技术资料

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

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

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons,resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells,grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids,confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons,while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity,trajectories,synaptic density,and communication pathways upon cell-cell contact,while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells,emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Mosaic organoids where pathological and healthy cells are grown together,reveal the rescue of phenotypes in pathological cells due to communication with healthy cells without harming them,as demonstrated by single-cell RNA-sequencing data. View Publication

Genomic imprinting controls parental allele-specific gene expression via epigenetic mechanisms. Abnormal imprinting at the GNAS gene causes multiple phenotypes,including pseudohypoparathyroidism type-1B (PHP1B),a disorder of multihormone resistance. Microdeletions affecting the neighboring STX16 gene ablate an imprinting control region (STX16-ICR) of GNAS and lead to PHP1B upon maternal but not paternal inheritance. Mechanisms behind this imprinted inheritance mode remain unknown. Here,we show that the STX16-ICR forms different chromatin conformations with each GNAS parental allele and enhances two GNAS promoters in human embryonic stem cells. When these cells differentiate toward proximal renal tubule cells,STX16-ICR loses its effect,accompanied by a transition to a somatic cell-specific GNAS imprinting status. The activity of STX16-ICR depends on an OCT4 motif,whose disruption impacts transcript levels differentially on each allele. Therefore,a biallelically active embryonic enhancer dictates GNAS imprinting via different chromatin conformations,underlying the allele-specific pathogenicity of STX16-ICR microdeletions. STX16 microdeletions cause pseudohypoparathyroidism type-1B only on the maternal allele. Here,the authors show that the allele-specific pathogenicity reflects differential conformations of a biallelically active enhancer dictating GNAS imprinting. View Publication

SummaryGABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs; generating embryoid bodies; and differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses.For complete details on the use and execution of this protocol,please refer to Pilotto et al.1 Graphical abstract Highlights•Steps described for generating GABAergic neurons from human iPSCs•Instructions for the enrichment of cerebellar GABAergic interneurons (cGNs)•Guide to calcium imaging of cGNs using genetically encoded calcium indicators Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. GABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human-cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs,and generating embryoid bodies,differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses. View Publication

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here,we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages,including yolk-sac (YS),AGM,fetal liver (FL),umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties,such as metabolism,self-renewal,differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly,we identified GRNs and key regulators controlling lymphoid potentiality,self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore,GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-024-00192-z. View Publication

BackgroundELMSAN1 (ELM2?SANT domain?containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex),playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However,the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage,we employed human?induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1.Methods and ResultsWe generated ELMSAN1?deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation,ELMSAN1 depletion inhibited pluripotency deactivation,decreased the expression of cardiac?specific markers,and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure,calcium handling,and ion channels was also noted in ELMSAN1?deficient cardiomyocytes derived from hiPSCs. Additionally,through a series of structural and functional assessments,we found that ELMSAN1?null hiPSC cardiomyocytes are immature,exhibiting incomplete sarcomere Z?line structure,decreased calcium handling,and impaired electrophysiological properties. Of note,we found that the cardiac?specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights,indicating maturation reduction with the energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes.ConclusionsIn this study,we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation,including cardiac differentiation,sarcomere formation,calcium handling,electrophysiological maturation,and proliferation. View Publication

IntroductionDual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal VGF gene network that regulates late-onset Alzheimer’s disease (AD). Importantly,decreased DUSP6 levels are correlated with an increased clinical dementia rating (CDR) in human subjects,and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model.MethodsTo investigate the role of DUSP6 in AD,we stereotactically injected AAV5-DUSP6 or AAV5-GFP (control) into the dorsal hippocampus (dHc) of both female and male 5xFAD or wild type mice,to induce overexpression of DUSP6 or GFP.ResultsBarnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load,Aß1–40 and Aß1–42 levels,and amyloid precursor protein processing enzyme BACE1,in male but not in female mice. Microglial activation,which was increased in 5xFAD mice,was significantly reduced by dHc DUSP6 overexpression in both males and females,as was the number of “microglial clusters,” which correlated with reduced amyloid plaque size. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulation of inflammatory and extracellular signal-regulated kinase pathways,while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. Gene ontology analysis of DEGs (p < 0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD.DiscussionIn summary,DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice,whereas reduced neuroinflammation and microglial activation were observed in both males and females,suggesting that DUSP6-induced reduction of microglial activation did not contribute to sex-dependent improvement in memory deficits. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression,however,correlated with the improvement of spatial memory deficits in male but not female 5xFAD. View Publication

Brain tumors are commonly treated with radiotherapy,but the efficacy of the treatment is limited by its toxicity to the normal tissue including post-irradiation contrast enhanced lesions often linked to necrosis. The poorly understood mechanisms behind such brain lesions were studied using cerebral organoids. Here we show that irradiation of such organoids leads to dose-dependent growth retardation and formation of liquid-filled cavities but is not correlated with necrosis. Instead,the radiation-induced changes comprise of an enhancement of cortical hem markers,altered neuroepithelial stem cell differentiation,and an increase of ZO1+/AQP1+/CLDN3+-choroid plexus (CP)-like structures accompanied by an upregulation of IGF2 mRNA,known to be expressed in CP and cerebrospinal fluid. The altered differentiation is attributed to changes in the WNT/BMP signaling pathways. We conclude that aberrant CP formation can be involved in radiation-induced brain lesions providing additional strategies for possible countermeasures. Human cerebral organoids provide insights into mechanisms behind the formation of choroid plexus (CP)-like structures that may contribute to radiation-induced brain image changes. View Publication

Cathepsin B (CatB),a protease in endosomal and lysosomal compartments,plays a key role in neuronal protein processing and degradation,but its function in brain development remains unclear. In this study,we found that CatB is highly expressed in the cortex of E12.5–E16.5 mice. Morphological analysis revealed significant defects in cortical development in CatB knockout (KO) mice,particularly in layer 6. In vitro experiments showed that CatB deficiency notably impaired neuronal migration and development. Behaviorally,CatB KO mice displayed prominent depressive-like behaviors,and electrophysiological recordings demonstrated significantly reduced neuronal activity in layer 6 of the medial prefrontal cortex. Mechanistically,proteomics analysis revealed that CatB KO affected neuronal migration and axonal growth,and decreased the expression of key transcription factors involved in neuronal development,particularly PEG3. Deficiency of PEG3 also significantly impaired neuronal migration and development. Our findings uncover a role for CatB in cortical development and suggest a mechanism linking CatB deficiency with depression and developmental defects through the destabilization of PEG3. Cathepsin B (CatB) is essential for cortical development. Its deficiency impairs neuronal migration,reduces PEG3 expression,and leads to layer 6 defects and depression-like behaviors,revealing a novel link between CatB and brain development. View Publication

ObjectiveGene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes,NDM) can provide unique insights into the development and function of human pancreatic beta-cells.MethodsWe performed genome sequencing in a cohort of 43 consanguineous individuals with NDM in whom all the known genetic causes had previously been excluded. We used quantitative PCR and RNA-sequencing in CRISPR-edited human induced pluripotent stem cells (iPSCs),and CUT&RUN-sequencing in EndoC-?H1 cells to investigate the effect of PAX4 loss on human pancreatic development.ResultsWe describe the identification of homozygous PAX4 loss-of-function variants in 2 individuals with transient NDM: a p.(Arg126?) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126?) variant causes nonsense mediated decay in CRISPR-edited iPSC-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN-sequencing in EndoC-?H1 cells and RNA-sequencing in PAX4-depleted islet stem cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion.ConclusionWe report the first human cases of complete loss of PAX4,establishing it as a novel cause of NDM and highlighting its role in human beta cell development. Both probands had transient NDM which remitted in early infancy but relapsed at the ages of 2.4 and 6.7 years,demonstrating that in contrast to mouse models,PAX4 is not essential for the development of human pancreatic beta-cells. Highlights•Homozygous loss-of-function variants in PAX4 are a novel genetic cause of transient neonatal diabetes.•PAX4 directly regulates genes involved in pancreatic beta cell development and glucose-sensitive insulin secretion.•The role of PAX4 in humans differs to that observed in mouse and is not essential for beta cell development. View Publication

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further,costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research,and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research,in platforms conducive to scale-up for clinical manufacturing.Image,graphical abstract View Publication