技术资料

Sac1 is a conserved phosphoinositide phosphatase,whose loss-of-function compromises cell and organism viability. Here,we employ acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 is degraded in ~1 h,paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour,and superseded by Golgi fragmentation,impaired glycosylation,and selective degradation of TGN proteins by ~4 h. The TGN disintegration results from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically,Sac1 mediated TGN membrane composition maintains an assembly-promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation are recapitulated in human differentiated trophoblasts,causing processing defects of chorionic gonadotropin,in line with loss-of-function intolerance of the human SACM1L gene. Collectively,our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange. This study employs auxin-inducible degradation of Sac1. The authors reveal that acute Sac1 depletion changes the Golgi membrane lipid composition,causing disassembly of the Golgi V-ATPase and eventually resulting in cargo processing defects. View Publication

BackgroundA growing body of evidence from primate embryos as well as in vitro systems supports the notion that amnion and primordial germ cell (PGC) lineage progressing cells share a common precursor.ResultsTo gain comprehensive transcriptomic insights into this critical but poorly understood precursor and its progeny,we examine the evolving transcriptome of a developing human pluripotent stem cell-derived model of amnion and PGC formation at the single cell level. This analysis reveals several continuous amniotic fate progressing states with state-specific markers. Additionally,a progenitor-like cell,that displays bi-potential characteristics for amnion and PGC-like cell lineages and is marked by CLDN10,is identified. Strikingly,we find that expression of CLDN10 is restricted to the amnion-epiblast boundary region in our human post-implantation amniotic sac model as well as in peri-gastrula cynomolgus macaque embryos; moreover,this boundary region presents amnion and PGC progenitor-like transcriptional characteristics. Furthermore,our loss of function analysis shows that CLDN10 promotes amniotic but suppresses PGC-like fate.ConclusionsOverall,based on the single cell transcriptomic resource in this study,we identify a CLDN10+ amnion and PGC progenitor-like population at the amnion-epiblast boundary of the primate peri-gastrula,and present additional molecular clues as to how amnion and PGC may be formed at the amnion-epiblast boundary in human peri-gastrula. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03751-y. View Publication

Immune rejection is one of the most serious challenges in allogeneic transplantation,including allogeneic induced pluripotent stem cell (allo-iPSC)-derived cell therapy. Beta-2-Microglobulin gene-knockout,human leukocyte antigen (HLA) class I-deficient iPSCs can evade immune rejection by host T cells,which occurs due to HLA mismatches. However,natural killer (NK) cells recognize HLA class Ⅰ-deficient cells and reject them,which is known as the missing-self response. Introducing chimeric HLA-E protein to HLA class Ⅰ-deficient iPSCs suppresses the missing-self response of NK cells expressing the inhibitory receptor NKG2A; however,technology to suppress NKG2A-negative NK cells is still required. Here,we developed novel agonists for the other inhibitory receptor,killer immunoglobulin receptor (KIR),on NK cells. We found that antibodies that bind to activating KIR enhance NK cell activation and developed selective agonists for inhibitory KIRs (KIR2DL1,KIR2DL2/3,and KIR3DL1). Introducing these selective inhibitory KIR agonists on T cells and HLA class Ⅰ-deficient iPSCs allowed them to evade immune rejection by NK cells. Additionally,we identified an NKG2A-selective agonist as an alternative to chimeric HLA-E,which stimulates the activating receptor NKG2C. This technology enhances immune tolerance in allo-iPSCs and facilitates the development of various iPSC-derived regenerative medicines. The online version contains supplementary material available at 10.1038/s41598-025-18394-z. Subject terms: Allotransplantation,NK cells View Publication

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD,though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD,while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST,amyloid precursor protein (APP),and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST,APP,and SNCA expression,pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases,we identified that miR‐153‐3p,as a master regulator,reduced a group of neurodegeneration related proteins: REST,amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC),while REST,by contrast,is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance. View Publication

Type 1 interferons (IFN-Is) exhibit significant antiviral,antitumor,and immunoregulatory properties,demonstrating substantial therapeutic potential. However,IFN-Is are pleiotropic cytokines,and the available data on their effect under specific pathological conditions are inconclusive. Furthermore,the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types,including human immune cells. In this study,we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4,SOX2,TRA 1-60,and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline,all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line,126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types,as well as for generating different types of cells with controllable IFN-β expression. View Publication

Seven female individuals with multiple congenital anomalies,developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T,p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined,and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways,we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling,transcriptomics and protein analysis,comparing healthy and variant cells. When comparing the two cell lines,we identified altered gene expression in neural cells expressing the variant,including genes regulating RNA polymerase II activity,transcription,pre-mRNA processing,and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development,forebrain differentiation,and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model,expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5. View Publication

IntroductionExposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species,along with the financial and ethical challenges associated with animal testing,the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously,we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay,ReproTracker,that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.MethodsTo further increase the predictability of the assay in identifying potential teratogens,we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens,including the compounds listed in the ICH S5 reference list.ResultsThe optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall,based on the current dataset,the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%),when compared to the previously described assay.DiscussionIn summary,trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker,making the assay an invaluable tool for early in vitro teratogenicity screening. View Publication

Prion diseases,such as sporadic Creutzfeldt-Jakob Disease (sCJD),are neurodegenerative disorders caused by misfolding of the prion protein (PrP). The D178N mutation in the PrP gene causes Fatal Familial Insomnia (FFI). Here we show that both sCJD and FFI prions can infect human cerebral organoids with or without the D178N mutation,and that the resulting infection is dictated by the inoculating prion and not the host organoid genotype. View Publication

Biomolecular condensates are found throughout a diversity of eukaryotic cell types and cellular compartments,playing roles in various cellular functions. A given protein generally forms functionally and compositionally heterogeneous condensates,but the underlying regulatory mechanisms are unknown. Here,we found that different RNA motifs modulate the formation of heterogeneous mRNA-protein condensates via riboregulation. Fragile X-related 1 (FXR1),an RNA-binding protein interacting with nuclear pores,assembles distinct localized subcellular mRNP condensates linked to cytosolic accumulation of G-quadruplex-containing pluripotent mRNAs and the localized translation of nucleoporin mRNAs at nuclear pores. The diverse locations of FXR1 condensates depend on the unique RNA-protein interaction modules of its two RNA binding domains,and the opposing effects of different RNA motifs on the affinity of FXR1 for nuclear pores. Notably,reduced FXR1 levels and impaired nuclear pore function lead to the nuclear accumulation of transcribed RNAs,facilitating fate transition in human embryonic stem cells. Preventing this decline would result in impaired hESC differentiation. Subject terms: RNA metabolism,Embryonic stem cells,RNA,RNA transport View Publication

The primary cilium is a signal transduction organelle whose dysfunction clinically causes ciliopathies in humans. RAB23 is a small GTPase known to regulate the Hedgehog signalling pathway and ciliary trafficking. Mutations of RAB23 in humans lead to Carpenter syndrome (CS),an autosomal recessive disorder clinically characterized by craniosynostosis,polysyndactyly,skeletal defects,obesity,and intellectual disability. Although the clinical features of CS bear some resemblance to those of ciliopathies,the exact relationship between the pathological manifestations of CS and the ciliary function of RAB23 remains ambiguous. Besides,the in vivo ciliary functions of RAB23 remain poorly characterised. Here,we demonstrate in vivo and in vitro Rab23 loss-of-function mutants modelling CS,including Rab23 conditional knockout (CKO) mouse mutants,CS patient-derived induced pluripotent stem cells (iPSCs),and zebrafish morphants. The Rab23-CKO mutants exhibit multiple developmental and phenotypical traits recapitulating the clinical features of human ciliopathies and CS,indicating a causal link between the loss of Rab23 and ciliopathy. In line with the ciliopathy-like phenotypes,all three different vertebrate mutant models consistently show a perturbation of primary cilia formation,intriguingly,in a context-dependent manner. Rab23-CKO mutants reveal cell-type specific ciliary abnormalities in chondrocytes,mouse embryonic fibroblasts,neural progenitor cells and neocortical neurons,but not in epithelial cells,cerebellar granule cells and hippocampus neurons. A profound reduction in ciliation frequency was observed specifically in neurons differentiated from CS patient iPSCs,whereas the patients’ fibroblasts,iPSCs and neural progenitor cells maintained normal ciliation percentages but shortened cilia length. Furthermore,Rab23-KO neural progenitor cells show perturbed ciliation and desensitized to primary cilium-dependent activation of the Hedgehog signaling pathway. Collectively,these findings indicate that the absence of RAB23 causes dysfunctional primary cilia in a cell-type distinctive manner,which underlies the pathological manifestations of CS. Our findings present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. Through the use of patient-derived iPSCs differentiated cells,we present direct evidence of primary cilia anomalies in CS,thereby confirming CS as a ciliopathy disorder. Author summaryRAB23 mutations lead to Carpenter syndrome (CS),which manifests multiple clinical features resembling those of ciliopathies,a spectrum of disorders caused by defective primary cilia. However,the in vivo ciliary functions of RAB23 remain ambiguous. We established multiple Rab23 loss-of-function mutants,including conditional knockout (CKO) mouse mutants,CS patient-derived induced pluripotent stem cells (iPSCs),and zebrafish morphants. These mutant models consistently show context-dependent primary cilia anomalies. Rab23-CKO mutants display profound ciliary abnormalities in neocortical neurons,but not in epithelial cells,cerebellar granule cells and hippocampus neurons. Aberrant cilia formation and shortened cilia were observed in the neurons and neural progenitor cells derived from CS patient iPSCs. Furthermore,Rab23-KO neural progenitor cells exhibit impeded primary cilium-dependent Hedgehog signaling pathway transduction. Our findings suggest that the cell-type distinctive dysfunctional primary cilia may underlie the pathological manifestations of CS. We present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. The results from patient-derived iPSCs differentiated cells reveal direct evidence of primary cilia anomalies in CS,confirming CS as a ciliopathy disorder. View Publication

The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4,Sox2,Klf4,and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and have the potential to differentiate into any adult human cell type,offering a versatile tool for research and potential therapeutic applications. However,the efficiency of differentiation protocols for generating complex structures with multiple cell types,Like kidney organoids,remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using the stepwise 2D monolayer-based protocol developed by Morizane et al. 2017. We found that treating hiPSCs with 1–2% DMSO affects gene expression of pluripotent transcription factors,the epigenetic landscape,and hiPSC colony morphology. Our findings also suggest DMSO treatment enhances the expression of the key metanephric mesenchyme nephron progenitor marker,SIX2 after 9 days of kidney organoid differentiation and helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to fully elucidate the mechanisms underlying these effects and refine the differentiation process for potential in vitro research applications in biomedical research and drug development. View Publication

Disturbances within the cerebrovascular system substantially contribute to the pathogenesis of age-related cognitive impairment and Alzheimer’s disease (AD). Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-β (Aβ) in the leptomeningeal and cortical arteries and is highly prevalent in AD,affecting over 90% of cases. While the ε4 allele of apolipoprotein E ( APOE ) represents the strongest genetic risk factor for AD,it is also associated with cerebrovascular dysregulations. APOE plays a crucial role in brain lipid transport,particularly in the trafficking of cholesterol and phospholipids. Lipid metabolism is increasingly recognized as a critical factor in AD pathogenesis. However,the precise mechanism by which APOE influences cerebrovascular lipid signatures in AD brains remains unclear. In this study,we conducted non-targeted lipidomics on cerebral vessels isolated from the middle temporal cortex of 89 postmortem human AD brains,representing varying degrees of CAA and different APOE genotypes: APOE ε2/ε3 (N = 9),APOE ε2/ε4 (N = 14),APOE ε3/ε3 (N = 21),APOE ε3/ε4 (N = 23),and APOE ε4/ε4 (N = 22). Lipidomics detected 10 major lipid classes with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) being the most abundant lipid species. While we observed a positive association between age and total acyl-carnitine (CAR) levels (p = 0.0008),the levels of specific CAR subclasses were influenced by the APOE ε4 allele. Notably,APOE ε4 was associated with increased PE (p = 0.049) and decreased sphingomyelin (SM) levels (p = 0.028) in the cerebrovasculature. Furthermore,cerebrovascular Aβ40 and Aβ42 levels showed associations with sphingolipid levels including SM (p = 0.0079) and ceramide (CER) (p = 0.024). Weighted correlation network analysis revealed correlations between total tau and phosphorylated tau and lipid clusters enriched for PE plasmalogen and lysoglycerophospholipids. Taken together,our results suggest that cerebrovascular lipidomic profiles offer novel insights into the pathogenic mechanisms of AD,with specific lipid alterations potentially serving as biomarkers or therapeutic targets for AD. The online version contains supplementary material available at 10.1007/s00401-025-02949-5. View Publication