技术资料

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

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation,but their mitochondrial regulators are poorly understood. Here we report that,proline dehydrogenase (PRODH),a mitochondria-localized proline metabolism enzyme,is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901,a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy,DNA damage,and apoptosis. Remarkably,MitoQ,a mitochondria-targeted antioxidant,effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs,indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly,PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus,we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production,and thereby safeguarding naive pluripotency of hESCs. Synopsis Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. PRODH is expressed in naive hESCs at a higher level compared to their primed counterparts.MEK inhibitor present in naive culture media upregulates PRODH by suppressing MYC.PRODH depletion boosts mtOXPHOS and ROS production in naive hESCs.PRODH promotes proteolytic degradation of the ETC complex components. Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. View Publication

SummaryGenome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified a plethora of risk loci. However,the disease variants/genes and the underlying mechanisms remain largely unknown. For a strong AD-associated locus near Clusterin (CLU),we tied an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. We identified a putative causal SNP of CLU that impacts neuron-specific chromatin accessibility to transcription-factor(s),with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. Transcriptomic analysis and functional studies in induced pluripotent stem cell (iPSC)-derived neurons co-cultured with mouse astrocytes show that neuronal CLU facilitates neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes cause astrocytes to uptake less glutamate thereby altering neuron excitability. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions. View Publication

BackgroundMicroglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells,such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2),as the strongest risk factors for Alzheimer’s disease (AD) highlights the importance of microglial biology in the brain. The sequence,structure and function of several microglial proteins are poorly conserved across species,which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs).MethodsIn this study,we identified,produced,and tested novel,selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models,as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo.ResultsWe proved their efficacy in human iPSC microglia in vitro,as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-? plaques in vivo in a model of AD.ConclusionsThis study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00725-9. View Publication

Fluorescence microscopy has undergone rapid advancements,offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However,the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here,we introduce sHAPR,a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings,enabling acquisition at the maximum camera readout rate,typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems,including high-throughput flow cytometry,cardiomyocyte contraction,and neuronal calcium waves,using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities,opening up new avenues for investigating high-speed biological phenomena. The authors introduce a highspeed acquisition technique,sHAPR,for rapid exploration of biodynamics using fluorescence microscopy. The method leverages sCMOS cameras and custom fibre optics to convert microscopy images into 1D recordings,enabling acquisition at the maximum camera readout rate. View Publication

SummaryThe neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11–q13. A deletion of the SNORD116 cluster,encoding small nucleolar RNAs,or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS,respectively. By investigation of their subcellular localization,we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2,a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases,we identified protein interaction partners for WT or mutant MAGEL2,in particular the survival motor neuron protein (SMN),involved in spinal muscular atrophy,and the fragile-X-messenger ribonucleoprotein (FMRP),involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism,while regulation of transcription was mainly observed for WT MAGEL2. Hence,we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus,including the SNORD116 cluster. Thereby,we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116,SNORD115,and SNORD109A,as well as protein-coding genes MKRN3 and SNRPN,thus bridging the gap between PWS and SYS. Graphical abstract Mutations within MAGEL2 from chromosomal region 15q11–q13 cause Schaaf-Yang syndrome,which is phenotypically related to Prader-Willi syndrome,caused by deletion of the SNORD116 cluster within the same locus. We correlate mutations within MAGEL2 to spinal muscular atrophy and autism and also demonstrate its influence on the abundance of SNORD116. View Publication

Propionic acidemia (PA) is a metabolic disorder caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC) due to mutations in the PCCA or PCCB genes,which encode the two PCC subunits. PA may lead to several types of cardiomyopathy and has been linked to cardiac electrical abnormalities such as QT interval prolongation,life-threatening arrhythmias,and sudden cardiac death. To gain insights into the mechanisms underlying PA-induced proarrhythmia,we recorded action potentials (APs) and ion currents using whole-cell patch-clamp in ventricular-like induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from a PA patient carrying two pathogenic mutations in the PCCA gene (p.Cys616_Val633del and p.Gly477Glufs*9) (PCCA cells) and from a healthy subject (healthy cells). In cells driven at 1 Hz,PCC deficiency increased the latency and prolonged the AP duration (APD) measured at 20% of repolarization,without modifying resting membrane potential or AP amplitude. Moreover,delayed afterdepolarizations appeared at the end of the repolarization phase in unstimulated and paced PCCA cells. PCC deficiency significantly reduced peak sodium current (INa) but increased the late INa (INaL) component. In addition,L-type Ca2+ current (ICaL) density was reduced,while the inward and outward density of the Na+/Ca2+ exchanger current (INCX) was increased in PCCA cells compared to healthy ones. In conclusion,our results demonstrate that at the cellular level,PCC deficiency can modify the ion currents controlling cardiac excitability,APD,and intracellular Ca2+ handling,increasing the risk of arrhythmias independently of the progressive late-onset cardiomyopathy induced by PA disease. View Publication