons. Thus,a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons,possibly contributing to disease-associated behaviors. Graphical abstract Highlights•Subpopulations of GABAAR-responding neurons exist in mouse and human neuronal networks•DS networks exhibit a larger fraction of neurons with depolarizing GABA responses•Restoring physiological GABA-mediated inhibition rescues anxiety behavior in DS mice•Heterogeneous GABAergic responses coexist in control and DS human iPSC neurons Behavioral neuroscience; Developmental neuroscience; Cellular neuroscience 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