技术资料

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

SUMMARYUnderstanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here,we present COMET,a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins,containing two to six effector domains,allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly,we keep full proteins or domains intact,maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription,we demonstrate cell type specific transcriptional modulation,and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering,emphasizing COMET as both a method for transcriptional effector discovery,and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry. View Publication

Ribosome profiling,which is based on deep sequencing of ribosome footprints,has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However,the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here,we overcome these hurdles through the development of “Ribo-FilterOut”,which is based on the separation of footprints from ribosome subunits by ultrafiltration,and “Ribo-Calibration”,which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript,the translation initiation rate,and the overall number of translation events before its decay,all in a genome-wide manner. Moreover,our method reveals the allocation of ribosomes under heat shock stress,during aging,and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome. Ribosome profiling faces issues with rRNA contamination and measurements of ribosome numbers on transcripts. Here,the authors develop Ribo-FilterOut and Ribo-Calibration,methods which can be used to estimate kinetic and stoichiometric parameters of translation under various conditions. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication

Huntington’s disease and other disorders of the basal ganglia create challenges for biomolecule-based medicines given the poor accessibility of these deep brain structures following intracerebral or intravascular delivery. Here,we found that low dose,low volume delivery of unbiased AAV libraries into the globus pallidus allowed recovery of novel capsids capable of broad access to key deep brain and cortical structures relevant for human therapies. One such capsid,AAV-DB-3,provided transduction of up to 45% of medium spiny neurons in the adult NHP striatum,along with substantial transduction of relevant deep layer neurons in the cortex. Notably,AAV-DB-3 behaved similarly in mice as in NHPs and potently transduced human neurons derived from induced pluripotent stem cells. Thus,AAV-DB-3 provides a unique AAV for network level brain gene therapies that translates up and down the evolutionary scale for preclinical studies and eventual clinical use. To date,brain gene therapies require high vector doses. Here,authors devised an AAV capsid screen and found variants with unprecedented potency for transduction of deep brain and cortical neurons and human iPSC-neurons with cell tropism relevant for Huntington’s and Parkinson’s disease. View Publication

AbstractBackgroundParkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra,which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed,a decrease in Treg number and function has been reported in PD. In this context,pramipexole,a dopaminergic receptor agonist used to treat PD symptoms,has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells.MethodsTreg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28?activated and non?activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co?cultured with in vitro?differentiated human dopaminergic neurons in a cytotoxicity assay with 6?hydroxydopamine (6?OHDA). The role of interleukin?10 (IL?10) was investigated by co?culturing activated IL?10?producing Treg cells with neurons. To further investigate the effect of treatment on Tregs,gene expression in pramipexole?treated,CD3/CD28?activated Treg cells was determined by Fluidigm analysis.ResultsPramipexole?treated CD3/CD28?activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6?OHDA. Pramipexole?treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL?10 release and the activation of genes associated with regulation and neuroprotection.ConclusionAnti?CD3/CD28?activated Treg cells protect dopaminergic neurons against 6?OHDA?induced damage. In addition,activated,IL?10?producing,pramipexole?treated Tregs also induced a neuroprotective effect,and the supernatants of these co?cultures promoted axonal growth. Pramipexole?treated,activated Tregs altered their gene expression in a concentration?dependent manner,and enhanced TGF??related dopamine receptor regulation and immune?related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD. Pramipexole?treated,activated regulatory T cells protect dopaminergic neurons against 6 OHDA damage and promote primary neurite length. This could be due to the production of the regulatory cytokine IL?10 and an increased expression of genes related to regulation and neuroprotection. View Publication

Patient specific induced pluripotent stem cells (iPSCs) derived ? cells represent an effective means for disease modeling and autologous diabetes cell replacement therapy. In this study,an AG73-5%gelatin methacryloyl (GelMA) /2% alginate methacrylate (AlgMA) hydrogel was employed to generate pancreatic progenitor (PP) organoids and improve stem cell-derived ? (SC-?) cell differentiation protocol. The laminin-derived homolog AG73,which mimics certain cell?matrix interactions,facilitates AKT signaling pathway activation to promote PDX1+/NKX6.1+ PP organoid formation and effectively modulates subsequent epithelial–mesenchymal transition (EMT) in the endocrine lineage. The 5%GelMA/2%AlgMA hydrogel mimics the physiological stiffness of the pancreas,providing the optimal mechanical stress and spatial structure for PP organoid differentiation. The Syndecan-4 (SDC4)-ITGAV complex plays a pivotal role in the early stages of pancreatic development by facilitating the formation of SOX9+/PDX1+ bipotent PPs. Our findings demonstrate that AG73-GelMA/AlgMA hydrogel-derived SC-? cells exhibit enhanced insulin secretion and accelerated hyperglycemia reversal in vivo. This study presents a cost-effective,stable,and efficient alternative for the comprehensive 3D culture of SC-? cells in vitro by mitigating the uncertainties associated with conventional culture methods. View Publication

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous,a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult,resulting in inaccuracies. In this study,we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts,human induced PSCs,and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time,with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2?4 and 2?3 hours after starvation,respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs,focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types,including undifferentiated PSCs,differentiated cells,and cells undergoing cellular reprogramming,and addresses critical issues,such as differences in basal metabolic levels and sensitivity to normalization,providing valuable insights into cellular energetics. View Publication

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia,platelet dysfunction,and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation,the DNA methylation status in FPDMM remains unknown,largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here,using genome editing techniques,we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks),consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs,with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1,an ETS family member,was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation,and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation. View Publication

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However,the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation,the determinants and functional consequences have largely remained unclear. Here,we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally,our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis. Synopsis Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. Dynamic changes in RNA and protein levels are strongly correlated during all stages of neuronal differentiation.Nanopore sequencing (ONT-seq) during human neurogenesis reveals 12,019 non-annotated RNA isoforms,a large number of which are differentially expressed during differentiation.70% of new RNA isoforms result from the use of alternative transcription start sites (TSSs) or polyadenylation (pA) sites and exon skipping.RNA isoform changes underlie protein isoform changes during human neurogenesis as revealed by integrating ONT-seq,RNA-seq and proteomics time course data.RNA motif enrichment,RNA-seq and available CLIP-seq data uncover a set of RNA binding proteins (RBPs) as potential determinants of differentiation stage-specific global isoform changes. Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. View Publication

Advancing cardiac tissue engineering requires innovative fabrication techniques,including 3D bioprinting and tissue maturation,to enable the generation of new muscle for repairing or replacing damaged heart tissue. Recent advances in tissue engineering have highlighted the need for rapid,high-resolution bioprinting methods that preserve cell viability and maintain structural fidelity. Traditional collagen-based bioinks gel slowly,limiting their use in bioprinting. Here,we implement TRACE (tunable rapid assembly of collagenous elements),a macromolecular crowding-driven bioprinting technique that enables the immediate gelation of collagen bioinks infused with cells. This overcomes the need for extended incubation,allowing for direct bioprinting of engineered cardiac tissues with high fidelity. Unlike methods that rely on high-concentration acidic collagen or fibrin for gelation,TRACE achieves rapid bioink stabilization without altering the biochemical composition. This ensures greater versatility in bioink selection while maintaining functional tissue outcomes. Additionally,agarose slurry provides stable structural support,preventing tissue collapse while allowing nutrient diffusion. This approach better preserves complex tissue geometries during culture than gelatin-based support baths or polydimethylsiloxane (PDMS) molds. Our results demonstrate that TRACE enables the bioprinting of structurally stable cardiac tissues with high resolution. By supporting the fabrication of biomimetic tissues,TRACE represents a promising advancement in bioprinting cardiac models and other engineered tissues. View Publication

Copy number variations of the human CNTN6 gene,resulting from megabase-scale microdeletions or microduplications in the 3p26.3 region,are frequently implicated in neurodevelopmental disorders such as intellectual disability and developmental delay. However,duplication of the full-length human CNTN6 gene presents with variable penetrance,resulting in phenotypes that range from neurodevelopmental disorders to no visible pathologies,even within the same family. Previously,we obtained a set of induced pluripotent stem cell lines derived from a patient with a CNTN6 gene duplication and from two healthy donors. Our findings demonstrated that CNTN6 expression in neurons carrying the duplication was significantly reduced. Additionally,the expression from the CNTN6 duplicated allele was markedly lower compared to the wild-type allele. Here,we first introduce a system for correcting megabase-scale duplications in induced pluripotent stem cells and secondly analyze the impact of this correction on CNTN6 gene expression. We showed that the deletion of one copy of the CNTN6 duplication did not affect the expression levels of the remaining allele in the neuronal cells. View Publication