技术资料

Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases,thereby contributing to cognitive impairment. Among other factors,DNA damage strongly promotes cellular aging,however,the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro,ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype expression,reduced BBB integrity,and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line,EC-KO mice showed more P21+ cells,augmented expression of angiogenic markers,and a concomitant increase in the number of brain ECs and pericytes. Moreover,EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter,suggesting a regional susceptibility. Collectively,our results underline the role of endothelial aging as a driver of impaired BBB function,endothelial sprouting,and increased immune cell migration into the brain,thereby contributing to impaired brain homeostasis as observed during the aging process. View Publication

Organoids are self-assembled 3D cellular structures that resemble organs structurally and functionally,providing in vitro platforms for molecular and therapeutic studies. Generation of organoids from human cells often requires long and costly procedures with arguably low efficiency. Prediction and selection of cellular aggregates that result in healthy and functional organoids can be achieved by using artificial intelligence-based tools. Transforming images of 3D cellular constructs into digitally processable data sets for training deep learning models requires labeling of morphological boundaries,which often is performed manually. Here,we report an application named OrganoLabeler,which can create large image-based data sets in a consistent,reliable,fast,and user-friendly manner. OrganoLabeler can create segmented versions of images with combinations of contrast adjusting,K-means clustering,CLAHE,binary,and Otsu thresholding methods. We created embryoid body and brain organoid data sets,of which segmented images were manually created by human researchers and compared with OrganoLabeler. Validation is performed by training U-Net models,which are deep learning models specialized in image segmentation. U-Net models,which are trained with images segmented by OrganoLabeler,achieved similar or better segmentation accuracies than the ones trained with manually labeled reference images. OrganoLabeler can replace manual labeling,providing faster and more accurate results for organoid research free of charge. View Publication

BackgroundChronic ischemic limb disease often leads to amputation,which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases,but studies with primary human SMCs have been limited by a lack of availability. Here,we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers,or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D?+?2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D?+?2D shaker protocol) or in a PBS bioreactor (the 3D?+?2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA],smooth muscle protein 22 [SM22],and Calponin-1),and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay),contraction in response to the treatment with a prostaglandin H2 analog (U46619),and tube formation on Geltrex,as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D?+?2D protocols yielded?>?5.6?×?107 hiPSC-SMCs/differentiation,which was?~?nine-fold more than that produced via 2D differentiation,and flow cytometry analyses confirmed that?>?98% of the 3D?+?2D-differentiated hiPSC-SMCs expressed SMA,?>?81% expressed SM22,and?>?89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D?+?2D shaker protocol also displayed typical SMC-like migratory,contraction,and tube-formation activity in-vitro and significantly improved measurements of perfusion,vessel density,and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D?+?2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice. 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

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

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

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

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

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment,and confirming their activity through orthogonal functional assays is crucial. Here,we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~ 35% of the tested enhancers,with most showing temporal-specific activity,suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community. View Publication

SummaryBackgroundPresence of nerves in tumours,by axonogenesis and neurogenesis,is gaining increased attention for its impact on cancer initiation and development,and the new field of cancer neuroscience is emerging. A recent study in prostate cancer suggested that the tumour microenvironment may influence cancer progression by recruitment of Doublecortin (DCX)-expressing neural progenitor cells (NPCs). However,the presence of such cells in human breast tumours has not been comprehensively explored.MethodsHere,we investigate the presence of DCX-expressing cells in breast cancer stromal tissue from patients using Imaging Mass Cytometry. Single-cell analysis of 372,468 cells across histopathological images of 107 breast cancers enabled spatial resolution of neural elements in the stromal compartment in correlation with clinicopathological features of these tumours. In parallel,we established a 3D in vitro model mimicking breast cancer neural progenitor-innervation and examined the two cell types as they co-evolved in co-culture by using mass spectrometry-based global proteomics.FindingsStromal presence of DCX + cells is associated with tumours of higher histological grade,a basal-like phenotype,and shorter patient survival in tumour tissue from patients with breast cancer. Global proteomics analysis revealed significant changes in the proteomic landscape of both breast cancer cells and neural progenitors in co-culture.InterpretationThese results support that neural involvement plays an active role in breast cancer and warrants further studies on the relevance of nerve elements for tumour progression.FundingThis work was supported by the 10.13039/501100005416Research Council of Norway through its Centre of Excellence funding scheme,project number 223250 (to L.A.A),the 10.13039/100008730Norwegian Cancer Society (to L.A.A. and H.V.),the Regional Health Trust Western Norway (Helse Vest) (to L.A.A.),the 10.13039/501100008728Meltzer Research Fund (to H.V.) and the 10.13039/100000002National Institutes of Health (NIH)/10.13039/100000057NIGMS grant R01 GM132129 (to J.A.P.). View Publication

BackgroundThe simultaneous differentiation of human pluripotent stem cells (hPSCs) into both endodermal and mesodermal lineages is crucial for developing complex,vascularized tissues,yet poses significant challenges. This study explores a method for co-differentiation of mesoderm and endoderm,and their subsequent differentiation into pancreatic progenitors (PP) with endothelial cells (EC).MethodsTwo hPSC lines were utilized. By manipulating WNT signaling,we optimized co-differentiation protocols of mesoderm and endoderm through adjusting the concentrations of CHIR99021 and mTeSR1. Subsequently,mesoderm and endoderm were differentiated into vascularized pancreatic progenitors (vPP) by adding VEGFA. The differentiation characteristics and potential of vPPs were analyzed via transcriptome sequencing and functional assays.ResultsA low-dose CHIR99021 in combination with mTeSR1 yielded approximately 30% mesodermal and 70% endodermal cells. Introduction of VEGFA significantly enhanced EC differentiation without compromising PP formation,increasing the EC proportion to 13.9%. Transcriptomic analyses confirmed the effectiveness of our protocol,showing up-regulation of mesodermal and endothelial markers,alongside enhanced metabolic pathways. Functional assays demonstrated that vPPs could efficiently differentiate into insulin-producing ?-cells,as evidenced by increased expression of ?-cell markers and insulin secretion.ConclusionOur findings provide a robust method for generating vPPs,which holds significant promise for regenerative medicine applications,particularly in diabetes treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04120-5. View Publication

AbstractBackgroundRodent models have been widely used to investigate skin development,but do not account for significant differences in composition compared to human skin. On the other hand,two-dimensional and three-dimensional engineered skin models still lack the complex features of human skin such as appendages and pigmentation. Recently,hair follicle containing skin organoids (SKOs) with a stratified epidermis,and dermis layer have been generated as floating spheres from human-induced pluripotent stem cells (hiPSCs).MethodsThe current study aims to investigate the generation of hiPSCs-derived SKOs using an air-liquid interface (ALI) model on transwell membranes (T-SKOs) and compares their development with conventional floating culture in low-attachment plates (F-SKOs).ResultsMature SKOs containing an epidermis,dermis,and appendages are created in both T-SKO and F-SKO conditions. It was found that the hair follicles are smaller and shorter in the F-SKO compared with T-SKOs. Additionally,the ALI conditions contribute to enhanced hair follicle numbers than conventional floating culture.ConclusionsTogether,this study demonstrates the significant influence of transwell culture on the morphogenesis of hair follicles within SKOs and highlights the potential for refinement of skin model engineering for advancing dermatology and skin research. View Publication