参考文献

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

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common genetic form of stroke. It is caused by a cysteine-altering variant in one of the 34 epidermal growth factor-like repeat (EGFr) domains of Notch3. NOTCH3 pathogenic variants in EGFr 1–6 are associated with high disease severity,whereas those in EGFr 7–34 are associated with late stroke onset and increased survival. However,whether and how the position of the NOTCH3 variant directly affects the disease severity remains unclear. In this study,we aimed to generate human-induced pluripotent stem cells (hiPSCs) from patients with CADASIL with EGFr 1–6 and 7–34 pathogenic variants to evaluate whether the NOTCH3 position affects the cell phenotype and protein profile of the generated hiPSCs lines. Six hiPSCs lines were generated: two from patients with CADASIL with EGFr 1–6 pathogenic variants,two from patients with EGFr 7–34 variants,and two from controls. Notch3 aggregation and protein profiles were tested in the established six hiPSCs lines. Cell analysis revealed that the NOTCH3 variants did not limit the cell reprogramming efficiency. However,EGFr 1–6 variant position was associated with increased accumulation of Notch3 protein in pluripotent stem cells and proteomic changes related with cytoplasmic reorganization mechanisms. In conclusion,our analysis of hiPSCs derived from patients with CADASIL support the clinical association between the NOTCH3 variant position and severity of CADASIL.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12017-025-08840-6. 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

Genomic instability is the main cause of abnormal embryo development and abortion. NLRP7 dysfunctions affect embryonic development and lead to Hydatidiform Moles,but the underlying mechanisms remain largely elusive. Here,we show that NLRP7 knockout affects the genetic stability,resulting in increased DNA damage in both human embryonic stem cells and blastoids,making embryonic cells in blastoids more susceptible to apoptosis. Mechanistically,NLRP7 can interact with factors related to alternative splicing and DNA damage response,including DDX39B,PRPF8,THRAP3 and PARP1. Moreover,NLRP7 dysfunction leads to abnormal alternative splicing of genes involved in homologous recombination in human embryonic stem cells,Such as Brca1 and Rad51. These results indicate that NLRP7-mediated Alternative splicing is potentially required for the maintenance of genome integrity during early human embryogenesis. Together,this study uncovers that NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. View Publication