技术资料

Under specific conditions,cultured human embryonic stem cells (hESCs) corresponding to primed post-implantation epiblasts can be converted back to a ‘naïve pluripotency’ state that resembles the pre-implantation epiblasts. The core pluripotency factor OCT4 is known to be crucial in regulating different states of pluripotency,but its potential regulatory role in human naïve pluripotency remains unexplored. In this study,we systematically mapped out phosphorylation sites in OCT4 protein that are differentially phosphorylated between two states of pluripotency,and further identified ATR as a key kinase that phosphorylated OCT4 in naïve but not primed hESCs. The kinase activity levels of ATR in naïve hESCs were higher than those in primed hESCs. Ablating cellular ATR activity significantly halted the induction of naïve hESCs from their primed counterparts,and increased early apoptotic death of naïve hESCs upon UV and CPT treatment. Thus,our work reveals the importance of ATR activity in safeguarding human naïve pluripotency,and implicates a potential association of OCT4 phosphorylation,DNA damage sensing and repairing system in regulating different states of pluripotency during early development. View Publication

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus strains that cause severe infections. Bicomponent PVL kills phagocytes depending on cell surface receptors,such as complement 5a receptor 1 (C5aR1). How the PVL-receptor interaction enables assembly of the leukocidin complex,targeting of membranes,and insertion of a pore channel remains incompletely understood. Here,we demonstrate that PVL binds the anionic phospholipids,phosphatidic acid,and cardiolipin,under acidic conditions and targets lipid bilayers that mimic lysosomal and mitochondrial membranes,but not the plasma membrane. The PVL–lipid interaction was sufficient to enable leukocidin complex formation as determined by neutron reflectometry and the rupture of model membranes,independent of protein receptors. In phagocytes,PVL and its C5aR1 receptor were internalized depending on sphingomyelin and cholesterol,which were dispensable for the interaction of the toxin with the plasma membrane. Internalized PVL compromised the integrity of lysosomes and mitochondria before plasma membrane rupture. Preventing the acidification of organelles or the genetic loss of PVL impaired the escape of intracellular S. aureus from macrophages. Together,the findings advance our understanding of how an S. aureus toxin kills host cells and provide key insights into how leukocidins target membranes. Staphylococcus aureus secretes toxins,such as Panton-Valentine leukocidin (PVL),to kill immune cells,including macrophages. This study shows that PVL binds phosphatidic acid and cardiolipin in acidic conditions,targeting lysosomal and mitochondrial membranes (but not the plasma membrane) to promote bacterial escape. View Publication

Human induced pluripotent stem cells (iPSCs) have great potential in research,but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here,we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD,NANOG,SPP1),endoderm (CER1,EOMES,GATA6),mesoderm (APLNR,HAND1,HOXB7),and ectoderm (HES5,PAMR1,PAX6). Using these genes,we develop a machine learning-based scoring system,“hiPSCore”,trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time,subjectivity,and resource use,thus enhancing iPSC quality for scientific and medical applications. Quality control,including pluripotency testing of human iPSCs lacks standardization. Here,authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality. View Publication

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity. Calcium is an important signal transducer that regulates key cellular processes,but its role in regulating CM proliferation is incompletely understood. Here we show a robust pathway for new calcium signaling-based cardiac regenerative strategies. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC) induced the CM cell cycle. Furthermore,overexpression of Ras-related associated with Diabetes (RRAD),an endogenous inhibitor of LTCC,induced CM cell cycle activity in vitro,in human cardiac slices,and in vivo. Mechanistically,LTCC inhibition by RRAD or nifedipine induced CM cell cycle by modulating calcineurin activity. Moreover,ectopic expression of RRAD/CDK4/CCND in combination induced CM proliferation in vitro and in vivo,improved cardiac function and reduced scar size post-myocardial infarction. View Publication

SummaryTissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here,we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells,the intervention,composed of one-carbon metabolites,reduces some dedifferentiation markers without losing the lineage identity,thus inducing limited reprogramming into a more flexible cell state. Moreover,the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall,our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones. Graphical abstract Highlights•Early cell transitions in differentiation include metabolites,supporting identity changes•Cell-transition biochemicals can be leveraged to induce plasticity•1C-metabolite supplementation streamlines cell-identity changes in vitro•1C-metabolite in vivo administration impacts acetylation genes,aiding muscle regeneration Hernandez-Benitez et al. identify a metabolomic wave conserved in the early transition of cells differentiating in vitro,and they leverage this finding to customize an in vivo supplementation that facilitates the transition of cell phenotypes when needed,like in regeneration after an injury. View Publication

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs),yet their role in MNs remains unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for other hematopoietic lineages. Ddx41 knockout in early erythropoiesis is embryonically lethal,while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4),which co-distribute with DDX41 on the erythroid genome. DDX41 directly binds to and resolves G4,which is significantly compromised in MN-associated DDX41 mutants. G4 accumulation induces erythroid genome instability,ribosomal defects,and p53 upregulation. However,p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel,genome instability also activates the cGas-Sting pathway,impairing survival,as cGas deficiency rescues the lethality of hematopoietic-specific Ddx41 knockout mice. This is supported by data from a DDX41-mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 resolvase,essential for erythroid genome stability and suppressing the cGAS-STING pathway. Germline DDX41 mutations are linked to myeloid neoplasms,but their roles in the disease is unclear. Here,the authors show that DDX41 resolves G-quadruplex structures to maintain erythroid genome stability and prevent cGAS-mediated cell death. These functions are lost in disease-associated variants. View Publication

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here,we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency,encompassing an eight-day time course with modulation of WNT,BMP,and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm,neural crest,and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas,evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together,this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology. Shen et al. report a method for multiplexing isogenic iPSCs for single-cell RNA-seq. With it,they created an atlas of in vitro differentiation and identified TMEM88 as a regulator of cardiovascular development,impacting blood pressure in adult mice. View Publication

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson’s disease,however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5’-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson’s patient midbrain cultures accumulate glucose and uridine-5’-triphosphate,while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme,GFPT2,through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate,while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological ?-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity,and its failure in Parkinson’s disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement. Reduced glucose flux via the hexosamine pathway contributes to lysosomal dysfunction and protein accumulation in Parkinson patient iPSC-neurons. Enhancing the hexosamine pathway rescues lysosome activity and restores proteostasis. View Publication

AbstractThe fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here,we implemented a 3D model of cardiac vasculogenesis,incorporating endothelial cells (EC),stromal cells,and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues,resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis,including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs,the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast,supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly,enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization,illustrating the use of this approach in the engineering of enhanced,perfusable,microfluidic models of the myocardium. View Publication

AbstractNeurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially reduce accumulation of neurological deficits and conversion to progressive disease forms. Diet and the associated gut microbiome are currently being discussed as crucial environmental risk factors that determine disease onset and subsequent progression. In people with multiple sclerosis,supplementation of the short-chain fatty acid propionic acid,as a microbial metabolite derived from the fermentation of a high-fiber diet,has previously been shown to regulate inflammation accompanied by neuroprotective properties. We set out to determine whether the neuroprotective impact of propionic acid is a direct mode of action of short-chain fatty acids on CNS neurons. We analysed neurite recovery in the presence of the short-chain fatty acid propionic acid and butyric acid in a reverse-translational disease-in-a-dish model of human-induced primary neurons differentiated from people with multiple sclerosis-derived induced pluripotent stem cells. We found that recovery of damaged neurites is induced by propionic acid and butyric acid. We could also show that administration of butyric acid is able to enhance propionic acid-associated neurite recovery. Whole-cell proteome analysis of induced primary neurons following recovery in the presence of propionic acid revealed abundant changes of protein groups that are associated with the chromatin assembly,translational,and metabolic processes. We further present evidence that these alterations in the chromatin assembly were associated with inhibition of histone deacetylase class I/II following both propionic acid and butyric acid treatment,mediated by free fatty acid receptor signalling. While neurite recovery in the presence of propionic acid is promoted by activation of the anti-oxidative response,administration of butyric acid increases neuronal ATP synthesis in people with multiple sclerosis-specific induced primary neurons. In human multiple sclerosis-specific neurons,differentiated via induced pluripotent stem cells,Gisevius et al. display neuroregeneration mediated by the short-chain fatty acids propionic and butyric acid. Intracellularly,free fatty acid receptor signalling leads to inhibition of histone deacetylase activity,thereby altering the oxidative stress response and cellular protein biosynthesis. Graphical Abstract Graphical Abstract View Publication

SUMMARY Increasing evidence suggests that the mechanics of chromatin and nucleoplasm regulate gene transcription and nuclear function. However,how the chromatin and nucleoplasm sense and respond to forces remains elusive. Here,we employed a strategy of applying forces directly to the chromatin of a cell via a microinjected 200-nm anti-H2B-antibody-coated ferromagnetic nanoparticle (FMNP) and an anti-immunoglobulin G (IgG)-antibody-coated or an uncoated FMNP. The chromatin behaved as a viscoelastic gel-like structure and the nucleoplasm was a softer viscoelastic structure at loading frequencies of 0.1–5 Hz. Protein diffusivity of the chromatin,nucleoplasm,and RNA polymerase II (RNA Pol II) and RNA Pol II activity were upregulated in a chromatin-stretching-dependent manner and stayed upregulated for tens of minutes after force cessation. Chromatin stiffness increased,but the mechanomemory duration of chromatin diffusivity decreased,with substrate stiffness. These findings may provide a mechanomemory mechanism of transcription upregulation and have implications on cell and nuclear functions. Graphical abstract In brief Rashid et al. show that chromatin and nucleoplasm in cells behave as viscoelastic materials. Chromatin stretching mediates the mechanomemory of chromatin and nucleoplasm diffusivity as well as of RNA polymerase II activity. The mechanomemory of RNA polymerase II activity provides a mechanism for sustained transcription upregulation tens of minutes after force cessation. View Publication

ABSTRACTLoss of Cdx2 in vivo leads to stunted development of the allantois,an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here,we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT),heterozygous (CDX2-Het),and homozygous null CDX2 (CDX2-KO) genotypes,differentiating these cells in a 2D gastruloid model,and subjecting these cells to single-nucleus RNA and ATAC sequencing,we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile,suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo,we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together,these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo. Summary: Using 2D human gastruloids,CDX2 is shown to dose-dependently influence genes related to tissue permeability,cell-cell adhesions,and cytoskeletal architecture during extraembryonic mesoderm development. View Publication