产品说明

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity,which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular,photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs),undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy,however its role in photoreceptor maturation and function is unknown. In this study retinal organoids,a valuable model system,were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR,western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%,abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone,inner segment and connecting cilium basal body markers,increased expression of genes associated with stress response and the ubiquitin-proteasome system,and downregulation of autophagy. Importantly,the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context,opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression,corroborating the pathogenesis of late-onset mild maculopathy. View Publication

Haloperidol is a typical antipsychotic used to treat schizophrenia and induces dopamine D2 receptor antagonism. Long-term use of haloperidol can reduce brain size in animals and humans; however,the underlying mechanism of this effect remains unclear. Notch1 signaling regulates the development and function of the nervous system by balancing stem cell proliferation and differentiation. Therefore,we investigated the effects of long-term exposure to haloperidol on human-derived brain organoids,which served as sophisticated in vitro models of human brain development. Long-term exposure to haloperidol reduced the size of brain organoids and decreased the ventricular zone and Notch1 signaling. When propionate,which protects against haloperidol-induced toxicity,was combined with haloperidol,it rescued both the overall size of brain organoids and Notch1 expression levels. Additionally,treatment with valproic acid,a Notch1 activator,partially restored the size of brain organoids and the thickness of the ventricular layer. Taken together,these data suggest that long-term exposure to haloperidol impairs neurodevelopment via Notch1 signaling in brain organoids. These findings contribute to our understanding of antipsychotic drug safety and provide information for new neurodevelopmental toxicity assessments.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-08855-w. View Publication

SummaryRibosomopathies arise from the disruptions in ribosome biogenesis within the nucleolus,which is organized via liquid-liquid phase separation (LLPS). The roles of LLPS in ribosomopathies remain poorly understood. Here,we generated human induced pluripotent stem cell (hiPSC) models of ribosomopathy caused by mutations in small nucleolar RNA (snoRNA) gene SNORD118. Mutant hiPSC-derived neural progenitor cells (NPCs) or neural crest cells (NCCs) exhibited ribosomopathy hallmark cellular defects resulting in reduced organoid growth,recapitulating developmental delay in patients. SNORD118 mutations in NPCs disrupted nucleolar morphology and LLPS properties coupled with impaired ribosome biogenesis and a translational downregulation of fibrillarin (FBL),the key LLPS effector acting via the intrinsically disordered region (IDR) motif. IDR-depleted FBL failed to rescue NPC defects,whereas a chimeric FBL with swapped IDR motif from an unrelated protein mitigated ribosomopathy and organoid growth defects. Thus,SNORD118 human iPSC models revealed aberrant phase separation and nucleolar functions as potential pathogenic mechanisms in ribosomopathies. Graphical abstract Highlights•SNORD118 mutant hiPSC-derived cells and organoids recapitulate the ribosomopathy defects•Mutations impair ribosome biogenesis and translation of phase separation effector FBL•Phase separation and nucleolar organization are defective in SNORD118 mutant cells•Impaired phase separation causes ribosomopathy and growth defects in hiPSC models Natural sciences; Biological sciences; Cell biology; Stem cell research View Publication

Mutations in FUS and TARDBP cause amyotrophic lateral sclerosis (ALS),but the precise mechanisms of selective motor neuron degeneration remain unresolved. To address if pathomechanisms are shared across mutations and related to either gain- or loss-of-function,we performed single-cell RNA sequencing across isogenic induced pluripotent stem cell-derived neuron types,harbouring FUS P525L,FUS R495X,TARDBP M337V mutations or FUS knockout. Transcriptional changes were far more pronounced in motor neurons than interneurons. About 20% of uniquely dysregulated motor neuron transcripts were shared across FUS mutations,half from gain-of-function. Most indicated mitochondrial impairments,with attenuated pathways shared with mutant TARDBP M337V as well as C9orf72-ALS patient motor neurons. Mitochondrial motility was impaired in ALS motor axons,even with nuclear localized FUS mutants,demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS,uncoupled from protein mislocalization. These early mitochondrial dysfunctions unique to motor neurons may affect survival and represent therapeutic targets in ALS. In this study,the authors performed single-cell RNA-sequencing across various isogenic mutant FUS and TDP43 neurons. Mitochondrial dysfunction emerged as pathway unique to motor neurons demonstrating shared toxic gain of-function mechanisms,uncoupled from protein mislocalization. View Publication

Lateral Meningocele Syndrome (LMS),a disorder associated with NOTCH3 pathogenic variants,presents with neurological,craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs,induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs,NOTCH36692-93insC and isogenic controls,free of chromosomal aberrations as determined by human CytoSNP850 array,were cultured under conditions of neural crest,mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1,HEY1,2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL,BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70–80% in mesenchymal cells and by 45–55% in osteogenic cells and NOTCH3 mRNA by 15–30% and 20–40%,respectively. In conclusion,a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS. View Publication

ABSTRACTProneural genes are conserved drivers of neurogenesis across the animal kingdom. How their functions have adapted to guide human-specific neurodevelopmental features is poorly understood. Here,we mined transcriptomic data from human fetal cortices and generated from human embryonic stem cell-derived cortical organoids (COs) to show that NEUROG1 and NEUROG2 are most highly expressed in basal neural progenitor cells,with pseudotime trajectory analyses indicating that NEUROG1-derived lineages predominate early and NEUROG2 lineages later. Using ChIP-qPCR,gene silencing and overexpression studies in COs,we show that NEUROG2 is necessary and sufficient to directly transactivate known target genes (NEUROD1,EOMES,RND2). To identify new targets,we engineered NEUROG2-mCherry knock-in human embryonic stem cells for CO generation. The mCherry-high CO cell transcriptome is enriched in extracellular matrix-associated genes,and two genes associated with human-accelerated regions: PPP1R17 and FZD8. We show that NEUROG2 binds COL1A1,COL3A1 and PPP1R17 regulatory elements,and induces their ectopic expression in COs,although NEUROG2 is not required for this expression. Neurog2 similarly induces Col3a1 and Ppp1r17 in murine P19 cells. These data are consistent with a conservation of NEUROG2 function across mammalian species. Summary: Analysis of human cortical organoids reveals that NEUROG1 lineages prevail early and NEUROG2 lineages later,and that NEUROG2 targets include COL genes and PPP1R17,a human-accelerated region-associated gene. View Publication

SummaryNeonatal mouse hearts have transient renewal capacity,which is lost in juvenile and adult stages. In neonatal mouse hearts,myocardial infarction (MI) causes an initial loss of cardiomyocytes. However,it is unclear which type of regulated cell death (RCD) occurs in stressed cardiomyocytes. In the current studies,we induced MI in neonatal and juvenile mouse hearts and showed that ischemic cardiomyocytes primarily undergo ferroptosis,a non-apoptotic and iron-dependent form of RCD. We demonstrated that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine effects and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. The fibrogenic activity of CFs,typically considered detrimental to heart function,is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition,Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI. Graphical abstract Highlights•Neonatal and juvenile mouse cardiomyocytes mainly undergo ferroptosis after MI•Cardiac fibroblasts protect cardiomyocytes through paracrine effect•Cardiac fibroblasts interact with cardiomyocytes to share iron burden•Pitx2 pathway protects cardiomyocytes from ferroptosis and controls fibrosis Cardiovascular medicine; Physiology; Cell biology View Publication

BackgroundEmbryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs,which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation. How this primed enhancer state is established and maintained and how it affects the regulation of developmental gene networks remains poorly understood.ResultsHere,we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of postgastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation,marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases,lineage-specific enhancers are epigenetically marked already in the zygote,weeks before their activation during lineage specification. Moreover,we outline a generalizable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function.ConclusionsOur findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03658-8. View Publication

Epicardium,the most outer mesothelium,exerts crucial functions in fetal heart development and adult heart regeneration. Here we use a three-step manipulation of WNT signalling entwined with BMP and RA signalling for generating a self-organized epicardial organoid that highly express with epicardium makers WT1 and TCF21 from human embryonic stem cells. After 8-days treatment of TGF-beta following by bFGF,cells enter into epithelium-mesenchymal transition and give rise to smooth muscle cells. Epicardium could also integrate and invade into mouse heart with SNAI1 expression,and give birth to numerous cardiomyocyte-like cells. Single-cell RNA seq unveils the heterogeneity and multipotency exhibited by epicardium-derived-cells and fetal-like epicardium. Meanwhile,extracellular matrix and growth factors secreted by epicardial organoid mimics the ecology of subepicardial space between the epicardium and cardiomyocytes. As such,this epicardial organoid offers a unique ground for investigating and exploring the potential of epicardium in heart development and regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01339-w. View Publication

SUMMARY GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology,we deleted EXOC2,which encodes an essential exocyst subunit,in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2,suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to,surprisingly,the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA,making it a potential therapeutic target in C9ORF72-ALS/FTD. In brief Halim et al. deleted the gene EXOC2 from patient stem cells and then differentiated them into motor neurons. They found that several amyotrophic lateral sclerosis-related phenotypes were rescued in patient neurons when EXOC2 was deleted or knocked down by a drug. This study identifies EXOC2 as a potential therapeutic target. Graphical Abstract 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

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